Tag: vertical farming

TGB1 and TGB2 also localized with membrane vesicles even though extensive membrane proliferation was not observed. However, ectopically expressed TGB3 elicited formation of a complex and well-defined ER network that is closely associated with, or houses, thick actin cables and TGB proteins.Virus movement and membrane reorganization, which is especially obvious in the perinuclear region, was disrupted by mutations in the central membranespanning domain of TGB3. Our findings thus suggest that host membrane associations are involved in several aspects of BSMV movement that merit future study. In addition to differences in requirements of the coat protein for cell-to-cell movement of hordeiviruses and potexviruses, a number of variations are evident in TGB1 protein structure, biochemical activities, interference with host gene silencing, and movement functions of the virgaviruses and the TGB-containing flexiviruses . A recent paper illuminating a TGB1 requirement for formation of X bodies associated with PVX highlights another major difference in functions of the TGB1 proteins of the hordeiviruses and the potexviruses that relates to BSMV actin remodeling. In contrast to BSMV, in which TGB3 expression has a major effect on actin architecture, PVX TGB1 is essential for X-body formation and functions in extensive actin and membrane remodeling . The multilayered membranous X-body is an important organelle that is required for normal levels of viral RNA replication and virion accumulation. Nevertheless, in plants and protoplasts infected with PVX mutants unable to express TGB1, morphogenesis of X bodies fails to occur, yet low levels of PVX replication can be detected and small amounts of virus particles accumulate . However, ectopic expression of PVX TGB1 results in massive remodeling of host actin and endomembranes, greenhouse ABS snap clamp and recruitment of these structures, as well as TGB2 and TGB3, to sites near the nucleus. Subsequently, X-bodies develop into complex multilayered membrane organelles adjacent to the nucleus, that selectively incorporate TGB proteins, ribosomes, viral RNA and virions to specific sites within the granular vesicular bodies .

These differences between the two viruses are further illustrated by conventional electron microscopic observations showing that structures corresponding to PVX X-bodies are not present in BSMV-infected cells, and that BSMV replicates in membrane vesicles formed from the chloroplast outer membrane . Our current results add to a growing list of major differences in the movement processes of TGB-encoding viruses . We previously reported that cytochalasin D treatment failed to affect TGB1 localization in BSMV infected protoplasts and as a result, postulated that cytoskeletal interactions of the protein were relatively minor. However, the experiments presented here reveal both actin remodeling and changes to ER structure as a consequence of BSMV infection and transient expression of TGB3 and TGB2/3. Our observations also provide evidence that the subcellular localization of the TGB proteins depends on actin cytoskeleton interactions. To investigate these interactions in more detail, we used LatB to inhibit actin polymerization in cells infiltrated with TGBreporter proteins. In contrast to cytochalasin D used in our earlier experiments , LatB can be up to 100-fold more potent than cytochalasins, and functions by shortening and thickening of actin filaments. After LatB treatment, the DsRed:Talin patterns in N. benthamiana infiltrated epidermal leaf cells exhibited a major shift from a filamentous actin network to thick cablelike structures.From these experiments, we conclude that actin cytoskeleton modifications are required for BSMV movement and that TGB3 has a critical role in cytoskeleton remodeling during movement. In contrast to the BSMV LatB experiments described above, experiments with the closely related PSLV TGB3 have resulted in different conclusions about mechanisms functioning in PD targeting . In the case of BSMV, actin cytoskeleton disruption by LatB interfered with CW localization of TGB3, and TGB1 when coexpressed with TGB2/3, whereas PSLV TGB3 CW localization was not dramatically affected by LatB treatment . These disparate results highlight fundamental differences in the mechanisms of subcellular transit of BSMV and PSLV. Such differences between related viruses may occur more often than previously realized, as illustrated by a previous report describing differences in the movement of two tobamoviruses .

In this direct comparison, movement of TMV is strongly inhibited by LatB treatment, whereas movement of the related TVCV is unaffected by LatB treatment. These results argue strongly that more than one mechanism may be operative in some closely related viruses, and our collective results suggest that BSMV and PSLV may fit within this category. Evidence for TGB3 associations with the Golgi membranes during coexpression of DsRed:TGB3 and the STGFP Golgi marker indicates that Golgi derived vesicles and DsRed:TGB3 co-localize with the CW after plasmolysis. BFA interference with Golgi stack integrity resulted in a major collapse of vesicles localized in close proximity to the CW, but BFA appears to have only limited effects on BSMV localization or PSLV TGB3 associations with “peripheral bodies” . Nevertheless, differences in the BSMV and PSLV LatB cytoskeleton disruption experiments suggest that different mechanisms may function in some TGB3 interactions culminating in PD targeting. Other than the preliminary experiments shown above, which suggest that BSMV infection does not result in obvious changes to microtubules, we have not extensively investigated possible direct interactions of BSMV TGB proteins with microtubules. However, in other experiments with the related Potato mop-top virus , colchicine treatments were used to disrupt tubulin polymerization and microtubule integrity . Colchicine can affect multiple metabolic and regulatory processes affecting a large number of functions that might interfere with TGB1 localization to the CW. However, the PD associations of PMTV mutants provided evidence for an association between microtubules and PMTV TGB1. Of particular interest, cells were observed for several days after transient expression of the three TGB proteins in ratios corresponding to those occurring during virus infection. During this period, a defined series of kinetic events were noted, beginning with PMTV TGB1 nucleolar interactions and proceeding through cytoplasmic granules to the CW. Thus, the effects of BSMV and PMTV on microtubule remodeling, seem to differ, and these experiments reinforce our suggestion that multiple pathways may operate in CW targeting during TGB1 expression of the virgaviruses. Unfortunately, individual events involved in viral movement from subcellular sites of replication to the PD and adjacent cells are difficult to dissect experimentally, and many of these problems have been discussed previously .

The infection front where important events are coordinated is a moving boundary consisting of a limited number of cells undergoing a series of asynchronous steps, so relatively few studies have probed events at this stage of infection. Variations in delivery protocols also contribute to experimental differences or artifacts that can lead to aberrant subcellular trafficking effects. In this regard,flower pot wholesale examples of the effects of over expression of PSLV TGB3 has been described recently in which anomalous cell death, membrane abnormalities and disrupted Golgi functions occur during transient infection . Third, pharmacological approaches can be quite variable in the hands of different researchers. Finally, a more diverse array of approaches, including infectivity studies applied to different hosts might provide interesting insights into alternative strategies employed by BSMV and other hordeiviruses. Although it would be preferable to investigate movement in the natural BSMV cereal hosts, these plants present technical difficulties that are difficult to circumvent. Fortunately, BSMV, unlike PSLV, is able to infect N. benthamiana, so we have been able to compare cytological and biochemical experiments with infectivity results in this host.Rust fungi are an order of >7000 species of highly specialized plant pathogens with a disproportionately large impact on agriculture, horticulture, forestry, and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. Asian soybean rust caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is a prime example of the damage that can be caused by rust fungi. It is a critical challenge for food security and one of the most damaging plant pathogens of this century. The disease is ubiquitously present in the soybean growing areas of Latin America, where 210 million metric tons of soybean are projected to be produced in 2022/23 , and on average representing a gross production value of U.S. $ 115 billion per season . A low incidence of this devastating disease can already affect yields and, if not managed properly, yield losses are reported of up to 80%. Chemical control in Brazil to manage the disease started in the 2002/03 growing season. In the following season, ~20 million hectares of soybeans were sprayed with fungicides to control this disease. The cost of managing P. pachyrhizi exceeds $2 billion USD per season in Brazil alone. The pathogen is highly adaptive and individually deployed resistance genes have been rapidly overcome when respective cultivars have been released. Similarly, the fungal tolerance to the main classes of site-specific fungicides is increasing, making chemical control less effective. Another remarkable feature for an obligate biotrophic pathogen is its wide host range, encompassing 153 species of legumes within 54 genera to date. Epidemiologically, this is relevant as it allows the pathogen to maintain itself in the absence of soybean on other legume hosts, such as overwintering on the invasive weed Kudzu in the United States. Despite the importance of the pathogen, not much was known about its genetic makeup as the large genome size , coupled to a high repeat content, high levels of heterozygosity and the dikaryotic nature of the infectious urediospores of the fungus have hampered whole genome assembly efforts. In this work, we provide reference quality assemblies and genome annotations of three P. pachyrhizi isolates. We uncover a genome with a total assembly size of up to 1.25 Gb.

Approximately, 93% of the genome consists of TEs, of which two super families make up 80% of the TE content. The three P. pachyrhizi isolates collected from South America represent a single clonal lineage with high levels of heterozygosity. Studying the TEs in detail, we demonstrate that the expansion of TEs within the genome happened over the last 10 My and accelerated over the last 3 My, and did so in several bursts. Although TEs are tightly controlled during sporulation and appressoriaformation, we can see a clear relaxation of repression during the in planta life stages of the pathogen. Due to the nested TEs, it is not possible at present to correlate specific TEs to specific expanded gene families. However, we can see that the P. pachyrhizi genome is expanded in genes related to amino acid metabolism and energy production, which may represent key lifestyle adaptations. Overall, our data unveil that TEs that started their proliferation during the radiation of the Leguminosae play a prominent role in the P. pachyrhizi’s genome and may have a key impact on a variety of processes such as host range adaptation, stress responses and plasticity of the genome. The high-quality genome assembly and transcriptome data presented here are a key resource for the community. It represents a critical step for further in-depth studies of this pathogen to develop new methods of control and to better understand the molecular dialogue between P. pachyrhizi and its agriculturally relevant host, Soybean.The high repeat content and dikaryotic nature of the P. pachryrhizi genome poses challenges to genome assembly methods. Recent improvements in sequencing technology and assembly methods have provided contiguous genome assemblies for several rust fungi. Here, we have expanded the effort and provided reference-levelgenome assemblies of three P. pachyrhizi isolates using long-read sequencing technologies. All three isolates were collected from different regions of South America. We have used PacBio sequencing for the K8108 and MT2006 isolates and Oxford Nanopore for the UFV02 isolate to generate three high-quality genomes . Due to longer read lengths from Oxford nanopore, the UFV02 assembly is more contiguous compared to K8108 and MT2006 and is used as a reference in the current study . The total genome assembly size of up to 1.25 Gb comprising two haplotypes, makes the P. pachyrhizi genome one of the largest fungal genomes sequenced to date . Analysis of the TE content in the P. pachyrhizi genome indicates ~93% of the genome consist of repetitive elements, one of the highest TE contents reported for any organism to date . This high TE content may represent a key strategy to increase genetic variation in P. pachyrhizi. The largest class of TEs are class 1 retrotransposons, that account for 54.0% of the genome.

High frequency irrigation systems involve fastidious planning and complex designs, so that timely and accurate additions of water and fertilizer can result in sustainable irrigation. At the same time these production systems are becoming more intensive, in an effort to optimise the return on expensive and scarce resources such as water and nutrients. Advanced fertigation systems combine drip irrigation and fertilizer application to deliver water and nutrients directly to the roots of crops, with the aim of synchronising the applications with crop demands , and maintaining the desired concentration and distribution of ions and water in the soil . Hence a clear understanding of water dynamics in the soil is important for the design, operation, and management of irrigation and fertigation under drip irrigation . However, there is a need to evaluate the performance of these systems, because considerable localised leaching canoccur near the drip lines, even under deficit irrigation conditions . The loss of nutrients, particularly nitrogen, from irrigation systems can be expensive and pose a serious threat to receiving water bodies . Citrus is one of the important horticultural crops being grown under advanced fertigation systems in Australia. Fertigation delivers nutrients in a soluble form with irrigation water directly into the root-zone, thus providing ideal conditions for rapid uptake of water and nutrients. Scholberg et al. demonstrated that more frequent applications of a dilute N solution to citrus seedlings doubled nitrogen uptake efficiency compared with less frequent applications of a more concentrated nutrient solution. Delivery of N through fertigation reduces N losses in the soil-plant system by ammonia volatilisation and nitrate leaching . However, poor irrigation management, i.e., an application of water in excess of crop requirements,hydroponic nft channel plus the storage capacity of the soil within the rooting depth, can contribute to leaching of water and nutrients below the rootzone.

Therefore, optimal irrigation scheduling is important to maximise the uptake efficiencies of water and nutrients . Most of the citrus production along the Murray River corridor is on sandy soils, which are highly vulnerable to rapid leaching of water and nutrients. Nitrogen is the key limiting nutrient and is therefore a main component of fertigation. An increasing use of nitrogenous fertilizers and their subsequent leaching as nitrate from the root zone of cropping systems is recognised as a potential source of groundwater contamination, because the harvested crop seldom takes up more than 25–70% of the total applied fertilizer . Several researchers have reported substantial leaching of applied N under citrus cultivation in field conditions . Similarly, in lysimeter experiments, Boaretto et al. showed 36% recovery of applied nitrogen by orange trees, while Jiang and Xia reported N leaching of 70% of the initial N value, and found denitrification and leaching to be the main processes for the loss of N. These studies suggest that knowledge of the nitrogen balance in cropping systems is essential for designing and managing drip irrigation systems and achieving high efficiency of N fertilizer use, thereby limiting the export of this nutrient as a pollutant to downstream water systems. Quantifying water and nitrogen losses below the root zone is highly challenging due to uncertainties associated with estimating drainage fluxes and solute concentrations in the leachate, even under well-controlled experimental conditions . Moreover, direct field measurements of simultaneous migration of water and nitrogen under drip irrigation is laborious, time-consuming and expensive . Hence simulation models have become valuable research tools for studying the complex and interactive processes of water and solute transport through the soil profile, as well as the effects of management practices on crop yields and on the environment .

In fact, models have proved to be particularly useful for describing and predicting transport processes, simulating conditions which are economically or technically impossible to carry out in field experiments . Several models have been developed to simulate flow and transport processes, nutrient uptake and biological transformations of nutrients in the soil . HYDRUS 2D/3D has been used extensively for evaluating the effects of soil hydraulic properties, soil layering, dripper discharge rates, irrigation frequency and quality, timing of nutrient applications on wetting patterns and solute distribution because it has the capability to analyse water flow and nutrient transport in multiple spatial dimensions . In the absence of experimental data we can use multidimensional models solving water flow and nutrient transport equations to evaluate the multi-dimensional aspect of nitrate movement under fertigation . However, earlier simulation studies have reported contradictory results on nitrate distribution in soils. For example, Cote et al. reported that nitrate application at the beginning of an irrigation cycle reduced the risk of leaching compared to fertigation at the end of the irrigation cycle. On the other hand, Hanson et al. reported that fertigation at the end of an irrigation cycle resulted in a higher nitrogen use efficiency compared to fertigation at the beginning or middle of an irrigation cycle. These studies very well outlined the importance of numerical modelling in the design and management of irrigation and fertigation systems, especially when there is a lack of experimental data on nutrient transport in soils. However, there is still a need to verify the fate of nitrate in soils with horticultural crops and modern irrigation systems. Therefore, a lysimeter was established to observe water movement and drainage under drip irrigated navel orange, and to calibrate the HYDRUS 2D/3D model against collected experimental data. The model was then used, in the absence of experimental data on nitrate, to develop various modelling scenarios to assess the fate of nitrate for different irrigation and fertigation schemes.The study was conducted on a weighing lysimeter assembled and installed at the Loxton Research Centre of the South Australian Research and Development Institute. The lysimeter consisted of a PVC tank located on 1.2 m × 1.2 m pallet scales fifitted with 4 × 1 tonne load-cells, and connected to a computerised logging system which logged readings hourly.

A specially designed drainage system placed at the bottom of the lysimeter consisted of radially running drainage pipes,nft growing system which were connected to a pair of parallel pipes, which facilitated a rapid exit of drainage water from the lysimeter. These pipes were covered in a drainage sock and buried in a 25-cmlayer of coarse washed river sand at the base of the lysimeter, which ensured easy flushing of water through the drainage pipe. A layer of geo-textile material was placed over the top of the sand layer to prevent roots growing down into it, as this layer was intended to be only a drainage layer. A healthy young citrus tree was excavated from an orchard at the Loxton Research Centre and transplanted into the lysimeter. A soil profile approximately 85 cm deep was transferred to the tank with the tree and saturated to remove air pockets and to facilitate settling. The final soil surface was around 10 cm below the rim of the tank. Soil samples were collected from0 to 20, 20 to 40, 40 to 60, 60 to 85, and 85 to 110 cm depths to measure bulk density and to carry out particle size analysis. Two months after transplanting, the lysimeter was installed amongst existing trees in the orchard. Measurements were initiated after about six months, in order to enable the plant to adjust to the lysimeter conditions. The lysimeter was equipped with Sentek® EnviroSCAN® logging capacitance soil water sensors installed adjacent to the drip line at depths of 10, 20, 40, 60, and 80 cm to measure changes in the volumetric soil water content. Drainage water was directed through flexible piping into a large bin installed below ground level. The experimental site was approximately 240 m from an established weather station, which measured air temperature, relative humidity, wind speed , rainfall, and net radiation.Irrigation was applied using 3 pressure compensated emitters with a discharge rate of 4 L h−1. Emitters were located on a circle 25 cm away from the tree trunk at an equal distance from each other . The irrigation schedule was based on the average reference evapotranspiration during the last 10 years at the site, multiplied by the crop coefficient taken from Sluggett . The cumulative crop evapotranspiration during the 29 day experimental period was equal to 65.3 mm, and daily ETC varied from 1.68 to 3.39 mm. Irrigation was initiated on 16 August 2010 and terminated on 13 September, 2010. Irrigation and rainfall were recorded daily and drainage volume was measured 3 times per week throughout the trial period. Daily irrigation was applied in 5 short pulses using an automated irrigation controller, with 2 h breaks between irrigation pulses. The amount of irrigation water applied was slightly higher than ETC for the period. A total of 70 mm of rainfall fell during the experimental period, including a single event of 52 mm on 3 September 2010.The simulation domain was represented by a 110-cm deep and 100-cm wide cylindrical cross section. Drip irrigation was modelled as a circular line source 25 cm from the centre of the lysimeter with a uniform water flux along the drip line.

This simplification was made to enable HYDRUS to model this problem in a 2D axi-symmetrical mode , rather than in a full 3D mode, which would be computationally much more demanding. Additionally, since the surface wetted area and input flux densities under drippers were dynamic, an option that we would not be able to model with HYDRUS in a 3D mode, we assumed that the simplification of the problem to axi-symmetrical 2D was adequate. Moreover, the drainage system laid out in the lysimeter also supported the use of an axi-symmetrical domain as the drainage pipes run in a circular fashion to collect and flush drainage water out of the lysimeter. The transport domain was discretized into 3294 finite elements, with a very fine grid around the dripper and near the outflow , with gradually increasing element spacing farther from these two locations . Simulations were carried out over a period of 29 days.Since most soils on which citrus is grown in South Australia are coarse textured soils with good drainage, high oxygen levels, low organic matter, and low microbial populations, denitrification and mineralisation was assumed to be negligible in this study. Similarly, the soil adsorption of nitrate was also considered to be negligible since both nitrate and solid surfaces are negatively charged. Plant uptake of non-adsorbing nutrients like nitrate is controlled mainly by mass flow of water uptake . Therefore, it was assumed that nitrate was either passively taken up by the tree with root water uptake or moved downward with soil water. Spatial distribution of nitrate in the transport domain was thus simulated using the convection–dispersion equation for a nonreactive tracer. The longitudinal dispersivity was considered to be 5 cm, with the transverse dispersivity being one-tenth of this . Similar values of these parameters have been used in other studies .Citrus trees in this region are fertilised from early September till March, and in drip systems fertilizers are mostly applied with the second irrigation pulse for the day. All fertigation scenarios reported here are hypothetical. Fertigation was assumed to be supplied with the same quantity of water as in irrigations without fertigation and to conform to the 2D axi-symmetrical domain. For the initial scenario, fertigation pulses were applied from 30 August 2010 at the rate of one fertigation pulse each day. These were followed by 2 days without fertigation and then another daily fertigation pulses. The resultant dose of N for the period from August till September was calculated based on recommended fertilizer application practices for 5–6 year old orange tree. The seasonal recommended dose of nitrogen for an orange tree of this age is 139 g N applied from September to March . Hence for the seasonal simulation, nitrogen was assumed to be applied in equal monthly doses , in similar pulses as described for the experimental period. The simulation was run for 300 days in order to evaluate the fate of seasonally applied nitrogen fertilizer in citrus. Further scenarios examining the impact of timing of nitrogen application on the efficiency of nitrogen uptake simulated a fertilizer application either at the beginning , middle , or end of the daily irrigation scheme. Since the daily irrigation consisted of 5 pulses, fertigation was applied during the 2, 3 and 4 irrigation pulse in the PF1, PF2 and PF3 scenarios, respectively. It is a common practice that the initial and final irrigation pulses are fertilizer free to ensure a uniform fertilizer application and flushing of the drip lines. In addition to these simulations, two continuous fertigation scenarios were also performed to compare pulsed and continuous fertigation.

The flow rate of an NFT (Nutrient Film Technique) hydroponic system depends on various factors, including the size of the system, the number of plants, and the specific requirements of the plants being grown. However, I can provide you with a general guideline for the flow rate in an NFT system.

In an NFT system, a thin film of nutrient solution continuously flows over the roots of the plants, providing them with water and nutrients. The flow rate is typically measured in liters per hour (L/h) or gallons per hour (GPH).

A common recommendation for the flow rate in an NFT system is around 1-2 liters per minute per square meter of growing channel. This means that for every square meter of NFT channel, the system should provide a flow rate of 60-120 liters per hour (or approximately 15-30 gallons per hour). This guideline ensures that there is a sufficient supply of water and nutrients to the plants’ roots while also allowing for efficient drainage and oxygenation.

Keep in mind that this is a general guideline, and the flow rate can be adjusted based on the specific needs of your plants, the environmental conditions, and the stage of growth. It’s always a good idea to consult specific resources or seek advice from experienced hydroponic growers to determine the optimal flow rate for your particular setup.

Hydroponic farming offers various methods for growing plants without soil. Here are some of the different ways to practice hydroponic farming:

1. Nutrient Film Technique (NFT): In this method, a thin film of nutrient-rich water continuously flows over the roots of the plants, allowing them to absorb nutrients while also receiving oxygen. The plants are usually placed in troughs or channels with a slight slope to facilitate the flow of the nutrient solution.
2. Deep Water Culture (DWC): DWC involves suspending plant roots in a nutrient solution with the support of a floating platform or raft. The roots are submerged in the oxygenated solution, providing constant access to nutrients and oxygen.
3. Drip System: Drip irrigation systems deliver nutrient-rich water directly to the plant’s root zone through small tubes or emitters. The solution is dripped or sprayed onto the medium or roots, allowing the plants to absorb the nutrients. Excess solution is collected and recirculated.
4. Aeroponics: Aeroponics is a method where the plants’ roots are suspended in air, and a nutrient mist is sprayed directly onto the roots. This method ensures maximum oxygenation for the roots and efficient nutrient uptake.
5. Wick System: The wick system is a passive hydroponic method where a wick, such as a cotton rope, transfers the nutrient solution from a reservoir to the plant’s growing medium. The wick draws up the solution, providing moisture and nutrients to the roots.
6. Ebb and Flow (Flood and Drain): This method involves periodically flooding the growing tray or container with nutrient solution and then allowing it to drain away. The flooding and draining cycles provide oxygen to the roots while ensuring they receive the necessary nutrients.
7. Vertical Farming: Vertical hydroponic systems utilize vertical space to grow plants in stacked layers or towers. This method maximizes the use of limited space and allows for high-density cultivation.
8. Nutrient Film Technique (NFT): The NFT system uses a constant flow of nutrient-rich water in a shallow channel, allowing the plant roots to access the solution while being exposed to air. The nutrient film provides continuous nutrient supply and oxygenation.

These are just a few examples of the many hydroponic farming methods available. Each method has its own advantages and considerations, and the choice depends on factors such as plant type, available space, resources, and personal preference.

A strawberry hydroponic gutter system is a method of growing strawberries using a hydroponic system that utilizes gutters as the growing medium. Hydroponics is a technique for cultivating plants without soil, where the plants receive all the necessary nutrients through a water-based solution.

In a hydroponic gutter system specifically designed for strawberries, a series of gutters are set up at an incline. The gutters typically have holes or channels where the strawberry plants are placed. The plants’ roots hang down into the flowing nutrient-rich water solution.

Here are the key components and considerations for a strawberry hydroponic gutter system:

1. Gutters: The gutters used in the system can be made of various materials such as PVC, metal, or plastic. They should be designed to hold the plants securely while allowing the roots to access the nutrient solution.
2. Nutrient Solution: The nutrient solution contains a balanced mix of essential nutrients that strawberries need for healthy growth. It typically includes macronutrients (such as nitrogen, phosphorus, and potassium) and micronutrients (such as iron, zinc, and calcium). The solution is regularly pumped or flowed through the gutters, ensuring the roots have access to the nutrients.
3. Substrate: A substrate or growing medium may be used in the gutter system to support the strawberry plants. Common options include coconut coir, perlite, vermiculite, or rockwool. The substrate holds moisture and provides stability for the plants.
4. Irrigation: An automated irrigation system is necessary to supply the nutrient solution to the gutters. This can be accomplished using drip irrigation, where small tubes or emitters deliver a regulated amount of nutrient solution to each plant or gutter.
5. Lighting: Depending on the location and available natural light, supplemental artificial lighting may be needed to ensure the strawberries receive sufficient light for photosynthesis. LED grow lights are commonly used in hydroponic systems to provide the specific light spectrum needed for plant growth.
6. Climate control: Controlling the temperature, humidity, and ventilation is crucial for optimal strawberry growth. Proper airflow and ventilation help prevent diseases and provide an environment suitable for plant development.
7. Planting and maintenance: Strawberry plants are typically propagated from runners or small transplants and placed into the gutter system. Regular monitoring of pH levels, nutrient concentrations, and overall plant health is necessary. Pruning, removing dead leaves, and ensuring pollination (if not using self-pollinating varieties) are also important for productive plants.

A hydroponic gutter system offers several advantages for growing strawberries. It allows for high-density planting, efficient use of space, and easier access to the plants for maintenance and harvesting. Additionally, since hydroponics avoids soil, the risk of soil-borne diseases and pests is minimized.

However, it’s important to note that designing and managing a hydroponic gutter system requires some expertise in hydroponic gardening. It may be beneficial to consult resources, attend workshops, or seek guidance from experienced growers to ensure the best results.

Low resolution phylogenetic trees made using the chloroplast regions mentioned above have been reported for other taxa, including Curcuma and Sisyrinchium . The inadequate resolution may be due to the lower substitution rates and lack of variation found in single plastid regions. Thus, we do not recommend single plastid regions as DNA barcodes for this the genus. Among the candidate barcode genes, the Consortium for the Barcode of Life Plant Working Group suggested that rbcL, matK, and the rbcL+matK combination should be sufficient for a plant barcode, and that this combination should be supplemented with additional markers as required . In addition, Kress et al. and Chase et al. proposed that trnH-psbA can be used in two-locus or three-locus barcode systems to improve resolution. For instance, two of the three combinations of the three chloroplast loci tested in this study, rbcL+trnH-psbA and rbcL+matK+trnH-psbA exhibited higher discriminatory performance than any single marker. Consequently, this highlights the need to use chloroplast multi-locus barcodes to improve the resolution of species identification in Pulsatilla. The nuclear ITS region provided the highest inter-and intraspecific divergences and had a higher success rate for the correct identification of species in TAXONDNA . However, as for the treebuilding method,large pot with drainge the discriminatory performance of ITS is not satisfactory, as its highest resolution is 39.02% .

As evidenced by previous studies, the multi-locus barcode is one of the combinations that demonstrated the highest species resolution rate, e.g., Aceraceae , Lysimachia , Oberonia , Rhodiola and Schisandraceae . However, in this study, addition of ITS to different kinds of combinations of chloroplast markers did not increase the resolution rate obviously . The resolution rate based on tree-building analyses was 51.22% for BI and 58.54% for PWG. In addition, we found no distinct barcoding gap. This phenomenon may be due to the one or more of several reasons. first, incomplete lineage sorting and non-homogeneous concerted evolution are likely to occur at the ITS locus . Second, the three chloroplast regions cannot compensate for the drawbacks of ITS because they are sourced from a different genome. Although the nuclear genome is inherited biparentally, the chloroplast genome is inherited uniparentally. Thus, the chloroplast genome experiences more complete lineage sorting than the ITS locus does. Third, hybridizations may cause conflicts between ITS and chloroplast loci, as well as problematic results in ITS phylogeny due to the possibility of homogenization to paternal copies in some lineages and maternal copies in others. A combination of DNA markers from different genomes— which have different modes of inheritance and conflicting phylogenies—can hinder our understanding of species delimitation and the evolutionary processes of speciation. Because of its myriad variable sites that can reliably distinguish species, resulting from a high mutation rate and rapid concerted evolution, we recommend ITS as a good single barcode for the genus Pulsatilla.Phylogenetic identification and species recognition are foundationally important for biology . The results of the phylogenetic analyses performed in this study may shed some light on the identification and taxonomy of the genus Pulsatilla . Here, we found that Pulsatilla formed a monophyletic group with high support. Moreover, the three recognized subgenera — i.e. subg. Pulsatilla, subg. Kostyczewianae, and subg.

Preonanthus— were resolved as distinct monophyletic groups, which is consistent with the recent phylogenetic result . Within subgenus Pulsatilla, our analyses found that P. camanella and P. ambigua were resolved as sister to one another with high support. These two species share many common morphological characters, such as almost fully expanded leaves at anthesis,black plastic planting pots and dense, long trichomes. The flowers of both species nod before anthesis . However, during anthesis, the sepals of P. camanella can easily be distinguished from those of P. ambigua by color . At the same time, the micro-morphological characters of the leaves are also different . Actinocytic and anomocytic stomata exist in both species, but most stomata in P. camanella are actinocytic, whereas most are anomocytic in P. ambigua. Thus, molecular data as well as micromorphological characters can distinguish between these two species relatively well. Both types of evidence may be helpful to accurately identify specimens that are damaged or lack sufficient diagnostic characters. In addition to its use in identifying specimens, DNA barcoding is also useful for resolving taxonomic uncertainty .They did not have distinct barcodes. The micro-morphological characters were also found to be the same, since both plants showed polygonal epidermal cells with striation, a dense distribution of stomata, and glabrous or sparsely short trichomes. In addition, the geographical distribution of these two species overlaps in Inner Mongolia. Taken together, these distinct lines of evidence collectively suggest that P. turczaninovii and P. tenuiloba are the same species. The discovery of hybridization, introgression, and/or incomplete lineage sorting among species is another useful application of DNA barcoding . The chloroplast region is inherited maternally, but the nuclear genome, including the ITS region, is inherited biparentally . Thus, if there are different results in different phylogenetic analyses from chloroplast and nuclear data, we speculate that these differences may be caused by hybridization and/or introgression among species, which could result in a non-monophyletic clade. In subg. Pulsatilla, we found several complex groups. The samples of P. chinensis and P. cernua in cluster III, were indistinguishable. In the Bayesian inference tree based on ITS sequences , the samples of P. cernua clustered in a clade along with sample P. chinensis. However, in the Bayesian inference tree based on the combination of chloroplast sequences , sample P. chinensis clustered in a clade with all other samples of P. chinensis. P. chinensisis a widespread species and has a geographical range that covers that of P. cernua; in addition, sample P. chinensis was collected near populations of P. cernua in Jilin Province, China. Hybridization or introgression might have occurred during the speciation of P. chinensisis and P. cernua. A similar situation was also found for sample P. ambigua108 and cluster I , suggesting hybridization may have occurred between P. ambigua and P. tenuiloba/P. turczaninovii.

In addition, as we strictly controlled cropload to a similar level each year by thinning flowers and young fruits, dry matter accumulation was not drastically different between the transgenic lines and the CK in our experiment.In response to a decreased sorbitol supply from source leaves, both the transcript level and the activity of SDH decreased in the transgenic fruit, which is consistent with previous findings in apple fruit cortex tissues fed with sorbitol and in shoot tips and fruit of the transgenic trees. As most of the fructose in apple fruit is converted from sorbitol by SDH, a significantly lower fructose level had been predicted in the transgenic fruit based on dramatically reduced import of sorbitol into the transgenic fruit and the associated lower SDH activity. However, the fructose level in the transgenic fruit was remarkably similar to that in the untransformed CK: no difference before 74 DAB and at harvest with only a slight difference detected at rapid fruit expansion between the transgenic fruit and the CK . This near homeostasis of fructose level in the transgenic fruit has clearly resulted from the response of the Sucrose cycle and the associated sugar transport system to increased availability of sucrose in the transgenic fruit, specifically, more sucrose is taken up into parenchyma cells in fruit after phloem unloading; more fructose is generated from sucrose breakdown by NINV and sucrose synthase and less fructose is phosphorylated by FK in the cytosol; and tonoplast sugar transporters are upregulated to take up more hexoses into the vacuole. In apple fruit, sucrose as well as sorbitol enters the parenchyma cells via the apoplastic pathway after being released from SE-CC complex. In many species that employ apoplastic unloading for sucrose in sink cells,draining plant pots sucrose is mainly converted to glucose and fructose by CWINV in the cell wall space and then transported into the parenchyma cells by hexose transporters. CWINV is typically considered as a sink-specific enzyme and its activity is usually very low in source leaves.

However, we found that, except for MdCWINV3 in 40- DAB fruit, the expression of MdCWINVs was much lower in the fruit than in the shoot tips where sucrose unloading is symplastic . In yeast cells expressing apple SOTs, sorbitol uptake is competitively inhibited by glucose and fructose but not by sucrose. So we postulate that most sucrose is directly transported into the parenchyma cells by plasma membrane-bound SUCs in apple fruit to avoid inhibition of sorbitol uptake by sucrose-derived glucose and fructose. Increased sucrose import into transgenic fruit did not alter the activity of CWINV but significantly elevated the transcript levels of both MdSUC1 and MdSUC4 , indicating that more sucrose is taken up into the parenchyma cells in the transgenic fruit. There are two pathways for sucrose breakdown in the cytosol of fruit parenchyma cells: conversion to fructose and glucose by NINV or to fructose and UDP-glucose by SUSY. The upregulation of transcript levels of MdNINV1, MdNINV3, and MdSUSY1-3 and activities of NINV and SUSY in the transgenic fruit , which is indicative of higher availability of sucrose in the cytosol, generates more fructose. This, combined with a lower FK2 transcript level and a lower FK activity , makes enough fructose available in the cytosol for accumulation in the vacuole of the transgenic fruit to largely compensate for the reduced level of sorbitolderived fructose. The higher NINV activity is also expected to elevate the glucose level in the cytosol, which may have led to higher transcript levels of MdHKs and a higher HK activity through glucose signaling and a higher dark respiration rate in the transgenic fruit . The higher HK activity detected in the transgenic fruit is similar to that of rice leaves in response to glucose manipulation. However, we found that increases in both HK activity and the glucose concentration did not enhance, but rather diminished, the accumulation of G6P in the transgenic fruit. This is likely due to a decrease in F6P flux from phosphorylation of fructose along with an increase in dark respiration such that more G6P was reversibly converted to F6P. Our result is consistent with the finding that glucose derived from sucrose contributes to the hexose phosphate pool more than fructose derived from sorbitol or sucrose in the apple fruit.

Despite a higher glucose flux going through dark respiration in the transgenic fruit, more glucose is still available for transport into vacuole for accumulation as indicated by the 3–6-fold increase in glucose concentration in the transgenic fruit at harvest. Higher fruit glucose levels have also been reported for these antisense plants by Teo et al.but to a lesser degree. It is interesting that greater import of sucrose did not significantly increase its concentration in transgenic fruit except at 74 DAB. We think that two factors may have contributed to this near homeostasis of sucrose in the transgenic fruit. first, upregulation of sucrose breakdown described above uses more sucrose. Second, downregulation of MdSPS3 and MdSPS6 transcript levels and SPS activity in the transgenic fruit makes less sucrose re-synthesized from F6P and UDP-glucose. While upregulation of SUSY in response to increased sucrose supply was observed in both fruit and shoot tips of the transgenic plants, NINV responded in the transgenic fruit but not in the shoot tips. The exact reason for this difference is not known, but differences in sucrose concentration and/or presence of different isoforms of NINV between fruit parenchyma cells and shoot tips might exist. Our findings on activities of SUSY, NINV, FK, HK, and SPS are not in agreement with those reported by Teo et al.. We believe that the discrepancy might be related to the difference in the way fruit samples were taken. In our study, it took about 2 min to cut and freeze fruit samples on site in the orchard, but in Teo et al.all harvested fruits were placed on ice before being transported to the laboratory and it was only after several quality indices were measured that the cortical tissues were frozen for further analysis. Because import of sorbitol and sucrose into fruit stops upon detachment from the tree, both enzyme activity and gene expression may be altered if they are not frozen in liquid nitrogen in a very short period of time. In addition, strict cropload CK in our study as reflected in much larger fruit might have made the difference between the transgenic fruit and the CK easier to be detected. Most of the hexoses and sucrose in fruit parenchyma cells are stored in the central vacuole that occupies >80% of the cell volume.

The uptake of these sugars into the vacuole is carried out by sugar transporters located on the tonoplast. Transcript levels of MdvGT1 and MdvGT2, both of which are vacuolar glucose transporters encoded by two Malus orthologs of AtvGT, were higher in the transgenic fruit than in the CK , suggesting that more glucose is transported into the vacuole of the transgenic fruit. This is consistent with the glucose concentration measured on bulk fruit samples . In addition, tonoplast monosaccharide transporters can transport both glucose and fructose into the vacuoles,drainage gutter and Arabidopsis TMT1 activity for fructose is approximately 30% of that for glucose. In the five Malus orthologs of TMT, it is possible that proteins encoded by MdTMT1 and/or MdTMT2 have high ability to transport fructose, and the enhanced expression by MdTMT1 in the transgenic fruit might indicate a regulatory response to the reduced flux of fructose derived from sorbitol. Alternatively, as fructose-specific TMTs have not been identified in fructose-accumulating fleshy fruits, the upregulation of MdTMT1 could be triggered by higher levels of glucose derived from sucrose in the transgenic fruit. In addition to hexoses, the vacuoles in ripening apple fruit accumulate a high concentration of sucrose. So far, no SUC has been identified to have proton-coupled antiport activity for loading sucrose into the vacuole, but AtTMT1/2 probably represents a proton-coupled antiporter capable of transporting both glucose and sucrose into the vacuole. A recent report on TMTs in sugar beet indicates that one of the two TMT2 proteins has developed specific affinity to sucrose and is responsible for sucrose accumulation in the taproots. The expression patterns of both MdTMT1 and MdTMT2 are in general agreement with that of sucrose accumulation in our apple fruit. It has been demonstrated that interruption of carbohydrate import into fruit by girdling or adjustment of cropload did not alter the fructose level in apple fruit. Contrasting light exposure did not appear to affect peel fructose level either. The data obtained from the transgenic fruit in this study provides further evidence for supporting the idea that the Sucrose cycle and the associated transport system operates to maintain the homeostasis of fructose in the apple fruit. From an evolutionary perspective, having fructose homeostasis in the apple fruit may help seed dispersal for this species because fructose is the sweetest among all the soluble sugars present in fleshy fruits. In conclusion, when sorbitol synthesis is decreased by antisense suppression of A6PR in the source leaves of apple trees, less sorbitol but more sucrose is transported from the leaves to the fruit. In response to the lower sorbitol/higher sucrose supply, sorbitol metabolism is down regulated, whereas breakdown of sucrose is upregulated in the transgenic fruit to compensate for the decreased flux of fructose derived from sorbitol.

This altered sugar metabolism, together with corresponding changes in the sugar transport system, leads to near homeostasis of fructose and sucrose and much higher levels of glucose and galactose in the transgenic fruit. This study clearly demonstrates the metabolic flexibility and the advantages of having two transport carbohydrates in sorbitol-synthesizing Rosaceae tree fruit species and the central role of the Sucrose cycle and the sugar transport system in determining sugar metabolism and accumulation in fleshy fruits.Five-year-old trees of the untransformed CK and transgenic lines of “Greensleeves” apple with antisense suppression of A6PR expression were used. A6PR activity in mature leaves of A27 and A04 was decreased to about 30% and 15% of that of CK, respectively. All trees were grafted onto M.26 root stocks and grown outdoors at Ithaca, NY, USA, under natural conditions, in 55-L plastic pots containing a sand:MetroMix 360 medium . There were five replicates for each genotype with three trees each arranged in a completely randomized design. The trees were trained as a spindle system at a density of 1.5 × 3.5 m2 . They were moved into a screen house for the entire bloom period to prevent pollen escape and hand-pollinated using mixed pollen of crab apple and several commercial varieties. The cropload of these trees was adjusted by hand-thinning to four fruits per cm2 trunk cross-sectional area at 10-mm king fruit size. During the growing season, the trees were supplied twice weekly with 15 mM N using Plantex® NPK with micro-nutrients .At 40 DAB , 74 DAB , 108 DAB , and 134 DAB , fruits were sampled from the south side of the tree canopy between 12 noon and 2:00 P.M. under full sun exposure. On each sampling date, five replicates per genotype with at least six fruits each from three trees were harvested. The fruits were immediately weighed, cut into small pieces after removing the core, and frozen in liquid nitrogen on-site. The entire process took approximately 2 min. To estimate the levels of transport carbohydrates, five replicates of leaf petioles and fruit pedicels with four each were covered with aluminum foil for 7 days prior to sampling on 75 DAB and were frozen in liquid nitrogen along with mature leaves. All samples were stored at −80 °C.Net CO2 assimilation rates of bourse shoot leaves were measured using a CIRAS-1 portable photosynthesis system with a broad leaf chamber at 5 tree developmental stages from 30 DAB to fruit harvest. On each sampling date, two bourse shoot leaves per replicate were measured at mid-day under full sun exposure and ambient temperature and relative humidity conditions. Fruit respiration was measured with the CIRAS-1 gas exchange system connected to a custom-made chamber, which accommodated the entire fruit for each sample. In the early afternoon on each of those four sampling dates, fruits under full sun exposure were detached from the trees and dark-adapted at 25 °C for 30 min before taking respiration measurements, and one fruit per replicate was measured.Although a large literature describes how recessions affect non-agricultural labor markets, few studies examine the effects of recessions in the seasonal agricultural labor market.

The extreme remodeling of pods by St and To eliminates pod shattering, but makes pods extremely difficult to thresh , and the alleles are therefore impractical for dry bean market classes. Since arid conditions are predicted to increase in coming decades , shattering-resistance alleles will be of increasing value for plant breeders. Despite this, little information exists on the degree of pod shattering in major market classes, the pattern of dominance and epistasis between resistance alleles, or the diversity available at each of these loci. Crucially, breeders also still lack genetic assays to evaluate the trait in segregating populations. Addressing these barriers will be critical to improve the productivity of a major source of nutrition globally. Three populations were evaluated in this study: a biparental population and two diversity panels, which represent each of the two domestication events of common bean. The biparental population was developed to study two shattering-related QTLs, their patterns of dominance and their interactions. Cultivars ‘Maylower’ and ‘Bill Z’ showed total resistance to pod shattering when field grown in Davis in 2017 . These varieties were among the most distantly related accessions in the MDP, with neither showing any evidence of admixturebetween ecogeographic races . May lower is a navy bean type , which possesses a SNP allele on Pv08 that is weakly associated with resistance to pod shattering in race Mesoamerica. Bill Z is a pinto bean type and has a SNP variant on Pv03 associated with strong PvPdh1-mediated shattering resistance common in race Durango. The population can therefore be used to determine if a reduction in pod shattering was independently selected in each of these ecogeographic races. An F3 population of 138 individuals was developed by hybridization between these cultivars. Each F3 individual was descended from a distinct F2 plant,best vertical garden system and all of the F2 s were the progeny of a single F1 developed by cross-pollinating Maylower and Bill Z. This 138-member Maylower x Bill Z population was used to validate the possible alleles on Pv03 and Pv08 and test any patterns of dominance and epistasis between the loci.

The two diversity panels were grown to evaluate the degree of pod shattering across diverse accessions of common bean. In 2016, 98 members of major market classes in the Andean Diversity Panel were ffield-grown in Davis, California, to evaluate each variety’s susceptibility to pod shattering. In 2017, 278 varieties of the BeanCAP Middle American Diversity Panel were similarly ffield-grown in Davis to evaluate pod shattering. At maturity, a sample of pods was harvested from each variety. Mature pods of all phenotyped varieties were harvested and then exposed to seven days of desiccation at 65 °C and a further seven days of re-equilibration to room temperature. The desiccation conditions for all varieties were identical, and desiccation was conducted using the same drying chamber. The proportion of pods dehiscing in this treatment was recorded, along with the market class of each variety. For evaluation of pod twists in the MxB population, all non-shattering pods were fractured by hand, and then, all pods were subjected to the desiccation treatment and re-equilibration again. The number of twists was counted for ten pods of each genotype, with “1” indicating a complete 360° rotation of the valve.DNA was extracted from young trifoliate leaves of the greenhouse-grown biparental MxB F3 generation, using a modiied CTAB protocol . DNA was quantiied with a NanoDrop spectrophotometer and genotyped using the BARCBean6K_3 BeadChip , yielding 5398 initial SNPs. SNPs that were missing or heterozygous in either parent or identical between the parents, were filtered from further analysis. The remaining SNPs were arranged into a linkage map using the ASMap R package . SNPs that did not map to one of the 11 major linkage groups were removed, leaving 1794 SNPs for QTL mapping. QTL mapping was conducted using the expectation maximization method in R/qtl . Phenotypes for QTL mapping were generated by harvesting all the pods from each greenhouse-grown F3 plant , then subjecting them to seven days at 65 °C and seven further days of re-equilibration to room temperature. Pods that had fractured to the tip of the beak due to this treatment were counted as shattered, while those with no opening or only issuring along the sutures were considered non-shattering.The maximum LOD score of 1000 randomized analyses of the data was used as a significance threshold. To test dominance, F3 individuals were subset by genotype at highly significant SNPs, and comparisons were made between groups by t-test.Next, the 43 SNPs within 100 kb of PvPdh1 in the MDP data set were analyzed to identify patterns of selection and diversity around the gene.

To simplify and visualize the data, principal component analysis was performed on the SNPs using R. Sequence variation was converted to integer values and the imputePCA function of the missMDA package was used to impute missing data . The genotype data were also sorted to identify unique haplotypes within the populations. The degree of similarity between the PCA and haplotype diversity was then compared. Individuals with missing data for SNPs distinguishing the haplotypes or haplotype clusters were not shown in plots and not numbered in plots as they could not be unambiguously placed within any haplotype group.The PvPdh1 putative causal polymorphism was used to develop a Cleaved Ampliied Polymorphic Sequence marker for efficient screening of breeding populations. The sequence surrounding the SNP was extracted using Phytozome 12 . Restriction enzymes that would differentially cut the alternative alleles were identified using RestrictionMapper version 3.0 . PCR primers were developed for the locus based on the sequence of accession G19833 , using the NCBI primer BLAST tool, and were then checked against the genome sequence of BAT93 to ensure that the sequences were identical and would successfully amplify membersof both major gene pools. The sequence surrounding the SNP was ampliied using the primers PDH1-TAQII-2F and PDH1-TAQII-2R . PCR was conducted with Takara ExTaq and included an initial elongation at 95 °C for three minutes, 44 cycles with denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, elongation at 72 °C for 60 s, and a final elongation of 72° for five minutes. PCR products were cleaved with ChimerX TaqII during a 65 °C degree incubation for seven hours, and run on a 2.5% agarose gel. The SNPs tightly linked to PvPdh1 in the MDP data set were then screened for other positions that could be useful for conversion to additional CAPS markers. The SNP closest to PvPdh1 in this data set, at Pv03 position 49,132,438 , is distinguishable by EcoRI and is highly correlated with pod shattering. Unlike the TaqII-based CAPS marker, the allele cleaved by EcoRI is the shattering-resistant variant, reducing the risk of falsely identifying a susceptible individual as resistant due to technical errors in digestion. The SNP distinguished by EcoRI is separated from the PvPdh1 causal polymorphism by less than 7 kb. The sequence surrounding this SNP was ampliied using the primers PDH1-ECORI-1F and PDH1-ECORI-1R . Marker development used the same methods as the TaqII-distinguishable marker, and the same PCR conditions successfully amplified both fragments.

The amplicons were then digested by Promega EcoRI at 37 °C for 15 min, and the PCR products were resolved on a 2.5% agarose gel. Major discrepancies in pod shattering exist between the major market classes of common bean . In the Andean gene pool, pod shattering is highest in the cranberry market class,vertical farming equipment with a mean value of 41% of pods shattering after desiccation. The purple speck/mottled market class has the greatest degree of shattering resistance among Andean beans, with only 3% of pods shattering after the same treatment. In Middle American beans, pod shattering is highest in the black and navy/small white market classes of race Mesoamerica, and lowest in the pinto , great northern and pink classes of race Durango. PvPdh1-mediated resistance to pod shattering is found almost exclusively in pinto, great northern, and pink market classes and is therefore associated with levels of pod shattering which may be the lowest of those of any major economic groups of common beans grown for grain.Three major SNP haplotype clusters were identified in the sequence surrounding the PvPdh1 gene . The most distinct of these included six individuals, several of which are of known Andean ancestry. The first principal component of the genetic data explained 64% of the variation and separated this group from the two other major clusters. The second principal component explained 25% of the variation and separated varieties belonging to race Mesoamerica from race Durango. Five individuals with missing data for the ten SNPs that distinguish these ecogeographic races were filtered from subsequent analyses. Additionally, cv. ‘Tepary 22′ and cv. ‘Jackpot’ exhibited highly unique haplotype patterns. Jackpot shows recombination in the region between the predominant haplotype of the Andean gene pool and race Durango, while P. acutifolius is a separate but closely related species . Races Durango and Mesoamerica differed in haplotype diversity around the PvPdh1 locus . The race Mesoamerica haplotype cluster includes six unique haplotypes. The most common of these includes 137 of the 148 varieties that can be clustered into a group unambiguously , without missing data in the SNP positions distinguishing the sub-groups. The race Mesoamerica haplotypes displayed 18% shattering on average. In contrast, race Durango varieties display only three haplotypes. The most common of these haplotypes includes 178 of 182 unambiguous varieties , with an average proportion shattering of 0.6% in this group. The two low-frequency race Durango haplotypes showed no shattering when field-grown in 2017 .The TaqII-based CAPS marker of the PvPdh1 causal polymorphism leads to cleavage of susceptible alleles, while resistant alleles are not cut. The total pre-digestion amplicon length in G19833 was 578 bp, comparable to the 580 bp amplicon of BAT93. After digestion, susceptible alleles were cleaved into fragments of 449 and 129 bp in G19833. While digestion was seen in all shattering-susceptible samples after digestion with TaqII, this enzyme led to only partial digestion in a minority of cases. The EcoRI-digestible CAPS marker was extremely robust, and never led to partial digestion. After digestion, this marker led to resistant alleles that were cut into fragments of approximately 332 bp and 310 bp, while susceptible alleles remained uncleaved at 642 bp in BAT 93 . Andean varieties showed comparable fragment sizes, such as 639 bp in G19833. The EcoRI-digestible CAPS marker never experienced partial digestion or ambiguity. The SNP used for this marker has a strongly significant correlation with pod shattering , and is one of the 10 SNPs contributing to the haplogroup differentiation between race Durango and race Mesoamerica . The median proportion of pod shattering among 97 varieties with the shattering susceptible allele was 0.14, equal to the maximum level of shattering seen in any of the 160 varieties carrying the resistant allele . Themedian proportion shattering of those varieties with the resistant allele was 0.00%. Testing for multiple origins of Middle American shattering resistance and allelic effects Only one major locus was identified with an effect on pod shattering in the Middle American domesticated gene pool of common bean. These results highlight the important role of the Pv03 PvPdh1 locus in this population. The lack of a major QTL on Pv08 in the MxB population suggests that Maylower has no shattering resistance allele on that chromosome which is not also found in the distantly related Bill Z. Because the Pv08 SNP identified through GWAS did not reach significance after a Bonferroni correction and only 11 of 280 members of the MDP possessed this SNP, our results indicate that this chromosome does not have a major, widespread role in regulating pod shattering in the Middle American domesticated gene pool. This does not preclude the possibility that the QTL has a role in shattering of Andean beans, the latter of which has been demonstrated with much greater conidence . The locus may also have a role in regulating pod shattering in a very small proportion of Middle American bean varieties, possibly through de novo mutation in race Mesoamerica or introgression from the Andean gene pool.

Several of the cache pits were made entirely of flat slabs of quartz diorite that evidently had fallen from the ceiling of the shelter. In each case, these tabular rocks were carefully overlapped to make as tight a construction as possible .Other slab-lined cache pits differed in that the rocks were placed in a mosaic fashion rather than overlapping. No small chinking rocks were noted in any of these features. One of these was constructed globular, as opposed to tabular, form. In some cases, smaller rocks were used to chink spaces between the larger rocks . One cache pit , constructed of irregularly shaped rocks, lacked any small rocks or predominantly of tabular rocks and was placed such that two massive boulders were part of the overall construction. Incorporated into its construction were two metate fragments, both of granitic material. The fill of the feature consisted of miscellaneous rocks and soil, a mano and two mano fragments, and one complete unifacial block metate. Some ofthe milling implements or other rocks from the fill may have been used in its construction. The majority of the cache pits excavated at Indian Hill Rock shelter were constructed of medium- to large-sized rocks of overall chinking, and there were large spaces between the rocks. Some rocks may have been taken from this feature and reused in the construction of the overlapping cache pit or for another purpose. One of the rocks used in the feature is a bifacial block metate; also present was a rock with a patch of red hematite paint. Feature 19 was a well-constructed rock-Hned basin. It was fairly shallow and apparently represented the floor of a cache pit. Construction material consisted of about 40 rocks,hydroponic vertical garden including one core-hammerstone, nine block metate fragments of granitic material , and one unifacial mano.

This feature was unique among the cache pits in that it contained so many large metate fragments. The situation at Indian Hill Rock shelter, then, is one in which rock-lined cache pits were built in sandy matrix using either flat slabs or other rocks, including metate fragments. Care was taken in some of the cache pits to overlap the slabs used in their construction, and in others to chink interstices between rocks. This suggests attempts to make the structures rodent-proof. We therefore conclude that bags or baskets of foodstuffs, rather than equipment, most likely were stored in these cache pits. We are inclined to classify these cache pits as group specific, permanent furnishings at Indian Hill Rock shelter. Each cache pit may have been used many times, each time carefully roofed over, perhaps with bunch grass to keep the sand out, and then rocks, and concealed beneath the sand that formed the floor of the shelter. Concealment may have been considered important in the event other groups used the shelter. We thus interpret the cache pits as more or less “permanent” site furnishings of whatever group used Indian HiU Rock shelter, but not publicly available site furniture. In all cases the contents of the cache pits had been removed in antiquity. In most cases, only the rock-paved floors of the cache pits remained intact, and most of the rocks that formed the walls were lacking. In some cases large rocks all but filled the cache pits, apparently having been discarded there upon abandonment of the cache. The roofing rocks and wall rocks, which may have extended to near the former ground surface, must have been pulled up out of the site matrix and used elsewhere for the same or another purpose. The ethnographic description of “pulling up” the site furniture , or “site furnishings,” in this case using it elsewhere at the site for the same or for another purpose, appears to describe the situation at Indian Hill Rock shelter. The levels from which cache pits are believed to have been dug have few or no ceramic sherds, but ceramics are common in overlying levels.

This distribution leads to the conclusion that ceramic ollas may have replaced rock-lined cache pits after the inception of ceramic technology in the region. Ollas may have been buried in the same sheltered sites that formerly contained rock lined cache pits. Rock-lined cache pits are reported from two sites, Chapman Rock shelter No. 1 and Resurrection Shelter . Four rock lined cache pits were excavated at Chapman Rock shelter No. 1. These cache pits were lined with basalt slabs, and metates were used in the construction of two of them. They ranged from ca. 75 to 95 cm. in diameter and from 45 to 60 cm. in depth. Two of these cache pits contained historic artifacts ; the third was built from a comparable level below the surface . Linings in these cache pits included such material as bunch grass; buckwheat plant parts, Joshua tree fiber, tule matting, and twined basketry. The fourth cache pit possibly may be older, as it was built far below the others, at about 1.2 m. below the present surface. This cache pit was not lined, but contained debitage, a small biface, a slate pendant fragment, a basalt mano, a bone bead, pinyon hulls, and twined basketry. A large, rock-lined cache pit was partially exposed during test excavations at Resurrection Shelter on the eastern slope of the Coso Range near Darwin Wash. The maximum diameter of this feature is estimated to be about 2 m., the depth about 40 cm. It was constructed of large, blocky rocks. None of these appeared to be metates, nor was any chinking with smaller rocks noted. The pit was lined with alternate layers of bunch grass plants and Joshua treebark. A covering of rocks was not discernible, but a large pile of rocks just inside the entrance of the shelter and next to this cache pit once may have covered it. Artifacts in and around the cache pit include basket fragments, a remnant of a fiber brush, fiber cordage, pinyon nut shells, fiber cordage wrapped with strips of skin, and painted reed fragments . The shelter is very small, and it may have been used only for storage. Some or all of the artifacts could have found their way into the cache when opened. Since pinyon trees grow several kilometers away at higher elevations, the nut shells apparently did not get into the cache pit accidentally. The available evidence suggests the cache served for storage of pine nuts rather than equipment. A detailed report by the investigators is in progress, and radiocarbon analyses are planned for selected materials from this cache pit .

Insufficient information is available to make a determination of the organizational strategy of the people that built the cache pits in the Coso Range. Ethnographically, the region was occupied by Panamint Shoshoni, who shifted from a dispersed forager strategy during the spring and summer to a clustered collector strategy during the fall and winter. Crown gall of grapevine reduces the grape yield by 20% to 40% and trunk diameter approximately by 9%, compared to uninfected healthy grapevines . The causal agent of the grapevine crown gall, Agrobacterium vitis, is a different biovar from other Agrobacterium spp. because it can induce a grapevine specific crown gall . A. vitis has host specificity-related genes, such as pehA, which encodes polygalacturonase that is hypothesized to facilitate A. vitis attachment and systemic colonization on susceptible grapevine tissues . Another host specificity-related gene function is tartrate utilization, and the host specificity is explained by the use of tartaric acid from specific organic acids in grapevines . Crown gall disease is one of the difficult diseases to eradicate by chemicals in vineyards. As a strategy to prevent crown gall disease without chemical sprays, vertical vegetable tower the use of resistance plant cultivars and antagonistic microbial species have been developed. Some grapevine species, mostly Vitis vinifera, are susceptible to crown gall disease, and thus, root stocks from resistant species or crossed seedlings are used by many vineyards to prevent crown gall disease on the scions of the susceptible grapevine cultivars . Another prevention strategy is the use of a non-tumorigenic A. vitis strain as host-specific biological control of grapevine crown gall, in which its pretreatment inhibits transformation by tumorigenic A. vitis strains on the grapevine, leading to the inhibition of crown gall induction . Recently, various platforms have been used to attempt to identify disease resistance-related metabolites in grapevine, including nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry . However, when using gas chromatography mass spectrometry , extracted metabolite-profiling data can annotate information from the metabolite libraries . Derivatization techniques are required to process GC-MS metabolite profiling . Silylation is the most suitable derivatization method for non-volatile metabolites, such as hydroxyl and amino compounds . One silylation agent, Nmethyl-N–trifluoro-acetamide , has been used to reveal the whole range of metabolites in plants . However, the highest advantages of GCMS analysis in metabolomics are noted as well-organized stable protocols from sampling to data analysis, covering relatively a broad range of compound classes .

Principal component analysis is an unsupervised method that attempts to create a model of the data without a priori information, which is able to provide an overview of the whole metabolite profile and find the differential metabolite profiling within the group using a qualitative analysis . Projections of latent structures analysis is a supervised analytical method that enhances the separation between groups, combining variable importance for projection scores to obtain a proper cutoff value and increase its performance . Orthogonal projections of latent structures analysis add a single component to the PLS analysis as a predictor of the model, and the other components describe the variation orthogonal to the first predictive component . OPLS discriminant analysis provides useful information about putative biomarkers using an S-plot that combines the covariation and correlation from the model in a scatter plot . The VIP score in PLS-DA and correlation value in OPLS-DA provide cutoff values for finding significant metabolites. This experimental method using GC-MS and multivariate data analysis can identify new aspects of pathogenicity-related metabolites. Genetic, physiological and morphological changes of the host plant to the infection of Agrobacterium species and in the crown gall development have been well documented in relation to plant responses to the tumorigenic bacterium before T-DNA transfer and during gall development . However, relatively little has been studied on those changes occurring in the non-tumorigenic plant tissues that have been influenced by the disease development , which may broaden the holistic understandings of plant responses to the crown gall pathogen infection and disease development. This may enhance their practical applicability in the disease control; e.g., use of crown gall-resistant root stocks. Therefore, in the present study, we examined metabolite profiling in the intermodal tissues of grapevine species in relation to plant responses to the crown gall development in grapevines induced by the infection of A. vitis using GC-MS and statistical multivariate data analysis. Grapevines used in our study were obtained from the National Clonal Germplasm Repository . For Vitis spp., 11 to 22 green shoot cuttings were collected from NCGR fields in June 2015 and placed in a perlite bed for rooting at 25o C with mist for one month. The green shoot cuttings with root systems were transferred to a pot with bed soil for pathogen inoculation test. The pathogen, kindly provided by Dr. Burr, Cornell University, was A. vitis. K305. The bacterium was cultured on potato-dextrose agar  at 25o C for three days. The bacterial cultures grown on PDA were collected by the agar surface scratching in sterile distilled water , and the population density was examined by dilution plate method , which was diluted with SDW to make a bacterial suspension with a concentration of 109 colony-forming units ml–1. For inoculation, a wound was made in the internode of the green shoot using a 2-mm-day drill and 10 µl of the bacterial suspension was injected into the wound using a micropipette. After inoculation, the wound site was immediately wrapped with sealing film and the green shoot cuttings were grown at 25o C in a greenhouse. Symptom development was examined every other day after inoculation throughout the experimental period. The GC-MS system consisting of a gas chromatograph and mass detector was used for GC-MS analysis of the metabolites extracted from the tissue samples.