Non-Destructive Monitoring of Crop Fresh Weight and Leaf Area with a Simple Formula and a Convolutional Neural Network.

Crop fresh weight and leaf area are considered non-destructive growth factors due to their direct relation to vegetative growth and carbon assimilation. Several methods to measure these parameters have been introduced; however, measuring these parameters using the existing methods can be difficult. Therefore, a non-destructive measurement method with high versatility is essential. The objective of this study was to establish a non-destructive monitoring system for estimating the fresh weight and leaf area of trellised crops. The data were collected from a greenhouse with sweet peppers (Capsicum annuum var. annuum); the target growth factors were the crop fresh weight and leaf area. The crop fresh weight was estimated based on the total system weight and volumetric water content using a simple formula. The leaf area was estimated using top-view images of the crops and a convolutional neural network (ConvNet). The estimated crop fresh weight and leaf area exhibited average R2 values of 0.70 and 0.95, respectively. The simple calculation was able to avoid overfitting with fewer limitations compared with the previous study. ConvNet was able to analyze raw images and evaluate the leaf area without additional sensors and features. As the simple calculation and ConvNet could adequately estimate the target growth factors, the monitoring system can be used for data collection in practice owing to its versatility. Therefore, the proposed monitoring system can be widely applied for diverse data analyses.

Translating CO[Formula: see text] variability in a plant growth system into plant dynamics.

Plant growth occurs owing to the continuous interactions between environmental and genetic factors, and the analysis of plant growth provides crucial information on plant responses. Recent agronomic and analytical methodologies for plant growth require various channels for capturing broader and more dynamic plant traits. In this study, we provide a method of non-invasive growth analyses by translating CO[Formula: see text] variability around a plant. We hypothesized that the cumulative coefficient of variation (CCV) of plant-driven ambient CO[Formula: see text] variation in a plant growth system could yield a numerical indicator that is connected to the plant growth dynamics. Using the system outside-plant growth system-plant coupled dynamic model, we found that the CCV could translate dynamic plant growth under environmental and biophysical constraints. Furthermore, we experimentally demonstrated the application of CCV by using non-airtight growth chamber systems. Our findings may enrich plant growth information channels and assist growers or researchers to analyze plant growth comprehensively.

Stone Wool Substrate Cover Incision Impacts on the Root-Zone Water Content, Temperature, and Yield of Tomato Cultures.

Standardized cultivation systems are crucial for establishing reproducible agronomic techniques. Especially stone wool-based cultivation is governed by standardized specifications and provides a controllable root-zone environment. However, the effects of stone wool cover incision on root-zone variability have rarely been studied. Therefore, in this study, we focused on the effect of the stone wool cover incision method on environmental variations and their subsequent effects on tomato plant productivity. Stone wool slab plastic covers represent a core component of this substrate system that can potentially affect the performance of water control techniques. We designed a cover incision method to create four different levels of drainage performances that were tested by cultivating tomato plants (Solanum lycopersicum "Dafnis"). The water content, root-zone temperature, and dissolved oxygen were measured and analyzed relative to the tomato yield. We found that the incision level with the lowest drainage performance showed a lower air-root zone temperature correlation slope than those of slabs with favorable drainage conditions. Furthermore, these slabs had low dissolved oxygen levels (3.2 mg/L); nevertheless, the tomatoes grown in the slabs with incision level showing the lowest drainage performance had greater fruit yield (6,748 g/plant) than those in the slabs with favorable drainage conditions (6,160 g/plant). Furthermore, the normalized yield separation timing between treatments coincided with the hotter air temperature (27°C average) periods. We noted that manipulating the cover incision process consequently entailed variations in the correlation slope between the air temperature and root-zone temperature in the substrate. Our results reveal another trade-off relationship in the conventional perspective on the drainage performance effects and provide insights into further optimization of crop production and water use in the stone wool-based system.

Theoretical and Experimental Analyses of Nutrient Control in Electrical Conductivity-Based Nutrient Recycling Soilless Culture System.

An electrical conductivity (EC)-based closed-loop soilless culture system is practical for in-field deployment. Literature on the closed-loop soilless culture nutrient management premise the limitations in managing recycled nutrients under dynamic changes in individual nutrient uptake concentrations. However, recent systems analysis studies predicting solutions for nutrient fluctuation stabilization in EC-based closed-loop soilless culture systems suggest that the system may have a deterministic side in nutrient variation. This study aims to derive a nutrient control principle in an EC-based nutrient recycling soilless culture system by theoretical and experimental analyses. An integrated model of solutes such as K+, Ca2+, and Mg2+ and water transport in growing media, automated nutrient solution preparation, and nutrient uptake was designed. In the simulation, the intrinsic characteristics of nutrient changes among open-, semi- closed-, and closed-loop soilless cultures were compared, and stochastic simulations for nutrient control were performed in the closed-loop system. Four automated irrigation modules for comparing nutrient changes among the soilless culture systems were constructed in the greenhouse. Sweet pepper plants were used in the experiment. In the experimental analysis, nutrient concentration conversion to the proportion between nutrients revealed distinctive trends of nutrient changes according to the treatment level of drainage recycling. Theoretical and experimental analyses exhibited that nutrient variations in open-, semi- closed-, and closed-loop soilless culture systems can be integrated as a function of nutrient supply to the system's boundary areas. Furthermore, stochastic simulation analysis indicated that the nutrient ratio in the soilless culture system reveals the nutrient uptake parameter-based deterministic patterns. Thus, the nutrient ratio in the closed-loop soilless culture could be controlled by the long-term feedback of this ratio. We expect that these findings provide theoretical frameworks for systemizing nutrient management techniques in EC-based closed-loop soilless culture systems.

Nutrient Dosing Framework for an Emission-Free Urban Hydroponic Production.

The urban hydroponic production system is accelerating industrialization in step with the potentials for reducing environmental impact. In contrast, establishing sustainable fertilizer dosing techniques still lags behind the pace of expansion of the system. The reproducibility of root-zone nutrient dynamics in the system is poorly understood, and managing nutrients has so far primarily relied on periodic discharge or dumping of highly concentrated nutrient solutions. Here, we assayed root-zone nutrient concentration changes using three possible nutrient dosing types. Three Brassica species were hydroponically cultivated in a controlled environment to apply the nutrient absorption and transpiration parameters to the simulation analysis. We found that nutrient dosing based on total ion concentration could provide more reproducible root-zone nutrient dynamics. Our findings highlight the nutrient absorption parameter domain in management practice. This simplifies conventional nutrient management into an optimization problem. Collectively, our framework can be extended to fertilizer-emission-free urban hydroponic production.

Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus.

A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.

Acanthamoeba healyi: expressed gene profiles with enhanced virulence after mouse-brain passage.

The virulence of Acanthamoeba can be attenuated by long-term in vitro cultivation, and can be recovered by serial mouse-brain passage via intranasal inoculation. Recovery is concomitant with changes in expression of virulence-related genes. To investigate the virulence factors of Acanthamoeba, expressed sequence tags (ESTs) from two kinds of cDNA libraries-long-term in vitro cultivated A. healyi (OLD) and three times mouse-brain passaged A. healyi (MBP)-were compared using reciprocal BLAST analysis, eukaryotic orthologous groups (KOG) assignment, and gene annotation. A total of 938 (OLD) and 1033 (MBP) ESTs were sequenced and resulted in the assembling of 718 OLD and 833 MBP unique sequences. Comparison of the KOG analysis revealed a relatively higher percentage of MBP ESTs in genes related to transcription (K group), amino acid transport and metabolism (E group), coenzyme transport and metabolism (H group), and secondary metabolites biosynthesis, transport and metabolism (Q group). However, a higher percentage of unidentified MBP ESTs (57.9%) than OLD ESTs (28.9%) was evidence of the limited understanding of virulence-related factors of Acanthamoeba. Characterization of the genes expressed during brain passage in mice will be useful in clarifying the pathogenesis of granulomatous amoebic encephalitis by Acanthamoeba.

Investigation of Klebsiella pneumoniae isolates producing SHV-12 and SHV-11 beta-lactamases in Korean hospitals.

Of 143 clinical isolates of Klebsiella pneumoniae collected from Korean non-tertiary hospitals, 24 (16.8%) showed an extended-spectrum beta-lactamase-positive phenotype. PCR and sequence analysis revealed the presence of TEM-116 (n=13), CTX-M-3 (n=5), CTX-M-14 (n=2), CTX-M-15 (n=3), and SHV-12 (n=16). Each of the 24 isolates encoded more than one beta-lactamase, and seven isolates (29%) harbored two different SHV-type beta-lactamase genes (blaSHV-11 and blaSHV-12) bounded by insertion sequence IS26 in a single transferable plasmid.

Construction of EST database for comparative gene studies of Acanthamoeba.

The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).

Long short-term memory for a model-free estimation of macronutrient ion concentrations of root-zone in closed-loop soilless cultures.

BACKGROUND: Root-zone environment is considered difficult to analyze, particularly in interpreting interactions between environment and plant. Closed-loop soilless cultures have been introduced to prevent environmental pollution, but difficulties in managing nutrients can cause nutrient imbalances with an adverse effect on crop growth. Recently, deep learning has been used to draw meaningful results from nonlinear data and long short-term memory (LSTM) is showing state-of-the-art results in analyzing time-series data. Therefore the macronutrient ion concentrations affected by accumulated environment conditions can be analyzed using LSTM. RESULTS: The trained LSTM can estimate macronutrient ion concentrations in closed-loop soilless cultures using environmental and growth data. The average training accuracy of six macronutrients was R2 = 0.84 and the test accuracy was R2 = 0.67 with RMSE = 1.48 meq L-1. The used values of input interval and time step were 1 h and 168 (1 week), respectively. The accuracy was improved when the input interval became shorter, but not improved when the LSTM consisted of a multilayer structure. Regarding training methods, the LSTM improved the accuracy better than the non-LSTM. The trained LSTM showed relatively adequate accuracies and the interpolated ion concentrations showed variations similar to those seen during traditional cultivation. CONCLUSIONS: We could analyze the nutrient balance in the closed-loop soilless culture, the model showed potential in estimating the macronutrient ion concentrations using environmental and growth factors measured in greenhouses. Since the LSTM is a powerful and flexible tool used to interpret accumulative changes, it is easily applicable to various plant and cultivation conditions. In the future, this approach can be used to analyze interactions between plant physiology and root-zone environment.

Forecasting Root-Zone Electrical Conductivity of Nutrient Solutions in Closed-Loop Soilless Cultures via a Recurrent Neural Network Using Environmental and Cultivation Information.

In existing closed-loop soilless cultures, nutrient solutions are controlled by the electrical conductivity (EC) of the solution. However, the EC of nutrient solutions is affected by both growth environments and crop growth, so it is hard to predict the EC of nutrient solution. The objective of this study was to predict the EC of root-zone nutrient solutions in closed-loop soilless cultures using recurrent neural network (RNN). In a test greenhouse with sweet peppers (Capsicum annuum L.), data were measured every 10 s from October 15 to December 31, 2014. Mean values for every hour were analyzed. Validation accuracy (R2) of a single-layer long short-term memory (LSTM) was 0.92 and root-mean-square error (RMSE) was 0.07, which were the best results among the different RNNs. The trained LSTM predicted the substrate EC accurately at all ranges. Test accuracy (R2) was 0.72 and RMSE was 0.08, which were lower than values for the validation. Deep learning algorithms were more accurate when more data were added for training. The addition of other environmental factors or plant growth data would improve model robustness. A trained LSTM can control the nutrient solutions in closed-loop soilless cultures based on predicted future EC. Therefore, the algorithm can make a planned management of nutrient solutions possible, reducing resource waste.

Sweet Pepper (Capsicum annuum L.) Canopy Photosynthesis Modeling Using 3D Plant Architecture and Light Ray-Tracing.

Canopy photosynthesis has typically been estimated using mathematical models that have the following assumptions: the light interception inside the canopy exponentially declines with the canopy depth, and the photosynthetic capacity is affected by light interception as a result of acclimation. However, in actual situations, light interception in the canopy is quite heterogenous depending on environmental factors such as the location, microclimate, leaf area index, and canopy architecture. It is important to apply these factors in an analysis. The objective of the current study is to estimate the canopy photosynthesis of paprika (Capsicum annuum L.) with an analysis of by simulating the intercepted irradiation of the canopy using a 3D ray-tracing and photosynthetic capacity in each layer. By inputting the structural data of an actual plant, the 3D architecture of paprika was reconstructed using graphic software (Houdini FX, FX, Canada). The light curves and A/C i curve of each layer were measured to parameterize the Farquhar, von Caemmerer, and Berry (FvCB) model. The difference in photosynthetic capacity within the canopy was observed. With the intercepted irradiation data and photosynthetic parameters of each layer, the values of an entire plant's photosynthesis rate were estimated by integrating the calculated photosynthesis rate at each layer. The estimated photosynthesis rate of an entire plant showed good agreement with the measured plant using a closed chamber for validation. From the results, this method was considered as a reliable tool to predict canopy photosynthesis using light interception, and can be extended to analyze the canopy photosynthesis in actual greenhouse conditions.

A transcription terminator in the groEx gene of symbiotic X-bacteria expressed in Escherichia coli.

The over-expressing groEx gene of symbiotic X-bacteria in Amoeba proteus has unique nucleotide motifs (Tx), containing two hairpins and a C-rich region at its 3'-end. To investigate the role of Tx as a transcription terminator, we mutated Tx and analyzed the effects on the expression of an upstream-located lacZ in E. coli. The level of beta-galactosidase (beta-gal) production in Tx deletion mutants was reduced to 23% of the control level. Site-directed mutation of the hairpin-1, C-rich region, and hairpin-2 reduced the beta-gal production to 28-64%, 33, and 20% of wild-type Tx, respectively. The amount of lacZ transcripts that were detected in RNA blots was proportional to the level of beta-gal. The Tx sequence had 97% termination efficiency in vivo, and the mutations in Tx resulted in read-through transcripts with significantly shortened half-life. In rho- E. coli, the level of the beta-gal production by Tx was reduced to 15% of that in rho+ E. coli. These results suggest that Tx is a Rho-dependent transcription terminator. Also, hairpin-2 is the most effective component among the three motifs of Tx for proper termination of the transcription and stability of mRNAs.

Acanthamoeba castellanii: gene profile of encystation by ESTs analysis and KOG assignment.

The trophozoite of Acanthamoeba transforms into a cyst, the resistant form under harmful environments such as starvation, cold and certain chemicals used in medical treatment. To investigate the factors mediating encystation, ESTs of encystation-induced A. castellanii were analysed and compared to those of trophozoites. Each EST was compared by the predicted proteins from the ESTs, to the cyst and the trophozoite by reciprocal BLAST analysis, KOG assignment, and gene annotation. In addition to the genes previously reported to encystation mediate such as cyst specific protein 21, protein kinase C, proteasome and heat shock protein, several genes like cullin 4, autophage protein 8 and ubiquitin-conjugating enzymes were identified to be related to encystation. Five kinds of proteinase genes were detected in cyst ESTs. The information of the genes expressed during encystation may open the way to further study on differentiation and resistance of cyst-forming pathogenic protozoa.

The dps gene of symbiotic "Candidatus Legionella jeonii" in Amoeba proteus responds to hydrogen peroxide and phagocytosis.

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots.

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.

Phylogenetic characterization of Legionella-like endosymbiotic X-bacteria in Amoeba proteus: a proposal for 'Candidatus Legionella jeonii' sp. nov.

The X-bacteria which initiated organismic association with the D strain of Amoeba proteus in 1966 as parasites have changed to obligate endosymbionts on which the host depends for survival. Owing to the difficulty in cultivating the bacteria in vitro, the identity of X-bacteria has not been determined. As the life cycle of X-bacteria is similar to that of Legionella spp. in soil amoebae, we applied the polymerase chain reaction method with specific primers aimed at Legionella spp. for the detection and cloning of 16S rRNA gene. The identity and intracellular localization of the endosymbiont were confirmed by the application of a specific fluorescently labelled 16S rRNA-targeted probe. In addition we cloned RNA polymerase beta-subunit gene (rpoB) of X-bacteria by genomic library tagging. A phylogenetic analysis of the 16S rRNA gene placed the bacterium within a unique monophyletic group containing all other members of the genus Legionella. Phylogeny from rpoB and mip genes further confirmed the taxonomic context of X-bacteria to be a Legionella sp. In all three phylogenic analyses, X-bacterium was placed apart from Legionella-like amoebal pathogens present in soil amoebae. Thus, we propose the name 'Candidatus Legionella jeonii' sp. nov. for the endosymbiotic X-bacteria in Amoeba proteus.

Hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis.

The cellular entry of Hantaan virus (HTN) occurs through interactions with beta(3) integrins as cellular receptors. However, the process of HTN infection following attachment to the cell surface is not well understood. Our data indicate that overexpression of a dominant-negative mutant dynamin inhibits HTN internalization and that compounds that block clathrin- but not caveolae-dependent endocytosis also reduce HTN infectivity. In addition, we show that HTN colocalizes with the clathrin heavy chain but not with caveolae. At the early phase of infection HTN colocalizes with EEA-1, an early endosome marker, and later, HTN colocalizes with LAMP-1, a lysosome marker. Cells treated with lysosomotropic agents are largely resistant to infection, suggesting that a low-pH-dependent step is required for HTN infection. These findings demonstrate that HTN enters cells via the clathrin-coated pit pathway and uses low-pH-dependent intracellular compartments for infectious entry.