Classification of Processing Damage in Sugar Beet (Beta vulgaris) Seeds by Multispectral Image Analysis.

The pericarp of monogerm sugar beet seed is rubbed off during processing in order to produce uniformly sized seeds ready for pelleting. This process can lead to mechanical damage, which may cause quality deterioration of the processed seeds. Identification of the mechanical damage and classification of the severity of the injury is important and currently time consuming, as visual inspections by trained analysts are used. This study aimed to find alternative seed quality assessment methods by evaluating a machine vision technique for the classification of five damage types in monogerm sugar beet seeds. Multispectral imaging (MSI) was employed using the VideometerLab3 instrument and instrument software. Statistical analysis of MSI-derived data produced a model, which had an average of 82% accuracy in classification of 200 seeds in the five damage classes. The first class contained seeds with the potential to produce good seedlings and the model was designed to put more limitations on seeds to be classified in this group. The classification accuracy of class one to five was 59, 100, 77, 77 and 89%, respectively. Based on the results we conclude that MSI-based classification of mechanical damage in sugar beet seeds is a potential tool for future seed quality assessment.

Effect of Verticillium dahliae Soil Inoculum Levels on Spinach Seed Infection.

Verticillium dahliae is a soilborne pathogen and a threat to spinach seed production. The aim of this study was to understand the relation between V. dahliae soil inoculum and infection in harvested seed. Quantitative polymerase chain reaction was used for quantification of the pathogen. Semifield experiments in which spinach was grown in soils with different inoculum levels enabled us to determine a threshold level for V. dahliae DNA of 0.003 ng/g of soil for seed infection to occur. Soils from production fields were sampled in 2013 and 2014 during and before planting, as well as the harvested seed. Seed from plants grown in infested soils were infected with V. dahliae in samples from both the semifield and open-field experiments. Lower levels of pathogen were found in seed from spinach grown in soils with a scattered distribution of V. dahliae (one or two positive of three soil subsamples) than in soils with a uniform distribution of the pathogen (three of three positive soil subsamples). Our results showed that infection of V. dahliae in harvested seed strongly depended on the presence of pathogen inoculum in the soil.

Changes in Lolium perenne transcriptome during cold acclimation in two genotypes adapted to different climatic conditions.

BACKGROUND: Activation of numerous protective mechanisms during cold acclimation is important for the acquisition of freezing tolerance in perennial ryegrass (Lolium perenne L.). To elucidate the molecular mechanisms of cold acclimation in two genotypes ('Veyo' and 'Falster') of perennial ryegrass from distinct geographical origins, we performed transcriptome profiling during cold acclimation using RNA-Seq. METHODS: We cold-acclimated plants from both genotypes in controlled conditions for a period of 17 days and isolated Total RNA at various time points for high throughput sequencing using Illumina technology. RNA-seq reads were aligned to genotype specific references to identify transcripts with significant changes in expression during cold acclimation. RESULTS: The genes induced were involved in protective mechanisms such as cell response to abiotic stimulus, signal transduction, redox homeostasis, plasma membrane and cell wall modifications, and carbohydrate metabolism in both genotypes. 'Falster' genotype, adapted to cold climates, showed a stronger transcriptional differentiation during cold acclimation, and more differentially expressed transcripts related to stress, signal transduction, response to abiotic stimulus, and metabolic processes compared to 'Veyo'. 'Falster' genotype also showed an induction of more transcripts with sequence homology to fructosyltransferase genes (FTs) and a higher fold induction of fructan in response to low-temperature stress. The circadian rhythm network was perturbed in the 'Veyo' genotype adapted to warmer climates. CONCLUSION: In this study, the differentially expressed genes during cold acclimation, potentially involved in numerous protective mechanisms, were identified in two genotypes of perennial ryegrass from distinct geographical origins. The observation that the circadian rhythm network was perturbed in 'Veyo' during cold acclimation may point to a low adaptability of 'Veyo' to low temperature stresses. This study also revealed the transcriptional mechanisms underlying carbon allocation towards fructan biosynthesis in perennial ryegrass.

Viability prediction of Ricinus cummunis L. seeds using multispectral imaging.

The purpose of this study was to highlight the use of multispectral imaging in seed quality testing of castor seeds. Visually, 120 seeds were divided into three classes: yellow, grey and black seeds. Thereafter, images at 19 different wavelengths ranging from 375-970 nm were captured of all the seeds. Mean intensity for each single seed was extracted from the images, and a significant difference between the three colour classes was observed, with the best separation in the near-infrared wavelengths. A specified feature (RegionMSI mean) based on normalized canonical discriminant analysis, were employed and viable seeds were distinguished from dead seeds with 92% accuracy. The same model was tested on a validation set of seeds. These seeds were divided into two groups depending on germination ability, 241 were predicted as viable and expected to germinate and 59 were predicted as dead or non-germinated seeds. This validation of the model resulted in 96% correct classification of the seeds. The results illustrate how multispectral imaging technology can be employed for prediction of viable castor seeds, based on seed coat colour.

Pollination and Plant Reproductive Success of Two Ploidy Levels in Red Clover (Trifolium pratense L.).

Plant reproduction in red clover requires cross-fertilization via insect pollination. However, the influences of visitation rate and timing on maximizing ovule utilization are yet to be determined. We aimed to study the influences of visitation rate, flowering stage, and self-incompatibility on reproductive success. We applied hand and honey bee pollination in the study of eight red clover cultivars with two ploidy levels released between 1964 and 2001. In hand pollination, increasing the visitation rates (from 10 to 80 pollinated florets per flower head) increased the seed number per flower head but reduced the seed number per pollinated floret. Different flowering stages (early, middle, and full flowering) did not influence the seed number per pollinated floret significantly. There was a marked difference in reproductive success depending on the ploidy level, with 0.52 seeds per pollinated floret in diploid and 0.16 in tetraploid cultivars. During the cultivar release history, seed number per pollinated floret seemed to decrease in diploid cultivars, whereas it increased in tetraploids. In honey bee pollination, diploid cultivars had more two-seeded florets than tetraploids. Different visitation rates and the stochastic nature of pollen transfer resulted in difficulties when the plant reproductive success between hand and bee pollination was compared. A maximum of 0.27 seeds per pollinated floret were produced in hand pollination compared to the 0.34 in honey bee pollination. In spite of this, hand pollination provided a valuable method for studying the pollination biology and reproduction of red clover. Future studies may employ hand pollination to unravel further aspects of the low reproductive success with the future perspective of improving seed number per pollinated floret in tetraploid red clover.

Multispectral and X-ray images for characterization of Jatropha curcas L. seed quality.

BACKGROUND: The use of non-destructive methods with less human interference is of great interest in agricultural industry and crop breeding. Modern imaging technologies enable the automatic visualization of multi-parameter for characterization of biological samples, reducing subjectivity and optimizing the analysis process. Furthermore, the combination of two or more imaging techniques has contributed to discovering new physicochemical tools and interpreting datasets in real time. RESULTS: We present a new method for automatic characterization of seed quality based on the combination of multispectral and X-ray imaging technologies. We proposed an approach using X-ray images to investigate internal tissues because seed surface profile can be negatively affected, but without reaching important internal regions of seeds. An oilseed plant (Jatropha curcas) was used as a model species, which also serves as a multi-purposed crop of economic importance worldwide. Our studies included the application of a normalized canonical discriminant analyses (nCDA) algorithm as a supervised transformation building method to obtain spatial and spectral patterns on different seedlots. We developed classification models using reflectance data and X-ray classes based on linear discriminant analysis (LDA). The classification models, individually or combined, showed high accuracy (> 0.96) using reflectance at 940 nm and X-ray data to predict quality traits such as normal seedlings, abnormal seedlings and dead seeds. CONCLUSIONS: Multispectral and X-ray imaging have a strong relationship with seed physiological performance. Reflectance at 940 nm and X-ray data can efficiently predict seed quality attributes. These techniques can be alternative methods for rapid, efficient, sustainable and non-destructive characterization of seed quality in the future, overcoming the intrinsic subjectivity of the conventional seed quality analysis.

Detection of live larvae in cocoons of Bathyplectes curculionis (Hymenoptera: Ichneumonidae) using visible/near-infrared multispectral imaging.

BACKGROUND: The multispectral (MS) imaging system is a non-destructive method with potential to reduce the labour and time required for quality control in the production of beneficial arthropods such as the parasitoid Bathyplectes curculionis. In Denmark, a project is being undertaken that focuses on the possible use of B. curculionis in augmentative control of Hypera weevil pests in white clover seed production where cocoons of the parasitoid remain as a by-product of seed processing. Only a fraction of the by-product contains live parasitoid larvae and an effective method is required detect live cocoons for later augmentative control of the pest. Therefore, this study aims to identify live larval cocoons of B. curculionis using the MS imaging system. RESULTS: Live and dead cocoons were identified using the canonical discriminant analysis (CDA) model with an accuracy of 91% and 80% (error rate 14%) in the training set, and a predicted accuracy of 89% and 81% (error rate 15%) in the test set. Reflectance from the near-infrared region was valuable in identifying live cocoons compared with that from the visible region. CONCLUSION: The MS imaging system is a rapid method for the separation of live and dead cocoons of B. curculionis. This study shows the potential of developing an MS imaging system to facilitate sorting of live and dead cocoons and optimize augmentative control of Hypera weevil pests. © 2018 Society of Chemical Industry.

Identification and Quantification of Loline-Type Alkaloids in Endophyte-Infected Grasses by LC-MS/MS.

Lolines, fungal metabolites of the grass-endophyte association, were identified and quantified using newly developed LC-MS/MS methods in endophyte-infected grasses belonging to the Lolium and Festuca genera after extraction with three different solvents using two extraction methods. The shaking extraction method with isopropanol/water was superior to the other methods due to its high sensitivity, high accuracy (recovery within or close to the range of 80-120%), and high precision (coefficient of variation of <10%). Seven loline alkaloids were identified and quantified using our newly established LC-MS/MS methods, and N-formylloline was the most abundant (5 mg/g dry matter), followed by N-acetylloline. These LC-MS/MS methods used the shortest sample handling time and the fewest sample preparation steps and proved to be good alternatives to existing GC and GC-MS analytical methods without compromising analytical efficiency. In conclusion, we developed for the first time a highly sensitive quantitative LC-MS/MS analytical method for the accurate and reproducible quantification and a LightSight-assisted LC-QTRAP/MS qualitative method for the tentative identification of loline-type alkaloids in endophyte-infected grasses.

Productivity and carbon footprint of perennial grass-forage legume intercropping strategies with high or low nitrogen fertilizer input.

A three-season field experiment was established and repeated twice with spring barley used as cover crop for different perennial grass-legume intercrops followed by a full year pasture cropping and winter wheat after sward incorporation. Two fertilization regimes were applied with plots fertilized with either a high or a low rate of mineral nitrogen (N) fertilizer. Life cycle assessment (LCA) was used to evaluate the carbon footprint (global warming potential) of the grassland management including measured nitrous oxide (N2O) emissions after sward incorporation. Without applying any mineral N fertilizer, the forage legume pure stand, especially red clover, was able to produce about 15 t above ground dry matter ha(-1) year(-1) saving around 325 kg mineral Nfertilizer ha(-1) compared to the cocksfoot and tall fescue grass treatments. The pure stand ryegrass yielded around 3t DM more than red clover in the high fertilizer treatment. Nitrous oxide emissions were highest in the treatments containing legumes. The LCA showed that the low input N systems had markedly lower carbon footprint values than crops from the high N input system with the pure stand legumes without N fertilization having the lowest carbon footprint. Thus, a reduction in N fertilizer application rates in the low input systems offsets increased N2O emissions after forage legume treatments compared to grass plots due to the N fertilizer production-related emissions. When including the subsequent wheat yield in the total aboveground production across the three-season rotation, the pure stand red clover without N application and pure stand ryegrass treatments with the highest N input equalled. The present study illustrate how leguminous biological nitrogen fixation (BNF) represents an important low impact renewable N source without reducing crop yields and thereby farmers earnings.

DNA barcoding and isolation of vertically transmitted ascomycetes in sorghum from Burkina Faso: Epicoccum sorghinum is dominant in seedlings and appears as a common root pathogen.

Molecular identification of fungal taxa commonly transmitted through seeds of sorghum in Western Africa is lacking. In the present study, farm-saved seeds, collected from four villages in Northern Burkina Faso, were surface sterilized and the distribution of fungal DNA in seeds and seven-day-old seedlings was analyzed by 18S ribosomal DNA (rDNA) amplicon sequencing. More than 99% of the fungal rDNA was found to originate from ascomycetes. The distribution of ascomycetes at species level was subsequently analyzed by barcoding of ITS2 rDNA. Eighteen Operational Taxonomic Units (OTUs) were identified from seedlings, compared to 29 OTUs from seeds. The top-eight most abundant ascomycete OTUs from seedlings were annotated as: Epicoccum sorghinum, Fusarium thapsinum, four different Curvularia spp., Exserohilum rostratum and Alternaria longissima. These OTUs were also present in amplicons from seed samples collected in Central Burkina Faso confirming a common occurrence. E. sorghinum was highly predominant in seedlings both measured by DNA analysis and by isolation. The dominance of E. sorghinum was particularly strong in roots from poorly growing seedlings. Pathogenicity of E. sorghinum isolates was compared to F. thapsinum by inoculation to seeds in vitro. Both fungal species caused significant inhibition of seedling growth (P<0.001) and Koch's postulates were fulfilled. Extensive, dark necrosis in roots was a typical symptom of E. sorghinum, whereas wilting of leaves was caused primarily by F. thapsinum. This study provides the first molecular approach to characterize the seedling mycoflora of sorghum in Western Africa and suggests E. sorghinum as a common root pathogen.

Fructan metabolism and changes in fructan composition during cold acclimation in perennial ryegrass.

Perennial ryegrass (Lolium perenne L.) produces high levels of fructans as a mixture of oligosaccharides and polysaccharides with different degrees of polymerization (DP). The present study describes the analysis of the compositional changes in the full spectrum of fructans, fructan distribution between above ground biomass (top) and the roots, and the transcription of candidate genes involved in fructan metabolism during cold acclimation in perennial ryegrass variety "Veyo" and ecotype "Falster" from distinct geographical origins. We observed changes in fructan composition and induction of low-DP fructans, especially DP = 4, in both the top and the roots of "Veyo" and "Falster" in response to low-temperature stress. The accumulation of DP > 50 fructans was only apparent in the top tissues where the Lp1-FFT expression is higher compared to the roots in both "Veyo" and "Falster." Our results also show the accumulation and depolymerization of fructans with different DP, together with the induction of genes encoding fructosyltransferases and fructan exohydrolases in both "Veyo" and "Falster" during cold acclimation, supporting the hypothesis that fructan synthesis and depolymerization occurring simultaneously. The ecotype "Falster," adapted to cold climates, increased total fructan content and produced more DP > 7 fructans in the roots than the variety "Veyo," adapted to warmer climates. This indicates that high-DP fructan accumulation in roots may be an adaptive trait for plant recovery after abiotic stresses.

Fructan accumulation and transcription of candidate genes during cold acclimation in three varieties of Poa pratensis.

Poa pratensis, a type species for the grass family (Poaceae), is an important cool season grass that accumulates fructans as a polysaccharide reserve. We studied fructan contents and expression of candidate fructan metabolism genes during cold acclimation in three varieties of P. pratensis adapted to different environments: Northern Norway, Denmark, and the Netherlands. Fructan content increased significantly during cold acclimation and varieties showed significant differences in the level of fructan accumulation. cDNA sequences of putative fructosyltransferase (FT), fructan exohydrolase (FEH), and cold acclimation protein (CAP) genes were identified and cloned. In agreement with a function in fructan biosynthesis, transcription of a putative sucrose:fructan 6-fructosyltransferase (Pp6-SFT) gene was induced during cold acclimation and fructan accumulation in all three P. pratensis varieties. Transcription of putative PpFEH and PpCAP genes was also induced by cold acclimation; however, transcription of these two genes was several-fold higher in the variety from Norway compared to the other two varieties. The results presented here suggest that Pp6-SFT is involved in fructan biosynthesis in P. pratensis. FEHs have previously been suggested to be involved in fructan biosynthesis and freezing tolerance, and induced expression of PpFEH during fructan accumulation could also suggest a role in fructan biosynthesis. However, based on the different PpFEH transcription rates among varieties and similar expression of PpFEH and PpCAP, we suggest that PpFEH is more likely to be involved in mediating freezing tolerance, e.g., by regulating the cell osmotic potential through fructan degradation.