Establishment of maize resistance to fungal diseases by host-induced gene silencing and site-directed mutagenesis

Maize is one of the most cultivated crops in the world. A disease called anthracnose accounts for up to 80% of the loss in maize production. It is caused by the hemibiotrophic fungus Colletotrichum graminicola. Unfortunately, the disease is notoriously difficult to combat, since host resistance mechanisms are hardly available. In the present investigation, the principle of host-induced gene silencing (HIGS) was employed to protect maize plants from C. graminicola infection. HIGS is an RNA-interefence (RNAi)-based process, wherein plant-produced short interfering RNAs (siRNA) are taken up by the fungus and trigger the silencing of cognate genes of the latter. In the present study, genes encoding fungicide targets were chosen as HIGS targets, namely C. graminicola -Tubulin 2 and Succinate dehydrogenase 1. RNAi vectors were designed using appropriate regions of these target genes. Transgenic plants expressing RNAi constructs were infected with C. graminicola, whereby the plants showed quantitative resistance. In addition to the HIGS approach, a further strategy was pursued, which consisted in knocking out a susceptibility factor against C. graminicola by means of targeted mutagenesis. This factor was the 9-LIPOXYGENASE LOX3 gene from maize, for which several mutated plants were generated by expression of RNA-directed Cas9 endonuclease. Homozygous lox3 mutants were tested in C. graminicola infection assays to analyze the consequences of their mutations. Quantification of fungal biomass revealed that the lox3 mutants were significantly less colonized by C. graminicola compared to the non-mutated wild-type. Corn common smut, another important fungal disease, is caused by the biotrophic pathogen Ustilago maydis. Transcriptional data (Doehlemann et al., 2008) collected during the course of infection with U. maydis showed that, depending on the infection, several members of the LOX gene family are upregulated, one of which is LOX3. Therefore, the available lox3 mutants were tested for their response to infection with U. maydis. The quantification of the disease symptoms showed that the lox3 mutants showed a moderate resistance against U. maydis infections. Furthermore, the quantification of the biomass of U. maydis revealed that the lox3 mutants were colonized by the fungus to a lesser extent compared to the wild-type. Furthermore, infection tests were performed using lox3 mutants independently produced by transposon insertion mutagenesis. These lines showed a resistance behavior similar to that of Cas9-induced mutants, by which the anticipated role of LOX3 for the interaction of maize and U. maydis was corroborated. From the literature it is known that U. maydis suppresses the accumulation of reactive oxygen species (ROS) to establish its biotrophic mode of pathogenesis. A ROS accumulation test revealed that lox3 mutants feature increased ROS accumulationcompared to the wild-type, suggesting that the immunity of the mutants triggered by pathogen-associated molecular pattern (PAMP) led to a reduction in the severity of fungal infection. This is the first study showing that lox3 mutants show moderate resistance to U. maydis. In view of these results, it is concluded that LOX3 is a susceptibility factor for U. maydis as wel

Impact of Camera Viewing Angle for Estimating Leaf Parameters of Wheat Plants from 3D Point Clouds

Estimation of plant canopy using low-altitude imagery can help monitor the normal growth status of crops and is highly beneficial for various digital farming applications such as precision crop protection. However, extracting 3D canopy information from raw images requires studying the effect of sensor viewing angle by taking into accounts the limitations of the mobile platform routes inside the field. The main objective of this research was to estimate wheat (Triticum aestivum L.) leaf parameters, including leaf length and width, from the 3D model representation of the plants. For this purpose, experiments with different camera viewing angles were conducted to find the optimum setup of a mono-camera system that would result in the best 3D point clouds. The angle-control analytical study was conducted on a four-row wheat plot with a row spacing of 0.17 m and with two seeding densities and growth stages as factors. Nadir and six oblique view image datasets were acquired from the plot with 88% overlapping and were then reconstructed to point clouds using Structure from Motion (SfM) and Multi-View Stereo (MVS) methods. Point clouds were first categorized into three classes as wheat canopy, soil background, and experimental plot. The wheat canopy class was then used to extract leaf parameters, which were then compared with those values from manual measurements. The comparison between results showed that (i) multiple-view dataset provided the best estimation for leaf length and leaf width, (ii) among the single-view dataset, canopy, and leaf parameters were best modeled with angles vertically at -45⸰_ and horizontally at 0⸰_ (VA -45, HA 0), while (iii) in nadir view, fewer underlying 3D points were obtained with a missing leaf rate of 70%. It was concluded that oblique imagery is a promising approach to effectively estimate wheat canopy 3D representation with SfM-MVS using a single camera platform for crop monitoring. This study contributes to the improvement of the proximal sensing platform for crop health assessment. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Agricultural Research Division 106th Annual Report 1992

It is a pleasure to provide the 106th Annual Report of the UNL Agricultural Research Division (ARD). This report contains lists of current faculty; active research projects; refereed journal articles, books and book chapters, and theses and dissertations published; germplasm/cultivars released; and patents awarded. Also included are brief descriptions of selected research projects, awards received by faculty for research accomplishments, and the financial report for the period July 1, 1991, through June 30,1992. This report was compiled in compliance with the intent of the law of the State of Nebraska that established the Nebraska Agricultural Experiment Station on March 31, 1887. Foreword ... 5 Administration ... 6 … Administrative Personnel ... 6 … Organizational Chart ... 7 … Administrative Units ... 8 … IANR Research Facilities ... 9 Research Highlights ... 10 Faculty ... 18 … Agricultural/Natural Resources Departments ... 18 … Home Economics Departments ... 23 … Off-Campus Research Centers ... 24 Faculty Awards and Recognition ... 26 Research Projects ... 30 … Agricultural/Natural Resources Departments ... 30 … Home Economics Departments ... 38 … Off-Campus Research Centers ... 39 … Interdisciplinary Centers ... 41 Publications ... 42 … Agricultural/Natural Resources Departments ... 43 … Home Economics Departments ... 59 … Off-Campus Research Centers ... 61 Patents ... 64 Variety and Germplasm Releases ... 65 Research Expenditures ... 6

Agricultural Research Division 106th Annual Report 1992

It is a pleasure to provide the 106th Annual Report of the UNL Agricultural Research Division (ARD). This report contains lists of current faculty; active research projects; refereed journal articles, books and book chapters, and theses and dissertations published; germplasm/cultivars released; and patents awarded. Also included are brief descriptions of selected research projects, awards received by faculty for research accomplishments, and the financial report for the period July 1, 1991, through June 30,1992. This report was compiled in compliance with the intent of the law of the State of Nebraska that established the Nebraska Agricultural Experiment Station on March 31, 1887. Foreword ... 5 Administration ... 6 … Administrative Personnel ... 6 … Organizational Chart ... 7 … Administrative Units ... 8 … IANR Research Facilities ... 9 Research Highlights ... 10 Faculty ... 18 … Agricultural/Natural Resources Departments ... 18 … Home Economics Departments ... 23 … Off-Campus Research Centers ... 24 Faculty Awards and Recognition ... 26 Research Projects ... 30 … Agricultural/Natural Resources Departments ... 30 … Home Economics Departments ... 38 … Off-Campus Research Centers ... 39 … Interdisciplinary Centers ... 41 Publications ... 42 … Agricultural/Natural Resources Departments ... 43 … Home Economics Departments ... 59 … Off-Campus Research Centers ... 61 Patents ... 64 Variety and Germplasm Releases ... 65 Research Expenditures ... 6

Novel insights into the infection mechanism of oomycete Pythium spp. in the host Arabidopsis thaliana

Phytopathogenic Pythium spp. cause seedling damping-off of a wide range of plant species worldwide and are traditionally considered necrotrophs. In this study, novel pathogenicity was discovered involving the oomycete Pythium cryptoirregulare and the model plant Arabidopsis thaliana. This pathogen was isolated from A. thaliana seedlings that were showing damping-off symptoms and was later identified as Pythium cryptoirregulare based on morphological and molecular characterization alongside reference species P. irregulare and P. ultimum var. ultimum. To examine its infection strategy, A. thaliana was inoculated with P. cryptoirregulare and studied using a microscopy approach. Viable colonized cells were observed based on neutral red uptake and the ability to undergo cell plasmolysis after infection. This biotrophic interaction contradicts the previously reported necrotrophic lifestyle of Pythium spp., which is characterized by killing the host cells prior to colonization. In addition, inhibition of root growth was detected prior to colonization by P. cryptoirregulare, suggesting that P. cryptoirregulare secreted growth inhibitors. Potentially, these inhibiting metabolites facilitate infection by delaying plant development and, thereby, extending the seedling stage that is targeted by this pathogen. Notably, P. cryptoirregulare culture filtrates disturbed transport and distribution of auxins, indicated by altered GFP expression in the A. thaliana lines PIN1-GFP, PIN2-GFP, PIN3-GFP, PIN7-GFP and DR5::GFP which visualize the auxin efflux. This disturbance was further confirmed by a reduced inhibitory effect on the auxin-insensitive A. thaliana mutants axr1-3, axr4-2, and aux1-7. Metabolic activity assay results suggested that P. cryptoirregulare secretes auxin-related metabolites that are involved in reprogramming plant growth. Overall, the characterization of P. cryptoirregulare as a novel pathogen on A. thaliana gives new insights into understanding the pathogenic mechanisms and interactions between oomycetes and plants

Genetic dissection of "Caulobacter crescentus" surface colonization

During its biphasic life cycle Caulobacter crescentus switches from a planktonic to surface attached life style. This transition requires the continuous remodeling of the cell poles through the temporally and spatially coordinated assembly and disassembly of polar organelles like the flagellum, pili, and an adhesive holdfast. A genetic screen for mutants affected in surface binding and colonization led to the identification of various genes required for motility, pili, and holdfast biogenesis, suggesting a specific role for all three organelles in C. crescentus surface colonization. Several novel holdfast genes were identified, which are potentially involved in the synthesis and regulation of the polysaccharidic component of the holdfast. Quantitative surface binding studies during the C. crescentus cell cycle revealed that optimal attachment coincides with the presence of flagellum, pili, and holdfast at the same pole. This indicated that accurate temporal control of polar appendices is critical for surface colonization of C. crescentus and represents the first example for developmentally controlled bacterial surface adhesion. We have used genetic and biochemical analyzes to demonstrate that di-cyclic guanosine monophosphate (c-di-GMP) is a central regulatory compound involved in the timing of C. crescentus pole development. Mutants lacking the diguanylatecyclase PleD show a dramatic delay of holdfast formation during swarmer cell differentiation. In contrast, cells lacking the GGDEF-EAL composite protein CC0091 show premature holdfast formation, while overexpression of CC0091 also leads to a delayed appearance of holdfast. The observation that CC0091 is a c-di-GMP specific phosphodiesterase indicated that the antagonistic activities of PleD and CC0091 could be responsible for the correct timing of holdfast formation and flagellum ejection. Finally, our genetic screen identified a candidate for the c-di-GMP effector protein, which mediates holdfast synthesis in response to fluctuating levels of c-di-GMP. The glycosyltransferase CC0095 is strictly required for holdfast formation and its overexpression leads to premature holdfast synthesis. This and the observation that CC0095 is able to bind c-di-GMP lead to the hypothesis that holdfast synthesis is regulated via allosteric control of the CC0095 glycosyltransferase. These data provide the first example of a developmental process being regulated by the bacterial second messenger, c-di-GMP

Renewable chitin based nanomaterials from fungi

Fungal cell walls are rich in structural fibers made of chitin and glucan. These fibers were covalently linked, forming a ready-made nanocomposite fabric which is potentially useful in the development of a single-sourced bioreinforcement material that combines the strength of chitin and the toughness of glucan. In this study, chitin-glucan material was extracted from common mushroom (Agaricus bisporus) and tree bracket fungi (Daedaleopsis confragosa). Animal chitin extracted from crab shells (Cancer pagurus) was used as a comparison. We adopted mild extraction process to preserve the native quality of chitin-glucan within the fungal source. Six themes will be discussed: (1) comparison between fungal-based and animal-based chitin, (2) the effect of grammage on fungal chitin film, (3) the effect of blending time on fungal chitin film, (4) composite preparation using combination of different fungal extracts, (5) preparation of high volume fraction fungal-based chitin laminates, and (6) utilization of fungal chitin nanofiber as a natural binder for loose flax fiber. Extracts from common mushroom were found to be (1) readily disintegrated into nanofiber dimension (10–20 nm wide, several micrometers long) without any post-mechanical treatment, (2) more hydrophobic than animal based chitin, and (3) possess a film forming capability. Extracts from tree bracket fungi assumes microfiber dimension (1–2 µm wide, several micrometers long) and require specialized equipment for further nanofibrillation. Chitin to glucan ratio in common mushroom was found to be nearly equal at 40:50, while glucan predominates in bracket fungi (chitin to glucan, 1:99). This translates to distinct mechanical properties, in which strong films having tensile strength of ~200 MPa were produced from common mushroom extract, while tough films with elongation at break exceeding 10% were produced from bracket fungi. The strength of common mushroom nanofibers is further extended as a reinforcement in epoxy laminated composite and as a binder for nonwoven flax preform.Open Acces

Determination of Time Dependent Stress Distribution on Potato Tubers at Mechanical Collision

This study focuses on determining internal stress progression and the realistic representation of time dependent deformation behaviour of potato tubers under a sample mechanical collision case. A reverse engineering approach, physical material tests and finite element method (FEM)-based explicit dynamics simulations were utilised to investigate the collision based deformation characteristics of the potato tubers. Useful numerical data and deformation visuals were obtained from the simulation results. The numerical results are presented in a format that can be used for the determination of bruise susceptibility magnitude on solid-like agricultural products. The modulus of elasticity was calculated from experimental data as 3.12 [MPa] and simulation results showed that the maximum equivalent stress was 1.40 [MPa] and 3.13 [MPa] on the impacting and impacted tubers respectively. These stress values indicate that bruising is likely on the tubers. This study contributes to further research on the usage of numerical-methods-based nonlinear explicit dynamics simulation techniques in complicated deformation and bruising investigations and industrial applications related to solid-like agricultural products

Genetic diversity assessment and evaluation of the concentration and stage of application of a male gametocide for hybrid development in sweet stem sorghum for bioethanol production.

Doctor of Philosophy in Plant Breeding. University of KwaZulu-Natal, Pietermaritzburg, 2018.Abstract available in PDF file