Institut für Phytomedizin

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Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum
(2022) Schlemmer, Timo; Lischka, Richard; Wegner, Linus; Ehlers, Katrin; Biedenkopf, Dagmar; Koch, Aline; Schlemmer, Timo; Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany; Lischka, Richard; Centre for BioSystems, Land Use and Nutrition, Institute of Phytopathology, Justus Liebig University, Giessen, Germany; Wegner, Linus; Intitute of Botany, Justus Liebig University, Giessen, Germany; Ehlers, Katrin; Intitute of Botany, Justus Liebig University, Giessen, Germany; Biedenkopf, Dagmar; Centre for BioSystems, Land Use and Nutrition, Institute of Phytopathology, Justus Liebig University, Giessen, Germany; Koch, Aline; Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany
Numerous reports have shown that incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto their leaves successfully protects them against invading pathogens exploiting the mechanism of RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown. It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their pathogens. Recently, we found that EVs isolated from host-induced gene silencing (HIGS) or spray-induced gene silencing (SIGS) plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from dsRNA-sprayed barley ( Hordeum vulgare ) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum . Encouraged by our previous finding that dropping barley-derived EVs on F. graminearum cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated F. graminearum with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of F. graminearum macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect F. graminearum growth and showed no gene silencing activity on F. graminearum CYP51 genes. Based on our findings, we concluded that either the amount of SIGS-derived siRNA was insufficient to induce target gene silencing in F. graminearum, indicating that the role of EVs in SIGS is minor, or that F. graminearum uptake of plant EVs from liquid cultures was inefficient or impossible.
Legume-derived phenolic acids influence Fusarium oxysporum f.sp. strigae compatibility and biocontrol potential in a Striga push–pull system
(2025) Assena, Mekuria Wolde; Schöne, Jochen; Rasche, Frank
Biological control of Striga hermonthica (Del.) Benth. includes push–pull, intercropping using legumes, and soil-borne fungi such as Fusarium oxysporum f.sp. strigae (FOS). Efficacy of both technologies is compromised by various environmental factors. Combining them could potentially overcome their limitations and enhance control of S. hermonthica. It was hypothesized that FOS inoculation is compatible with push–pull intercropping, allowing consistent S. hermonthica suppression. Effects of selected phenolic acids and crude root exudates from three legume species (Mucuna pruriens, Desmodium uncinatum, Crotalaria juncea) on FOS mycelial growth and spore germination were studied. Bioassays showed that most phenolic acids (e.g., caffeic, p-hydroxybenzoic, syringic) had no effect on FOS development, except for piperonylic (80 %, 38 %) and t-cinnamic (53 %, 33 %) acids, which strongly inhibited growth at concentrations of 1 and 0.5 mM, respectively. Crude root exudates from Crotalaria inhibited FOS mycelial growth at 1 (44 %) and 0.5 (32 %) mg ml-1. Desmodium and Mucuna exudates showed no inhibition. Greenhouse results showed that legume intercropping and FOS inoculation reduced S. hermonthica emergence, with their integration achieving over 95 % suppression and boosting maize biomass. Legume presence enhanced FOS proliferation, with Mucuna and Desmodium increasing FOS gene copy numbers by over 44 %. LC-MS analysis identified nine phenolic acids, with p-coumaric (85–128 µg g−1) and p-hydroxybenzoic (50–97 µg g−1) acid being most abundant; the latter positively correlated with FOS abundance. In this setting, legume intercropping with maize did not impair FOS proliferation in rhizosphere soil and effectively suppressed S. hermonthica, demonstrating compatibility of FOS inoculation with push–pull for effective S. hermonthica management.
Mating-type analysis in Diaporthe isolates from soybean in central Europe
(2025) Hosseini, Behnoush; Käfer, Lena Sophia; Link, Tobias Immanuel; Cai, Lei
Species of the genus Diaporthe have a mating-type system with the two mating types MAT1-1 and MAT1-2, like other ascomycetes. They can either be heterothallic, which means that any isolate only possesses one of the two mating types and needs a mating partner for sexual reproduction, or homothallic, which means that they possess both mating types and are self-fertile. For several Diaporthe species, no sexual reproduction has been observed so far. Using PCR with primers specific to the defining genes MAT1-1-1 and MAT1-2-1 , we determined the mating types of 33 isolates of Diaporthe caulivora , D. eres , D. longicolla , and D. novem from central Europe. In addition, we partially sequenced the mating-type genes of 25 isolates. We found that different D. longicolla isolates either possess MAT1-1-1 or MAT1-2-1, making the species heterothallic, which is in contrast to previous studies and the general assumption that D. longicolla only reproduces asexually. D. eres and D. novem were also found to be heterothallic. Using genomic sequence information and re-sequencing of DNA and RNA, we identified the MAT1-1-1 gene in D. caulivora and present here the full sequence of the mating-type locus of this homothallic species. Finally, we used sequence information from MAT1-1-1 and MAT1-2-1 , respectively, for improved phylogenetic resolution of our isolates.
Arthropods as possible vectors of grapevine trunk disease pathogens in German vineyards
(2025) Brandenburg, Elisa; Vögele, Ralf
The health of grapevines worldwide is highly threatened by grapevine trunk diseases (GTDs), which are caused by various fungal pathogens that colonize and destroy the vascular system of the vine wood. The esca disease complex is one of the most important GTDs. Typical symptoms are the characteristic "tiger stripe" patterns on leaves, gummosis, necrosis and white rot inside the vine wood as well as the sudden dying of individual shoots or even the entire vine. The ascomycetes Phaeomoniella chlamydospora and members of the genera Phaeoacremonium and Cadophora can cause the death of even young vines. Air currents and/or rain splashes carry the pathogenic spores to their entry points in the vine wood, which are mainly susceptible winter pruning wounds. The involvement of arthropods has also been described in South African studies. The aim of this dissertation was to determine and evaluate the risk of arthropod-mediated transmission of GTD pathogens for German vineyards. Over the course of two years (2019-2020), the spectrum of possible vectors was determined in two different vineyards and arthropod visits of presumably susceptible winter pruning wounds were documented using camera-based monitoring in April 2019. The presence of esca-related pathogens on the exoskeletons of various arthropods was examined using nested multiplex PCR. Of the total of 2099 samples, 35 %, 21 % and 7 % tested positive for Pa. chlamydospora, Phaeoacremonium spp. and C. luteo-olivacea, respectively. The European earwig (Forficula auricularia) and various spiders were the most common arthropods on vines, with frequencies of 30 % and 27 %, respectively. Under artificial conditions, it was confirmed that earwigs and black garden ants (Lasius niger) can transmit the pathogens Pa. chlamydospora and Pm. minimum to healthy potted vines. The potential of earwig feces as an inoculum source for Pa. chlamydospora and Pm. minimum was also discussed. Under artificial conditions, it was shown that the germination ability of the spores is not impaired by earwig gut passage and that healthy potted vines can be infected with contaminated earwig feces. However, fecal samples from earwigs collected in vineyards showed only a very low detection rate of at least esca pathogens. The number of Pa. chlamydospora spores on the exoskeletons of earwigs, ants and two jumping spider species collected in vineyards was determined by qPCR. The spore number was in 40 % of the samples that had previously tested positive for Pa. chlamydospora in the nested PCR below the detection limit of the qPCR. In 56 % of the samples analyzed by qPCR, fewer than 1000 spores were detected. Under natural conditions, a number of 100-2000 Pa. chlamydospora-spores is sufficient to infect susceptible wounds in the vine wood. In this work, the mycobiome of the surface of the exoskeleton of earwigs collected in vineyards was analyzed for the first time. Nine GTD-associated genera were detected, of which Phaeomoniella and Eutypa were found to have relative abundances of 6.6 % and 2.8 %, respectively. This could indicate a possible involvement of earwigs in the dissemination of esca and eutypiosis. The present investigations have shown that arthropod-mediated transmission of GTDs, especially esca pathogens, is theoretically possible in vineyards. However, the practical relevance of this fact seems low compared to the numbers of airborne spores of the pathogens, especially Pa. chlamydospora, detected during the presumed period of susceptibility of winter pruning wounds. In addition, the simultaneous occurrence of susceptible winter pruning wounds and potential vectors in the field is only present with late-pruned vines, while the risk of wounds during the growing season requires a more detailed assessment. The results of this work underline the importance of protecting all types of wounds in the vine wood in order to reduce the invasion of pathogens.
Kinetics and dynamics of plant toxins in sequestering insects
(2024) Betz, Anja; Petschenka, Georg
Many plants produce a variety of chemical compounds to protect themselves from herbivorous insects. However, numerous insects have developed adaptations to tolerate and even utilize these toxins. These adaptations can include behavioral changes (feeding on or avoiding toxic plant parts), detoxification (metabolizing or excreting the toxins), barriers (preventing uptake or protecting target sites), or sequestration (storing secondary plant compounds in the body for self-defense). A widely distributed group of phytochemicals are the cardiac glycosides, which have been extensively studied in chemical ecology. These toxic steroids act in a highly specific manner by inhibiting the Na+/K+-ATPase, an essential transmembrane cation carrier that is ubiquitously expressed in animal cells. A cardiac glycoside-resistant Na+/K+-ATPase was first described in the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini), which sequesters cardiac glycosides from its food plants. This led to the hypothesis that the evolution of a resistant Na+/K+-ATPase is linked to the sequestration of cardiac glycosides. It is now known that species from at least six insect orders possess Na+/K+-ATPases with reduced affinity for cardiac glycosides. This resistance is mediated by a few amino acid substitutions in the binding site, a mechanism referred to as "target site insensitivity." Interestingly, the ability to tolerate dietary cardiac glycosides does not necessarily require a resistant Na+/K+-ATPase. For example, the non-sequestering common crow (Euploea core, Lepidoptera: Danaini) can develop on cardiac glycoside-containing plants without expressing a resistant Na+/K+-ATPase. Although plant toxin sequestration has been documented in more than 275 insect species, the underlying kinetics and dynamics of toxin transport - such as the duration and mode of transport, target sites, barriers, and potential modified binding sites - remain largely unknown. Like the Danaini, milkweed bugs (Heteroptera: Lygaeinae) also have the ability to sequester cardiac glycosides. Remarkably, one species within this family, Spilostethus saxatilis, is able to sequester not only cardiac glycosides but also colchicine alkaloids from autumn crocus (Colchicum autumnale, Liliales: Colchicaceae). Colchicine binds to unpolymerized betatubulin at the interface with alpha-tubulin, thereby inhibiting microtubule formation. However, it is not known how colchicine resistance is achieved in S. saxatilis and whether there is also target site insensitivity. In my dissertation, I investigated the mechanisms behind the sequestration of cardiac glycosides in D. plexippus and colchicine in S. saxatilis within a phylogenetic framework. In D. plexippus, I was able to show that sequestration begins at the behavioral level, because unlike related species, caterpillars can increase their toxicity in the adult stage by drinking plant latex containing cardiac glycosides. I also found that a large portion of cardiac glycosides is lost during metamorphosis, suggesting that excessive toxin intake during the larval stage may be important. Furthermore, I could show that the uptake of the toxin by the intestinal epithelium is a very rapid process although probably not specific to D. plexippus, as uptake in nonsequestering lepidopterans does also occur. Regarding the sequestration of colchicine in S. saxatilis, I identified an amino acid substitution in the colchicine binding site of tubulin. Subsequently, an in vitro colchicine binding assays with crude tubulin extract of bug tissue and an in vivo feeding experiment with genetically modified Drosophila with one matching substitution in the colchicine binding site were conducted. Together both experiments suggest that this modification may play a crucial role in the sequestration of colchicine, indicating a novel natural target site resistance. My dissertation provides new insights into how insects adapt to toxic food plants and how they sequester their toxic metabolites. This contributes not only to a better understanding of how insects cope with natural plant toxins, but also to a better understanding of the physiological complexity of these adaptations.
Hohenheimer Lexikon der Agrarbiografien
„Persönlichkeiten aus Land- und Forstwirtschaft, Gartenbau und Veterinärmedizin“
(2025) Boettcher, Hartmut; Raupp, Manfred G.
Development and testing of a precision hoeing system for re-compacted ridge tillage in maize
(2024) Alagbo, Oyebanji O.; Saile, Marcus; Spaeth, Michael; Schumacher, Matthias; Gerhards, Roland
Ridge tillage (RT) is a conservation practice that provides several benefits such as enhanced root growth and reduced soil erosion. The objectives of this study were to develop an autosteered living mulch seeder and hoeing prototype for RT systems using RTK-GNSS (real-time kinematic global navigation satellite systems) created ridges as a guide. It was also aimed to compare weed control efficacy and crop response of ridge-hoeing compared to conventional hoeing in flat tillage (FT). It was further aimed to investigate the impact of a new RT technology (with ridge re-compaction) on maize root development, yield, soil temperature, and moisture compared to FT. Field experiments were conducted with maize in 2021 and 2022 in a two-factorial split-plot design with tillage (RT and FT) as main treatment and weed control (untreated, herbicide, twice hoeing, hoeing + living mulch) as sub-treatment factors. Weed density, coverage, biomass, crop density, weed control efficacy (WCE) and maize silage yield were assessed. Temperature loggers were installed within RT and FT to take temperature readings at 20 min. Soil moisture and root penetrability were measured every two weeks in each plot using soil samples and a penetrometer. The WCE and yield did not differ significantly between the tillage systems. Twice hoeing resulted in 71–80 % WCE, which was equal to herbicide treatment. Hoeing + living mulch achieved 70–72 % WCE. Different from previous studies with ridge tillage, temperatures in the compacted ridges did not consistently differ from the ridge valleys and flat seedbeds. Root penetration (against 1.4 MPa penetrometer cone index) was 40 % higher in RT than in FT. On average, RT maize produced more (53.6 g m−2) root biomass compared to FT. In summary, re-compacted ridges built along RTK-GNSS lines can allow post-emergent hoeing and living mulch seeding along ridges and also provide good growing conditions for maize.
A novel dataset of annotated oyster mushroom images with environmental context for machine learning applications
(2024) Duman, Sonay; Elewi, Abdullah; Hajhamed, Abdulsalam; Khankan, Rasheed; Souag, Amina; Ahmed, Asma
State-of-the-art technologies such as computer vision and machine learning, are revolutionizing the smart mushroom industry by addressing diverse challenges in yield prediction, growth analysis, mushroom classification, disease and deformation detection, and digital twinning. However, mushrooms have long presented a challenge to automated systems due to their varied sizes, shapes, and surface characteristics, limiting the effectiveness of technologies aimed at mushroom classification and growth analysis. Clean and well-labelled datasets are therefore a cornerstone for developing efficient machine-learning models. Bridging this gap in oyster mushroom cultivation, we present a novel dataset comprising 555 high-quality camera raw images, from which approximately 16.000 manually annotated images were extracted. These images capture mushrooms in various shapes, maturity stages, and conditions, photographed in a greenhouse using two cameras for comprehensive coverage. Alongside the images, we recorded key environmental parameters within the mushroom greenhouse, such as temperature, relative humidity, moisture, and air quality, for a holistic analysis. This dataset is unique in providing both visual and environmental time-point data, organized into four storage folders: “Raw Images”; “Mushroom Labelled Images and Annotation Files”; “Maturity Labelled Images and Annotation Files”; and “Sensor Data”, which includes time-stamped sensor readings in Excel files. This dataset can enable researchers to develop high-quality prediction and classification machine learning models for the intelligent cultivation of oyster mushrooms. Beyond mushroom cultivation, this dataset also has the potential to be utilized in the fields of computer vision, artificial intelligence, robotics, precision agriculture, and fungal studies in general.
Efficacy of various mechanical weeding methods - single and in combination - in terms of different field conditions and weed densities
(2021) Naruhn, Georg-Peter; Peteinatos, Gerassimos G.; Butz, Andreas F.; Möller, Kurt; Gerhards, Roland
Public awareness and environmental policies have increased interest in applying non-herbicide weed control methods in conventional farming systems. Even though mechanical weed control has been used for centuries in agricultural practice, continuous developments—both in terms of implements and automation technologies—are continuously improving the potential outcomes. Current mechanical weed control methods were evaluated for their weed control efficacy and effects on yield potential against their equivalent herbicide methods. Furthermore, not much is known about the correlation between weed control efficacy (WCE) of different mechanical methods at varying weed density levels. A total of six experiments in winter wheat (2), peas (2), and soybean (2) were carried out in the years 2018, 2019, and 2020 in southwestern Germany. Harrowing and hoeing treatments at different speeds were carried out and compared to the herbicide treatments and untreated control plots. Regarding the average WCE, the combination of harrowing and hoeing was both the strongest (82%) and the most stable (74–100%) mechanical treatment in the different weed density levels. Whereas, in average, hoeing (72%) and harrowing (71%) were on the same WCE level, but harrowing (49–82%) was more stable than hoeing (40–99%). The grain yields in winter wheat varied between 4.1 Mg∙ha−1 (control) and 6.3 Mg∙ha−1 (harrow), in pea between 2.8 Mg∙ha−1 (hoe slow) and 5.7 Mg∙ha−1 (hoe fast) and in soybean between 1.7 Mg∙ha−1 (control) and 4 Mg∙ha−1 (herbicide). However, there were no significant differences in most cases. The results have shown that it is not possible to pinpoint a specific type of treatment as the most appropriate method for this cultivation, across all of the different circumstances. Different field and weather conditions can heavily affect and impact the expected outcome, giving, each time, an advantage for a specific type of treatment.
Role of benzoic acid and lettucenin A in the defense response of lettuce against soil-borne pathogens
(2021) Windisch, Saskia; Walter, Anja; Moradtalab, Narges; Walker, Frank; Höglinger, Birgit; El-Hasan, Abbas; Ludewig, Uwe; Neumann, Günter; Grosch, Rita
Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.
Assessment of a postharvest treatment with pyrimethanil via thermo-nebulization in controlling storage rots of apples
(2021) Büchele, Felix; Neuwald, Daniel A.; Scheer, Christian; Wood, Rachael M.; Vögele, Ralf T.; Wünsche, Jens N.
Apples are very susceptible to infections from various fungal pathogens during the growing season due to prolonged exposure to environmental influences in the field. Therefore, a strict and targeted fungicide strategy is essential to protect fruit and trees. Increased environmental and health concerns and pathogen resistance have resulted in a rising demand to reduce fungicide usage and residues on marketed fruit. Thus, producers must develop new plant protection strategies to conform to the legal and social demands while still offering high-quality apples. This study assessed the efficacy of a post-harvest fungicide treatment with pyrimethanil via thermo-nebulization for controlling storage rots and compared the results to those of standard pre-harvest fungicide strategies. The results showed that a single post-harvest application of pyrimethanil successfully controlled storage rots and is comparable to strategies using multiple pre-harvest fungicide applications. The control of fungal rot was sustained even after 5 months of storage and 2 weeks of shelf life. Thermo-nebulization into the storage facility allowed for a lower dosage of fungicide to be used compared to pre-harvest applications, while still maintaining optimal rot control. Residue analyses showed that the post-harvest fungicide treatment did not exceed legal or retailer’s standards.
Exploring the effects of different stubble tillage practices and glyphosate application combined with the new soil residual herbicide cinmethylin against Alopecurus myosuroides Huds. in winter wheat
(2022) Messelhäuser, Miriam Hannah; Saile, Marcus; Sievernich, Bernd; Gerhards, Roland
Effective control of Alopecurus myosuroides Huds. (blackgrass) solely with a chemical treatment is not guaranteed anymore because populations exhibit resistance to almost all herbicide modes of action. Integrated weed management (IWM) against blackgrass is necessary to maintain high weed control efficacies in winter cereals. Four field experiments were conducted in Southwest Germany from 2018 to 2020 to control A. myosuroides with a combination of cultural and chemical methods. Stubble treatments, including flat, deep and inversion soil tillage; false seedbed preparation and glyphosate use, were combined with the application of the new pre-emergence herbicide cinmethylin in two rates in winter wheat. Average densities of A. myosuroides in the untreated control plots were up to 505 plants m−2. The combination of different stubble management strategies and the pre-emergence herbicide cinmethylin controlled 86–97% of A. myosuroides plants at the low rate and 95–100% at the high rate until 120 days after sowing. The different stubble tillage practices varied in their efficacy between trials and years. Most effective and consistent were pre-sowing glyphosate application on the stubble and stale seedbed preparation with a disc harrow. Stubble treatments increased winter wheat density in the first year but had no effect on crop density in the second year. Pre-emergence application of cinmethylin did not reduce winter wheat densities. Multiple tactics of weed control, including stubble treatments and pre-emergence application of cinmethylin, provided higher and more consistent control of A. myosuroides. Integration of cultural weed management could prevent the herbicide resistance development.
Agronomic and technical evaluation of herbicide spot spraying in maize based on high-resolution aerial weed maps - an on-farm trial
(2024) Allmendinger, Alicia; Spaeth, Michael; Saile, Marcus; Peteinatos, Gerassimos G.; Gerhards, Roland; Allmendinger, Alicia; Department of Weed Science, Institute for Phytomedicine, University of Hohenheim, 70599 Stuttgart, Germany; (A.A.);; Spaeth, Michael; Department of Weed Science, Institute for Phytomedicine, University of Hohenheim, 70599 Stuttgart, Germany; (A.A.);; Saile, Marcus; Department of Weed Science, Institute for Phytomedicine, University of Hohenheim, 70599 Stuttgart, Germany; (A.A.);; Peteinatos, Gerassimos G.; ELGO-DIMITRA, Leof Dimokratias 61, Agii Anargiri, 135 61 Athens, Greece;; Gerhards, Roland; Department of Weed Science, Institute for Phytomedicine, University of Hohenheim, 70599 Stuttgart, Germany; (A.A.);; Rossi, Vittorio
Spot spraying can significantly reduce herbicide use while maintaining equal weed control efficacy as a broadcast application of herbicides. Several online spot-spraying systems have been developed, with sensors mounted on the sprayer or by recording the RTK-GNSS position of each crop seed. In this study, spot spraying was realized offline based on georeferenced unmanned aerial vehicle (UAV) images with high spatial resolution. Studies were conducted in four maize fields in Southwestern Germany in 2023. A randomized complete block design was used with seven treatments containing broadcast and spot applications of pre-emergence and post-emergence herbicides. Post-emergence herbicides were applied at 2–4-leaf and at 6–8-leaf stages of maize. Weed and crop density, weed control efficacy (WCE), crop losses, accuracy of weed classification in UAV images, herbicide savings and maize yield were measured and analyzed. On average, 94% of all weed plants were correctly identified in the UAV images with the automatic classifier. Spot-spraying achieved up to 86% WCE, which was equal to the broadcast herbicide treatment. Early spot spraying saved 47% of herbicides compared to the broadcast herbicide application. Maize yields in the spot-spraying plots were equal to the broadcast herbicide application plots. This study demonstrates that spot-spraying based on UAV weed maps is feasible and provides a significant reduction in herbicide use.
CRISPR/SpCas9‐mediated double knockout of barley Microrchidia MORC1 and MORC6a reveals their strong involvement in plant immunity, transcriptional gene silencing and plant growth
(2021) Galli, Matteo; Martiny, Engie; Imani, Jafargholi; Kumar, Neelendra; Koch, Aline; Steinbrenner, Jens; Kogel, Karl‐Heinz
The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9‐mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium‐mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9‐induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo‐/heteromers with other HvMORCs and interact with components of the RNA‐directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.
Successful silencing of the mycotoxin synthesis gene TRI5 in fusarium culmorum and observation of reduced virulence in VIGS and SIGS experiments
(2022) Tretiakova, Polina; Voegele, Ralf Thomas; Soloviev, Alexander; Link, Tobias Immanuel
Crops constantly experience various biotic stresses during their life cycle, and Fusarium spp. remain one of the most serious groups of pathogens affecting plants. The ability to manipulate the expression of certain microorganism genes via RNAi creates the opportunity for new-generation dsRNA-based preparations to control a large number of diseases. In this study, we applied virus-induced gene silencing (VIGS), and spray-induced gene silencing (SIGS) to silence the trichothecene-producing gene TRI5 in F. culmorum as a means to reduce its aggressiveness on spring wheat. Treatment of the fungus with dsTRI5RNA in vitro reduced deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-A-DON) accumulations by 53–85% and 61–87%, respectively, and reduced TRI5 expression by 84–97%. VIGS decreased the proportion of infected wheat spikelets by 73%, but upregulation was observed for TRI5. SIGS on wheat leaves and ears using certain dsTRI5RNA amounts negatively impacted F. culmorum growth. However, when performing in vivo analyses of TRI5 mRNA levels, the upregulation of the gene was determined in the variants where fungal colonization was restricted, suggesting a compensatory reaction of the pathogen to RNAi.
Advances in site-specific weed management in agriculture: A review
(2022) Gerhards, Roland; Andújar Sanchez, Dionisio; Hamouz, Pavel; Peteinatos, Gerassimos G.; Christensen, Svend; Fernandez‐Quintanilla, Cesar
The developments of information and automation technologies have opened a new era for weed management to fit physical and chemical control treatments to the spatial and temporal heterogeneity of weed distributions in agricultural fields. This review describes the technologies of site‐specific weed management (SSWM) systems, evaluates their ecological and economic benefits and gives a perspective for the implementation in practical farming. Sensor technologies including 3D cameras, multispectral imaging and Artificial Intelligence (AI) for weed classification and computer‐based decision algorithms are described in combination with precise spraying and hoeing operations. Those treatments are targeted for patches of weeds or individual weed plants. Cameras can also guide inter‐row hoes precisely in the centre between two crop rows at much higher driving speed. Camera‐guided hoeing increased selectivity and weed control efficacy compared with manual steered hoeing. Robots combine those guiding systems with in‐row hoeing or spot spraying systems that can selectively control individual weeds within crop rows. Results with patch spraying show at least 50% saving of herbicides in various crops without causing additional costs for weed control in the following years. A challenge with these technologies is the interoperability of sensing and controllers. Most of the current SSWM technologies use their own IT protocols that do not allow connecting different sensors and implements. Plug & play standards for linking detection, decision making and weeding would improve the adoption of new SSWM technologies and reduce operational costs. An important impact of SSWM is the potential contribution to the EU‐Green Deal targets to reduce pesticide use and increase biodiversity. However, further on‐farm research is needed for integrating those technologies into agricultural practice.
New approaches to manage Asian soybean rust (Phakopsora pachyrhizi) using Trichoderma spp. or their antifungal secondary metabolites
(2022) El-Hasan, Abbas; Walker, Frank; Klaiber, Iris; Schöne, Jochen; Pfannstiel, Jens; Voegele, Ralf T.
Attempts have been made to determine the in vitro and in planta suppressive potential of particular Trichoderma strains (T16 and T23) and their secondary metabolites (SMs) against Asian soybean rust (ASR) incited by Phakopsora pachyrhizi. Aside from the previously identified SMs 6-pentyl-α-pyrone (6PAP) and viridiofungin A (VFA), the chemical structures of harzianic acid (HA), iso-harzianic acid (iso-HA), and harzianolide (HZL) were characterized in this study. Our results indicate that exposure of urediospores to 200 ppm 6PAP completely inhibits germination. A slightly higher dosage (250 ppm) of HZL and VFA reduces germination by 53.7% and 44%, respectively. Germ tube elongation seems more sensitive to 6PAP than urediospore germination. On detached leaves, application of conidia of T16 and T23 results in 81.4% and 74.3% protection, respectively. Likewise, 200 ppm 6PAP recorded the highest ASR suppression (98%), followed by HZL (78%) and HA (69%). Treatment of undetached leaves with 6PAP, HA, or HZL reduces ASR severity by 84.2%, 65.8%, and 50.4%, respectively. Disease reduction on the next, untreated trifoliate by T23 (53%), T16 (41%), HZL (42%), and 6PAP (32%) suggests a translocation or systemic activity of the SMs and their producers. To our knowledge, this study provides the first proof for controlling ASR using antifungal SMs of Trichoderma. Our findings strongly recommend the integration of these innovative metabolites, particularly 6PAP and/or their producers in ASR management strategies.
Precision chemical weed management strategies: A review and a design of a new CNN-based modular spot sprayer
(2022) Allmendinger, Alicia; Spaeth, Michael; Saile, Marcus; Peteinatos, Gerassimos G.; Gerhards, Roland
Site-specific weed control offers a great potential for herbicide savings in agricultural crops without causing yield losses and additional weed management costs in the following years. Therefore, precision weed management is an efficient tool to meet the EU targets for pesticide reduction. This review summarizes different commercial technologies and prototypes for precision patch spraying and spot spraying. All the presented technologies have in common that they consist of three essential parts. (1) Sensors and classifiers for weed/crop detection, (2) Decision algorithms to decide whether weed control is needed and to determine a suitable type and rate of herbicide. Usually, decision algorithms are installed on a controller and (3) a precise sprayer with boom section control or single nozzle control. One point that differs between some of the techniques is the way the decision algorithms classify. They are based on different approaches. Green vegetation can be differentiated from soil and crop residues based on spectral information in the visible and near-infrared wavebands (“Green on Brown”). Those sensors can be applied for real-time on/off control of single nozzles to control weeds before sowing after conservation tillage and in the inter-row area of crops. More sophisticated imaging algorithms are used to classify weeds in crops (“Green on Green”). This paper will focus on Convolutional Neural Networks (CNN) for plant species identification. Alternatively, the position of each crop can be recorded during sowing/planting and afterward herbicides can be targeted to single weeds or larger patches of weeds if the economic weed threshold is exceeded. With a standardized protocol of data communication between sensor, controller and sprayer, the user can combine different sensors with different sprayers. In this review, an ISOBUS communication protocol is presented for a spot sprayer. Precision chemical weed control can be realized with tractor-mounted sprayers and autonomous robots. Commercial systems for both classes will be introduced and their economic and environmental benefits and limitations will be highlighted. Farmers ask for robust systems with less need for maintenance and flexible application in different crops.
Effects of a two-factorial dynamic storage system on apple fruit metabolism, quality attributes, disorder incidences and biochemical properties
(2024) Büchele, Felix; Vögele, Ralf; Kittemann, Dominikus
Dynamic Controlled Atmosphere (DCA) technologies are designed to monitor the metabolism of fruit as a function of time and the oxygen partial pressure (pO2) in the storage atmosphere. By identifying signals sent by the fruit in response to low oxygen stress, this concept allows defining the lowest oxygen level tolerated by the fruit material and the specific and transient pO2, which is proposed to slow down the aerobic respiration of the fruit to a minimum and accordingly the ripening-related loss of fruit quality. This work examines a novel storage technology for pome fruit referred to as DCA-CD+, which can be considered the first generation of a two-factorial dynamic storage system. DCA-CD+ aims to define both a transient optimum pO2 and storage temperature in real-time, based on monitoring the carbon dioxide (CO2) production rate of the stored product. The CO2-release rate is proposed as a dependable indicator of low oxygen stress and an accurate depiction of the metabolic intensity of the fruit in response to temperature variations. The idea behind dynamic temperature adjustments is based on the assumption, that increased storage temperatures can reduce the energy usage of the refrigeration systems during long-term storage substantially, while also mitigating oxidative stress in the fruit, thus contributing to reducing the occurrence of storage disorders. The extremely low pO2 levels established in the storage atmosphere are suggested to counteract the ripening-inducing effects of this dynamic temperature approach. The assessment of DCA-CD+ in this work is based on a comparison to other postharvest conditions such as cold storage (RA) or static controlled atmosphere (CA). Furthermore, the interactions between storage atmosphere, temperature, and applications of the ethylene-inhibitor product 1-methylcyclopropene (1-MCP) are investigated. The conducted biochemical analyses highlight that apples stored under DCA-CD+ undergo repeated periods of hypoxia. Fruit adapt to the energy crisis induced by low oxygen stress by increasing their glycolytic flux, which is coupled to the activation of the fermentative pathway. Importantly, none of the examined apple cultivars in any of the experimental seasons exceeded critical thresholds for volatile fermentative products acetaldehyde (AA) and ethanol (EtOH), which could potentially be associated with the development of off-flavors or internal disorder symptoms. Consequently, the atmospheric conditions implemented did not result in any fruit damage associated with low oxygen stress in any of the tested scenarios. These findings suggest that DCA-CD+, specifically the use of carbon dioxide as an input value, is effective in identifying low oxygen stress in stored fruit and defining the pO2 for optimum quality conservation. pO2 setpoint calculation by the DCA-CD+ algorithm showed an interaction effect with the respective storage temperatures. Furthermore, the sensitivity of the stored fruit to temperature variation was found to be cultivar-dependent and transient during the storage period. Depending on the stored apple cultivar and season, DCA-CD+ calculated temperature setpoints reaching up to 3°C to 4°C, from the baseline temperature of 1°C. Temperature peaks were generally followed by a significant decrease in the calculated temperature setpoints, as an increased CO2 production rate signaled an intensifying fruit metabolism due to elevated storage temperature. The analysis of quality-defining parameters and disorder symptoms support the conclusion that DCA-CD+ allows for dynamic temperature adjustments without accelerating fruit ripening and the associated loss of quality. Preliminary findings indicate that this approach can reduce the energy usage of the cooling system in commercial storage rooms, without requiring cost-intensive additional installation of technology or renovations of room structural components. Lower pO2 setpoints were calculated at higher temperatures, suggesting that increasing the storage temperature can contribute to alleviating low oxygen stress in apples. Improved conservation of fruit quality attributes and a reduction in storage disorder incidences of DCA-CD+ in comparison to CA could be demonstrated in some instances, contingent on experimental season and apple cultivar. These benefits presumably become more pronounced with extended storage durations exceeding eight months. Ultimately, it can be argued that the complementary and interactive effects of dynamic temperature and oxygen in DCA-CD+ with 1-MCP application provide the highest potential for fruit quality conservation, limiting storage disorder, and reducing cooling-related energy usage. DCA-CD+ was demonstrated to potentially counteract detrimental effects of 1-MCP applications, e.g. an increased risk of carbon dioxide injuries. This work aimed to contribute to understanding the mechanisms behind the interference of the established temperature and atmospheric conditions in the DCA-CD+ system with the volatile aroma profile of apples. It was demonstrated that the synthesis capacity of volatile organic compounds (VOC) is primarily suppressed on a principal substrate level, and less in the later conversion of aldehydes to alcohols and esters. The pathways for the synthesis of linear volatiles originating from fatty acids were determined more responsive to low oxygen environments, in comparison to the pathways of branched volatiles derived from amino acids. Further insights were gained into the physiological mechanisms underlying the activation of the fermentative pathway, suggesting it functions as an adaptation mechanism not exclusively linked to low oxygen stress. Moreover, efforts were made to establish a connection between ripening and disorder-related modifications in cell structural components and associated alterations in the volatile profile, primarily highlighting the role of the lipoxygenase pathway. Statistical classification demonstrated that the repeated induction of low oxygen stress in DCA-CD+ storage resulted in a distinct volatile profile and a higher association with aroma defining compounds compared to CA storage. The observed increased EtOH accumulation is discussed to mitigate the ripening-inducing effects of the hormone ethylene, while also providing additional substrate for the synthesis of ethyl esters. Preliminary findings of this work indicate that storage temperatures can play a role in the aroma development of stored apples, even when low pO2 conditions are established. In summary, this study created a comprehensive documentation of the commercial viability of two-factorial dynamic storage systems for pome fruit and provided insights into the metabolomic responses of apples to extremely low oxygen levels, particularly in interaction with dynamic storage temperature adjustments.
Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles to assess their role in RNA spray-based crop protection
(2021) Schlemmer, Timo; Barth, Patrick; Weipert, Lisa; Preußer, Christian; Hardt, Martin; Möbus, Anna; Busche, Tobias; Koch, Aline
The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.

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