Beneficial Plant-Microbe Interactions to Improve Nutrient Uptake and Biotic Stress Response in Crops

Mutualism is a very common phenomenon among living organisms on earth. Legumes because of their high protein content, serve as a great nutrient resource for animals. This group of plants can form a mutualistic symbiosis with beneficial microbes. For example, Alfalfa (Medicago) and soybean (Glycine max) can get colonized with arbuscular mycorrhizal fungi (AMF) and rhizobia bacteria simultaneously forming a complex tripartite interaction for nutrient benefits. Most of the previous research evaluated individual symbionts, either rhizobia bacteria or AMF, but not both. There are only a few reports which discuss the nutrient exchange mechanisms in a tripartite interaction. Thus, there is a lack of fundamental understanding of how the resources are exchanged in tripartite interactions. Nitrogen (N) and phosphorus (P) are essential nutrients for plant growth; AMF can supply both P and N, while rhizobia bacteria can only supply N to their host plant. Both root symbionts can provide other benefits like abiotic and biotic stress tolerance. In return, the host plant distributes a substantial amount of its photosynthetic carbon (C) produced in the leaves to its root symbionts. However, the regulation mechanisms on C resources allocation by the host plant to its root symbionts is not well understood. In my first experiment, I hypothesized that the N-fixing capability of the rhizobia bacteria affects the C allocation pattern in a tripartite system with AMF. I evaluated C allocation to the symbionts under in a tripartite interaction with various nutrient access scenarios including the use of a rhizobial strain that lacks biological nitrogen fixation (BNF) capability and AMF having access to a labeled N source. The dual inoculation of N fixing rhizobia (Fix+) and AMF results in a synergistic increase in shoot biomass, enhanced N and P uptake in the sink (roots) but low delivery toward the source (leaves). On the other hand, tripartite interactions of Fix- rhizobia that lack biological N fixation activity and AMF lead to a significant increase in N uptake and delivery towards the source but a significant drop in carbon allocation towards Fix- rhizobia root. Consistent with these findings, we found changes in SUCROSE UPTAKE TRANSPORTER (SUT) and SUGAR WILL EVENTUALLY BE EXPORTED TRANSPORTER (SWEET) genes. These results provide substantial new information about how host plants control their carbon allocations under the different status of N demand in presence of rhizobia and AMF inoculation. During tripartite interactions, rhizobia bacteria are restricted to the host roots but extraradical mycelia (ERM) of AMF can go beyond, colonizing another host root. This leads to the development of common networks among two or more plants which are known as the common mycelial Network (CMN), creating a biological market for nutrient transport. The nitrogen-fixing capability of rhizobia bacteria can affect the transport of nitrogen (N) by AMF to host plants connected by CMNs. In the second experiment, I hypothesized that access of exogenous 15N to AMF would allocate more N to host plants colonized by Fix- rhizobia that lack BNF capability than those colonized by Fix+ rhizobia. We found that co-inoculation with Fix- rhizobia with AMF or non-mycorrhizal control plants resulted in elevated 15N enrichment in the shoot of the host plant. This suggests that AMF allocates most of the N they uptake from the soil to the host plant with a greater N demand due to the lack of access to fixed nitrogen. As expected, we found that AMF does not transfer as much N with host plants colonized by Fix+ rhizobia because their N demand can be fulfilled by the rhizobia bacteria. Plant diseases can be managed in various ways, including the use of disease-resistant and/or tolerant crop varieties, chemical controls, and biological controls. A diseaseresistant variety can lose its resistance due to the development of a new variant of the pathogen. Chemicals used in agriculture and other systems can have a very adverse effect on the environment. The use of Microbes for controlling plant diseases is safer and offers environmental sustainability compared to chemical pesticides. In my third experiment, I evaluated if AMF could mitigate the destructive effect of Soybean cyst nematode (SCN: Heterodera glycines), one of the most dreadful pests in soybean. Soybean plants infested with SCN do not show any aboveground symptoms in most of the cases, so the field gets unrecognized for a long time. Through the AMF symbiosis, plant hosts receive protection from pathogens as well among other benefits. In this experiment, we evaluated the effects of a commercially available AMF soil additive called MycoApply® (consists of an equal ratio of Glomus mossaea, Rhizophagus irregulare, G. etunicatum, G. aggregatum) under greenhouse and field conditions on the reproduction of SCN and the soybean growth and yield increase. We observed increased shoot weight for AMF-treated SCN susceptible variety (Williams-82) infested with SCN but no effect on the resistant variety, Jack (PI88788) in a greenhouse but no differences were found in SCN egg number. However, soybean seed yield was increased up to 40 % in mycorrhizal treated plots than nonmycorrhizal plots (they do have a natural community of AMF). Our results show that commercially available AMF inoculum can be used to increase soybean production even in the field infested with SCN. However, further investigation should be conducted to know the actual mechanism of how these fungi are able to increase soybean production without any change in AM colonization rate and reduction in SCN egg population in the soil. In summary, tripartite interactions of legumes with AM fungi and rhizobia bacteria led synergistically increase in plant growth independent of N fixing capability of rhizobia. However, delivery of N by AMF towards shoot increased when plants only have AMF for N source. Consistent with the biological market model, the host plant allocates a significant amount of C to benefit root symbionts. Similar trends were found when plants were interconnected via CMNs. On the other hand, AMF does not provide nutritional benefits but also can provide biotic stress tolerance such as enhanced SCN tolerance. All these indicated a bigger potential role for beneficial microbes in sustainable agriculture

Root Morphological and Physiological Bases to Understand Genotypic Control of Mineral Acquisition in Rice Grains

Rice (Oryza sativa L.) supports half of the human population. However, predominant rice consumption leads to malnutrition due to mineral deficiencies. The research goal was to support identification of genes responsible for the uptake/accumulation of potassium (K), iron (Fe), zinc (Zn) and molybdenum (Mo), thus promoting the breeding for rice with high grain concentrations of these elements. Prior studies identified rice genotypes with high grain-K, -Fe, -Zn or -Mo concentrations that were hypothesized to be due to differences in root traits. The research objective was to identify root traits associated with these elements. These traits could be bases for identifying genes. The first study determined if these genotypes showed similar accumulation patterns in leaves as in grains, which would hint at influences of the roots and enable identifying distinct root traits and possible genes in vegetative growth stages. The second study determined if root traits of high grain-Mo genotypes displayed an acid-tolerance mechanism as these genotypes originated from Malaysia where acidic soils strongly adsorb Mo making it unavailable for plants. The third study identified root trait differences of high grain-K, -Fe, -Zn and -Mo genotypes in hydroponics media, while the fourth determined root trait differences in these genotypes in sand-culture media including a 1-Naphthalene Acetic Acid (NAA) seed treatment for perturbation. The first study identified several high grain-Mo genotypes with similar Mo accumulation patterns in V4 to V6 stage-leaves as in grains, suggestive of a root influence. The second study established that gross morphological and physiological root traits of a high grain-Mo genotype were not part of an acid-tolerance mechanism. Neither the third nor fourth study identified root traits related to shoot K, Fe, Zn or Mo concentration, however positive associations of seedling vigor traits with several beneficial elements, including K, and negative associations with numerous toxic elements were established. Lack of correlation with root traits suggests other mechanisms (e.g. active uptake transporters) instead control the observed grain accumulation differences. Based on the fourth study, either direct effects of NAA on element uptake/transfer or indirect effects on soil pH and redox potential altered tissue Fe and Zn levels

Deciphering the rhizosphere bacteriome associated with biological control of tobacco black shank disease

IntroductionThe black shank disease seriously affects the health of tobacco plants. Conventional control methods have limitations in terms of effectiveness or economic aspects and cause public health concerns. Thus, biological control methods have come into the field, and microorganisms play a key role in suppressing tobacco black shank disease.MethodsIn this study, we examined the impact of soil microbial community on black shank disease basing on the structural difference of bacterial communities in rhizosphere soils. We used Illumina sequencing to compare the bacterial community diversity and structure in different rhizosphere soil samples in terms of healthy tobacco, tobacco showing typical black shank symptoms, and tobacco treated with the biocontrol agent, Bacillus velezensis S719.ResultsWe found that Alphaproteobacteria in the biocontrol group, accounted for 27.2% of the ASVs, was the most abundant bacterial class among three groups. Heatmap and LEfSe analyses were done to determine the distinct bacterial genera in the three sample groups. For the healthy group, Pseudomonas was the most significant genus; for the diseased group, Stenotrophomonas exhibited the strongest enrichment trend, and Sphingomonas showed the highest linear discriminant analysis score, and was even more abundant than Bacillus; for the biocontrol group, Bacillus, and Gemmatimonas were the largely distributed genus. In addition, co-occurrence network analysis confirmed the abundance of taxa, and detected a recovery trend in the network topological parameters of the biocontrol group. Further functional prediction also provided a possible explanation for the bacterial community changes with related KEGG annotation terms.DiscussionThese findings will improve our knowledge of plant-microbe interactions and the application of biocontrol agents to improve plant fitness, and may contribute to the selection of biocontrol strains

Population Genetics and Phylogenetic Context of Weed Evolution in the Genus Amaranthus: Amaranthaceae)

Agricultural weeds have evolved to compete aggressively with domesticated plants in agricultural environments. Although the evolution of invasiveness has been studied extensively in natural ecosystems, few comparable studies have been conducted using agricultural weeds. In this dissertation, I used the genus Amaranthus to examine agricultural weed evolution over different evolutionary time scales, ranging from fitness measurements within a single species to a genus-wide, macroevolutionary analysis. To explore the recent evolution of agricultural invasiveness, I studied a native Midwestern species, A. tuberculatus: waterhemp), which has become an aggressive agricultural weed only within the last several decades. I used microsatellite markers to investigate the present-day population structure of A. tuberculatus. To assess intraspecific variation in agricultural adaptation, I conducted a common garden study measuring the relative fitness of plants from across the species range in experimental soybean plots. I discovered two genetic subpopulations. The 20th century invasion of Midwestern agricultural fields was due to the eastward migration of the western genetic subpopulation, which has high competitive fitness in soybean fields and which may have been preadapted to the agricultural environment. Waterhemp has rapidly evolved resistance to multiple classes of herbicides. The role of native Midwestern riverbank populations in this process is unknown. I screened agricultural and riverbank populations of A. tuberculatus in Ohio for a common agricultural resistance mutation, using a combination of herbicide resistance phenotyping, PCR genotyping, and gene sequencing. I found that the most common agricultural mutation was indeed present in riverbank populations, suggesting that these native populations may serve as a reservoir of resistance alleles. Finally, I constructed a phylogeny for the genus Amaranthus to investigate traits associated with the evolution of weediness. Amaranthus is a worldwide genus of 70 species, with no previous generic phylogeny. I included 58 species and two outgroups, sequenced at four nuclear genes and two chloroplast regions, in my molecular phylogenetic analyses using maximum parsimony, maximum likelihood, and Bayesian inference. Weediness exhibits no phylogenetic signal in Amaranthus; however, using non-phylogenetic statistical tests, I found associations of weediness with several morphological and ecological traits in the genus

The effects of nutrient availability on the host plant resistance of gerbera to western flower thrips

Nutrition of host plants has been shown to have a direct effect on the productivity of numerous insect pests, including western flower thrips [(WFT) Frankliniella occidentalis (Pergande)] – a major pest on both horticulture and agronomic crops. Plants use constitutive and induced chemical defenses to aid in protection against phytophagous insects. Reductions in WFT abundance in response to decreased nutrient availability has been attributed to the reduced availability of nutrients required for WFT productivity. The goals of this research were to determine the effects of fertilization on chemical defenses, and subsequent effects on WFT feeding and abundance. More importantly, the effects of fertilization and WFT feeding on plant growth, development, physiology, and quality were determined to assess the viability of optimizing fertilization in order to increase host plant resistance in gerbera. Constitutive (i.e. phenolics) and induced (i.e. jasmonic acid) chemical defenses were enhanced when fertilization was reduced. Reducing fertilization increased the total phenolics and wound- and WFT-induced jasmonic acid (JA) accumulation in gerbera. The enhanced chemical defenses in lower fertility plants resulted in reduced WFT abundance and feeding damage. These results indicate that the strategy for some plant species under nutrient stress is to increase constitutive defenses, while maintaining, or possibly increasing inducible defenses instead of growth. Similar to 0X fertility plants (only supplied with initial fertilizer charge in commercial media), 0.3X (received 30% of recommended rate) gerberas had reduced biomass and greater chemical defenses compared to 1X plants, but these plants did not appear to be nutritionally stressed—and 0.3X plants without WFT were rated as marketable. Reducing fertilization by 70% (0.3X) did not affect flower dry mass (DM) or the rate of flowering, but the flower stalks (peduncles) were taller in response to the fertilizer reduction. Hence, reducing fertilization to a moderate level in gerbera production may reduce susceptibility to WFT, while producing marketable crops

Incorporating agronomic measures into integrated weed management strategies using pre-emergence herbicide cinmethylin to control Alopecurus myosuroides Huds.

Alopecurus myosuroides Huds. is one of the most problematic grass weeds in cereal production in Western Europe. This grass weed spread rapidly due to the repeated and intensive use of herbicides with the same mode of action and changes in arable cropping and tillage systems. Herbicide applications are the common agricultural practice for successful control of A. myosuroides due to its high flexibility and low cost. However, due to European and national restrictions and the growth of herbicide-resistant populations, farmers are forced to reduce herbicide use to minimize chemical impacts on the environment and food chain. As a holistic approach for reducing herbicide use, integrated weed management (IWM) is a diversification of the control strategy of A. myosuroides. In this thesis, several aspects of IWM were examined and combined to test for a successful A. myosuroides control strategy in winter cereals. Special attention was paid to cinmethylin, a pre-emergence herbicide with a new mode of action in winter cereals to control A. myosuroides. The first article comprised the development of an agar bioassay sensitivity test to determine sensitivity differences in A. myosuroides populations to pre-emergence herbicides containing flufenacet and the re-discovered substance cinmethylin. All of the tested populations did not show reduced sensitivity to cinmethylin, but differences in resistance factors were observed between the agar bioassay sensitivity test and the standard whole plant pot bioassay in the greenhouse. Nevertheless, it was possible for the most part to confirm the results for cinmethylin and flufenacet of the standardized greenhouse whole plant pot bioassay in the agar bioassay sensitivity tests and hence create a reliable, faster test system. The second article focused on cultural measures like cover crop mixtures, various stubble tillage methods and glyphosate treatments and their effect on total weed infestation in particular on A. mysouroides and volunteer wheat. Within two field experiments, the cover crop mixtures and the dual glyphosate application achieved a control efficacy of A. myosuroides of up to 100%, whereas stubble tillage and the single glyphosate treatment did not reduce A. myosuroides population significantly. The results demonstrated, that besides a double glyphosate application, well developed cover crop mixtures have a great ability for weed control, even for A. myosuroides. The third article also dealed with the combination of cultural measures (delayed seeding) and herbicide application and their influence on A. myosuroides control efficacy and yield response of winter wheat and triticale. Results indicate that cultural methods such as delayed seeding can reduce A. myosuroides populations up to 75%, although to achieve control efficacy of > 95%, supplementary herbicides should be used. In the fourth article, a two-year experiment on two experimental sites was set up with a special focus on stubble tillage methods, glyphosate application and the application of the pre-emergence herbicide cinmethylin in two rates. Control efficiencies of 99-100% were achieved by ploughing, double glyphosate application or via false seedbed preparation, each in combination with a cinmethylin application. In the last article, over a period of three years the new pre-emergence herbicide cinmethylin was tested in combination with stubble treatments and delayed drilling of winter annual cereals in winter wheat and winter triticale in Southwestern Germany. Cinmethylin controlled 58-99% of A. myosuroides plants until 120 days after sowing. Additive and synergistic effects of cinmethylin and delayed drilling were found for all studies. In this study, the focus was set on monitoring, cultural and direct weed control methods. Considering especially A. myosuroides, a diverse control strategy needs to be implemented to ensure a sustainable and reduced herbicide use, high control levels, minimized crop damage, safeguarded grain yields and reduced risk of resistance development. However, IWM measures imply increased system complexity, which may make their adoption by farmers difficult. Nevertheless, the results show that cinmethylin can be successfully used for weed control systems in combination with different stubble tillage methods, glyphosate application, delayed seeding, or herbicide sequences and mixtures, making it a valuable tool in integrated weed and resistance management strategies with its novel and unique mode of action.Alopecurus myosuroides Huds. ist eines der problematischsten Grasunkräuter im Getreideanbau in Westeuropa. Der verstärkte Anbau von einjährigen Winterkulturen, der wiederholte Einsatz von Herbiziden mit gleichen Wirkmechanismen, reduzierte Bodenbearbeitung und frühe Aussaattermine von Getreide führen zu steigenden Populationsdichten von A. myosuroides. In der landwirtschaftlichen Praxis wird häufig aufgrund ihrer hohen Flexibilität, Bekämpfungserfolge sowie den geringen Kosten auf Herbiziden zur erfolgreichen Bekämpfung von A. myosuroides zurückgegriffen. Durch die Zunahme an Herbizidresistenten Unkrautpopulationen, sowie durch europäische und nationale Beschränkungen sind die Landwirte gezwungen, den Herbizideinsatz zu reduzieren, um die negativen Auswirkungen auf die Umwelt und die Rückstände in Nahrungskette zu minimieren. Als ganzheitlicher Ansatz zur Reduzierung des Herbizideinsatzes und die damit verbundenen negativ Auswirkungen, stellt das integrierte Unkrautmanagement (IWM) eine Diversifizierung der Bekämpfungsstrategie von A. myosuroides dar. Um eine integrierte Bekämpfungsstrategie von A. myosuroides in Wintergetreide zu entwickeln, wurden im Rahmen dieser Arbeit verschiedene Aspekte von IWM einzeln untersucht bzw. kombiniert. Besondere Aufmerksamkeit wurde hierbei dem neuen Wirkstoff Cinmethylin geschenkt, welches durch seinen neuen Wirkungsmechanismus eine Erweiterung des Vorauflaufherbizidrepertoires in Wintergetreide zur Bekämpfung von A. myosuroides darstellt. Der erste Artikel befasst sich mit der Entwicklung eines Agar Biotests zur Bestimmung von Sensitivitätsunterschieden in A. myosuroides Populationen gegenüber den in Vorauflaufherbiziden enthaltenen Wirkstoffen Flufenacet und Cinmethylin. Alle getesteten Populationen zeigten keine reduzierte Sensitivität gegenüber Cinmethylin, dennoch wurden Unterschiede in den Resistenzfaktoren zwischen dem Agar Biotest und dem Standard Gewächshaus Biotest festgestellt. Die Ergebnisse des Gewächshaus Biotests konnten zum größten Teil für Cinmethylin und Flufenacet durch den Agar Biotest reproduziert werden und damit ein effizienteres Testsystem geschaffen werden. Der zweite Artikel konzentrierte sich auf vorbeugende Unkrautbekämpfungsmaßnahmen wie Zwischenfruchtmischungen, verschiedene Stoppelbearbeitungsmethoden und Glyphosatbehandlungen und deren Auswirkung auf die Unkrautbekämpfung, insbesondere auf A. myosuroides und Ausfallweizen. Die Ergebnisse zeigen, dass neben einer doppelten Glyphosatanwendung auch gut etablierte Zwischenfruchtbestände das Potential für einen hohen Unkrautbekämpfungserfolg von A. myosuroides bieten. Der dritte Artikel befasste sich mit der Kombination vorbeugender Unkrautbekämpfungsmaßnahmen (Früher-, Später-Saattermin) sowie dem Einsatz unterschiedlicher Herbizide. Die Ergebnisse zeigen, dass vorbeugende Bekämpfungsmaßnahmen wie die Spätsaat, die Bestandsdichte von A. myosuroides um bis zu 75% reduzieren konnte. Dennoch sollten zum Erreichen eines Bekämpfungserfolges von > 95% zusätzliche zur Wahl des Saattermines, Herbizide eingesetzt werden. Im vierten Artikel wurde über eine Periode von zwei Jahren auf zwei Standorten vier Feldversuche angelegt, bei dem die Kombination aus Stoppelbearbeitung, einer Glyphosatanwendung sowie die Anwendung des in Vorauflaufherbizid enthaltenen Wirkstoffs Cinmethylin in zwei Aufwandmengen im Vordergrund standen. Durch die Kombinationen von Cinmethylin mit pflügen, doppelter Glyphosatanwendung oder durch die Kombination mit einer falschen Saatbettbereitung konnten Bekämpfungserfolge von A. myosuroides von 99-100% erzielt werden. Im letzten Artikel wurde über einen Zeitraum von drei Jahren das neue Vorauflaufherbizid Cinmethylin in Kombination mit verschiedenen Stoppelbearbeitungsstrategien und einer verzögerten Aussaat von Wintergetreide in vier Feldversuchen mit Winterweizen und Wintertriticale im Südwesten Deutschlands getestet. Cinmethylin kontrollierte 120 Tage nach der Aussaat, 58-99 % der A. myosuroides-Pflanzen. Additive und synergistische Effekte von Cinmethylin und verzögerter Aussaat wurden in allen Studien festgestellt. In dieser Studie lag der Fokus auf dem Monitoring, vorbeugenden sowie chemischen Unkrautbekämpfungsmethoden. Konkret bedeutet dies, dass durch vorbeugende Bekämpfungsmaßnahmen vor der Aussaat der Hauptkultur ungünstige Wachstumsbedingungen für Unkräuter geschaffen wurden. IWM-Maßnahmen implizieren jedoch eine erhöhte Systemkomplexität, was ihre Akzeptanz durch die Landwirte erschweren kann. Die Ergebnisse dieser Studie zeigen, dass Cinmethylin in Kombination mit verschiedenen Stoppelbearbeitungsmethoden, Glyphosatanwendungen, Spätsaat oder Herbizid Sequenzen/-mischungen zu nachhaltigen Bekämpfungserfolg führt. Cinmethlyin stellt mit seiner neuartigen und einzigartigen Wirkungsweise eine wertvolle Ergänzung für ein integriertes Unkraut- und Resistenzmanagement dar

Toward a Sustainable Agriculture Through Plant Biostimulants

Over the past decade, interest in plant biostimulants has been on the rise, compelled by the growing interest of researchers, extension specialists, private industries, and farmers in integrating these products in the array of environmentally friendly tools to secure improved crop performance, nutrient efficiency, product quality, and yield stability. Plant biostimulants include diverse organic and inorganic substances, natural compounds, and/or beneficial microorganisms such as humic acids, protein hydrolysates, seaweed and plant extracts, silicon, endophytic fungi like mycorrhizal fungi, and plant growth-promoting rhizobacteria belonging to the genera Azospirillum, Azotobacter, and Rhizobium. Other substances (e.g., chitosan and other biopolymers and inorganic compounds) can have biostimulant properties, but their classification within the group of biostimulants is still under consideration. Plant biostimulants are usually applied to high-value crops, mainly greenhouse crops, fruit trees and vines, open-field crops, flowers, and ornamentals to sustainably increase yield and product quality. The global biostimulant market is currently estimated at about $2.0 billion and is expected to reach$3.0 billion by 2021 at an annual growth rate of 13%. A growing interest in plant biostimulants from industries and scientists was demonstrated by the high number of published peer-reviewed articles, conferences, workshops, and symposia in the past ten years. This book compiles several original research articles, technology reports, methods, opinions, perspectives, and invited reviews and mini reviews dissecting the biostimulatory action of these natural compounds and substances and beneficial microorganisms on crops grown under optimal and suboptimal growing conditions (e.g., salinity, drought, nutrient deficiency and toxicity, heavy metal contaminations, waterlogging, and adverse soil pH conditions). Also included are contributions dealing with the effect as well as the molecular and physiological mechanisms of plant biostimulants on nutrient efficiency, product quality, and modulation of the microbial population both quantitatively and qualitatively. In addition, identification and understanding of the optimal method, time, rate of application and phenological stage for improving plant performance and resilience to stress as well as the best combinations of plant species/cultivar × environment × management practices are also reported. We strongly believe that high standard reflected in this compilation on the principles and practices of plant biostimulants will foster knowledge transfer among scientific communities, industries, and agronomists, and will enable a better understanding of the mode of action and application procedures of biostimulants in different cropping systems