Molecular Characterization of Indigenous Rhizobia from Kenyan Soils Nodulating with Common Beans.

Kenya is the seventh most prominent producer of common beans globally and the second leading producer in East Africa. However, the annual national productivity is low due to insufficient quantities of vital nutrients and nitrogen in the soils. Rhizobia are symbiotic bacteria that fix nitrogen through their interaction with leguminous plants. Nevertheless, inoculating beans with commercial rhizobia inoculants results in sparse nodulation and low nitrogen supply to the host plants because these strains are poorly adapted to the local soils. Several studies describe native rhizobia with much better symbiotic capabilities than commercial strains, but only a few have conducted field studies. This study aimed to test the competence of new rhizobia strains that we isolated from Western Kenya soils and for which the symbiotic efficiency was successfully determined in greenhouse experiments. Furthermore, we present and analyze the whole-genome sequence for a promising candidate for agricultural application, which has high nitrogen fixation features and promotes common bean yields in field studies. Plants inoculated with the rhizobial isolate S3 or with a consortium of local isolates (COMB), including S3, produced a significantly higher number of seeds and seed dry weight when compared to uninoculated control plants at two study sites. The performance of plants inoculated with commercial isolate CIAT899 was not significantly different from uninoculated plants (p > 0.05), indicating tight competition from native rhizobia for nodule occupancy. Pangenome analysis and the overall genome-related indices showed that S3 is a member of R. phaseoli. However, synteny analysis revealed significant differences in the gene order, orientation, and copy numbers between S3 and the reference R. phaseoli. Isolate S3 is phylogenomically similar to R. phaseoli. However, it has undergone significant genome rearrangements (global mutagenesis) to adapt to harsh conditions in Kenyan soils. Its high nitrogen fixation ability shows optimal adaptation to Kenyan soils, and the strain can potentially replace nitrogenous fertilizer application. We recommend that extensive fieldwork in other parts of the country over a period of five years be performed on S3 to check on how the yield changes with varying whether conditions.

Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense.

Although phloem-feeding insects such as aphids can cause significant damage to plants, relatively little is known about early plant defenses against these insects. As a first line of defense, legumes can stop the phloem mass flow through a conformational change in phloem proteins known as forisomes in response to Ca2+ influx. However, specialized phloem-feeding insects might be able to suppress the conformational change of forisomes and thereby prevent sieve element occlusion. To investigate this possibility, we triggered forisome dispersion through application of a local heat stimulus to the leaf tips of pea (Pisum sativum), clover (Trifolium pratense) and broad bean (Vicia faba) plants infested with different pea aphid (Acyrthosiphon pisum) host races and monitored forisome responses. Pea aphids were able to suppress forisome dispersion, but this depended on the infesting aphid host race, the plant species, and the age of the plant. Differences in the ability of aphids to suppress forisome dispersion may be explained by differences in the composition and quantity of the aphid saliva injected into the plant. Various mechanisms of how pea aphids might suppress forisome dispersion are discussed.

Rhizobia Contribute to Salinity Tolerance in Common Beans (Phaseolus vulgaris L.).

Rhizobia are soil bacteria that induce nodule formation on leguminous plants. In the nodules, they reduce dinitrogen to ammonium that can be utilized by plants. Besides nitrogen fixation, rhizobia have other symbiotic functions in plants including phosphorus and iron mobilization and protection of the plants against various abiotic stresses including salinity. Worldwide, about 20% of cultivable and 33% of irrigation land is saline, and it is estimated that around 50% of the arable land will be saline by 2050. Salinity inhibits plant growth and development, results in senescence, and ultimately plant death. The purpose of this study was to investigate how rhizobia, isolated from Kenyan soils, relieve common beans from salinity stress. The yield loss of common bean plants, which were either not inoculated or inoculated with the commercial R. tropici rhizobia CIAT899 was reduced by 73% when the plants were exposed to 300 mM NaCl, while only 60% yield loss was observed after inoculation with a novel indigenous isolate from Kenyan soil, named S3. Expression profiles showed that genes involved in the transport of mineral ions (such as K+, Ca2+, Fe3+, PO43-, and NO3-) to the host plant, and for the synthesis and transport of osmotolerance molecules (soluble carbohydrates, amino acids, and nucleotides) are highly expressed in S3 bacteroids during salt stress than in the controls. Furthermore, genes for the synthesis and transport of glutathione and γ-aminobutyric acid were upregulated in salt-stressed and S3-inocculated common bean plants. We conclude that microbial osmolytes, mineral ions, and antioxidant molecules from rhizobia enhance salt tolerance in common beans.

CORK1, A LRR-Malectin Receptor Kinase, Is Required for Cellooligomer-Induced Responses in Arabidopsis thaliana.

Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical and chemical distortions, pH changes, and breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers that induce Ca2+-dependent CWI responses. We show that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis and are preferentially activated by cellotriose (CT). CORK1 is required for cellooligomer-induced cytoplasmic Ca2+ elevation, reactive oxygen species (ROS) production, mitogen-associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes, including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that CORK1 is required for CWI and immune responses activated by cellulose breakdown products.

Guardians of the phloem - forisomes and beyond.

The phloem is a highly specialized vascular tissue that forms a fundamentally important transport and signaling pathway in plants. It is therefore a system worth protecting. The main function of the phloem is to transport the products of photosynthesis throughout the whole plant, but it also transports soluble signaling molecules and propagates electrophysiological signals. The phloem is constantly threatened by mechanical injuries, phloem-sucking pests and parasites, and the spread of pathogens, which has led to the evolution of efficient defense mechanisms. One such mechanism involves structural phloem proteins, which are thought to facilitate sieve element occlusion following injury and to defend the plant against pathogens. In leguminous plants, specialized structural phloem proteins known as forisomes form unique mechanoproteins via sophisticated molecular interaction and assembly mechanisms, thus enabling reversible sieve element occlusion. By understanding the structure and function of forisomes and other structural phloem proteins, we can develop a toolbox for biotechnological applications in material science and medicine. Furthermore, understanding the involvement of structural phloem proteins in plant defense mechanisms will allow phloem engineering as a new strategy for the development of crop varieties that are resistant to pests, pathogens and parasites.

Distribution, Characterization and the Commercialization of Elite Rhizobia Strains in Africa.

Grain legumes play a significant role in smallholder farming systems in Africa because of their contribution to nutrition and income security and their role in fixing nitrogen. Biological Nitrogen Fixation (BNF) serves a critical role in improving soil fertility for legumes. Although much research has been conducted on rhizobia in nitrogen fixation and their contribution to soil fertility, much less is known about the distribution and diversity of the bacteria strains in different areas of the world and which of the strains achieve optimal benefits for the host plants under specific soil and environmental conditions. This paper reviews the distribution, characterization, and commercialization of elite rhizobia strains in Africa.

So similar yet so different: The distinct contributions of extrafascicular and fascicular phloem to transport and exudation in cucumber plants.

Cucurbits have been used as phloem research models for many decades because their exudates can be accessed with ease. However, cucurbit plants possess two distinct phloem systems known as the fascicular phloem (FP) and extrafascicular phloem (EFP). Therefore, the molecular composition and function of certain exudates can be misinterpreted due to their unclear origin. To characterize the anatomy and function of the different phloem systems more clearly, we generated specific antibodies against marker proteins (PP1 homologs) allowing the clear identification of the EFP at the organ, tissue and cellular levels by immunological staining. We also used detailed microscopy to determine common and unique anatomical features of the FP and EFP sieve elements (SEs) in cucumber (Cucumis sativus). The comparison of exudation rates and the dynamic viscosity, density and sugar content of the exudates from plants grown in the light and dark revealed the consistent composition and behavior of the EFP exudate even when photosynthesis was prevented, thus differing from the properties of the FP exudate. Furthermore, the analysis of phloem transport using a fluorescein disodium salt showed only wound-induced exudation of dye from the EFP, indicating the absence of transport in this tissue. Our results show that it is important to distinguish between the EFP and FP in cucurbits, particularly their differing behaviors in response to wounding.

The Cell Membrane of a Novel Rhizobium phaseoli Strain Is the Crucial Target for Aluminium Toxicity and Tolerance.

Soils with low pH and high aluminium (Al) contamination restrict common bean production, mainly due to adverse effects on rhizobia. We isolated a novel rhizobium strain, B3, from Kenyan soil which is more tolerant to Al stress than the widely used commercial strain CIAT899. B3 was resistant to 50 µM Al and recovered from 100 µM Al stress, while CIAT899 did not. Calcein labeling showed that less Al binds to the B3 membranes and less ATP and mScarlet-1 protein, a cytoplasmic marker, leaked out of B3 than CIAT899 cells in Al-containing media. Expression profiles showed that the primary targets of Al are genes involved in membrane biogenesis, metal ions binding and transport, carbohydrate, and amino acid metabolism and transport. The identified differentially expressed genes suggested that the intracellular γ-aminobutyric acid (GABA), glutathione (GSH), and amino acid levels, as well as the amount of the extracellular exopolysaccharide (EPS), might change during Al stress. Altered EPS levels could also influence biofilm formation. Therefore, these parameters were investigated in more detail. The GABA levels, extracellular EPS production, and biofilm formation increased, while GSH and amino acid level decreased. In conclusion, our comparative analysis identified genes that respond to Al stress in R. phaseoli. It appears that a large portion of the identified genes code for proteins stabilizing the plasma membrane. These genes might be helpful for future studies investigating the molecular basis of Al tolerance and the characterization of candidate rhizobial isolates that perform better in Al-contaminated soils than commercial strains.

Studying the Function of Phytoplasma Effector Proteins Using a Chemical-Inducible Expression System in Transgenic Plants.

Phytoplasmas are bacterial pathogens that live mainly in the phloem of their plant hosts. They dramatically manipulate plant development by secreting effector proteins that target developmental proteins of their hosts. Traditionally, the effects of individual effector proteins have been studied by ectopic overexpression using strong, ubiquitously active promoters in transgenic model plants. However, the impact of phytoplasma infection on the host plants depends on the intensity and timing of infection with respect to the developmental stage of the host. To facilitate investigations addressing the timing of effector protein activity, we have established chemical-inducible expression systems for the three most well-characterized phytoplasma effector proteins, SECRETED ASTER YELLOWS WITCHES' BROOM PROTEIN 11 (SAP11), SAP54 and TENGU in transgenic Arabidopsis thaliana. We induced gene expression either continuously, or at germination stage, seedling stage, or flowering stage. mRNA expression was determined by quantitative reverse transcription PCR, protein accumulation by confocal laser scanning microscopy of GFP fusion proteins. Our data reveal tight regulation of effector gene expression and strong upregulation after induction. Phenotypic analyses showed differences in disease phenotypes depending on the timing of induction. Comparative phenotype analysis revealed so far unreported similarities in disease phenotypes, with all three effector proteins interfering with flower development and shoot branching, indicating a surprising functional redundancy of SAP54, SAP11 and TENGU. However, subtle but mechanistically important differences were also observed, especially affecting the branching pattern of the plants.

New phytoplasma effector: 50 shades of green.

Phytoplasmas are pathogenic bacteria that manipulate their plant hosts in dramatic ways. In issue 20 Vol. 184 of Cell, Huang and colleagues report on a phytoplasma effector protein that brings about manifold effects by a surprising mechanism. Are resistant plants on the horizon now?

Isolation and Characterization of High-Efficiency Rhizobia From Western Kenya Nodulating With Common Bean.

Common bean is one of the primary protein sources in third-world countries. They form nodules with nitrogen-fixing rhizobia, which have to be adapted to the local soils. Commercial rhizobial strains such as Rhizobium tropici CIAT899 are often used in agriculture. However, this strain failed to significantly increase the common bean yield in many places, including Kenya, due to the local soils' low pH. We isolated two indigenous rhizobial strains from the nodules of common bean from two fields in Western Kenya that have never been exposed to commercial inocula. We then determined their ability to fix nitrogen in common beans, solubilize phosphorus, and produce indole acetic acid. In greenhouse experiments, common bean plants inoculated with two isolates, B3 and S2 in sterile vermiculite, performed better than those inoculated with CIAT899 or plants grown with nitrogen fertilizer alone. In contrast to CIAT899, both isolates grew in the media with pH 4.8. Furthermore, isolate B3 had higher phosphate solubilization ability and produced more indole acetic acid than the other two rhizobia. Genome analyses revealed that B3 and S2 are different strains of Rhizobium phaseoli. We recommend fieldwork studies in Kenyan soils to test the efficacy of the two isolates in the natural environment in an effort to produce inoculants specific for these soils.

Specialized 16SrX phytoplasmas induce diverse morphological and physiological changes in their respective fruit crops.

The host-pathogen combinations-Malus domestica (apple)/`Candidatus Phytoplasma mali´, Prunus persica (peach)/`Ca. P. prunorum´ and Pyrus communis (pear)/`Ca. P. pyri´ show different courses of diseases although the phytoplasma strains belong to the same 16SrX group. While infected apple trees can survive for decades, peach and pear trees die within weeks to few years. To this date, neither morphological nor physiological differences caused by phytoplasmas have been studied in these host plants. In this study, phytoplasma-induced morphological changes of the vascular system as well as physiological changes of the phloem sap and leaf phytohormones were analysed and compared with non-infected plants. Unlike peach and pear, infected apple trees showed substantial reductions in leaf and vascular area, affecting phloem mass flow. In contrast, in infected pear mass flow and physicochemical characteristics of phloem sap increased. Additionally, an increased callose deposition was detected in pear and peach leaves but not in apple trees in response to phytoplasma infection. The phytohormone levels in pear were not affected by an infection, while in apple and peach trees concentrations of defence- and stress-related phytohormones were increased. Compared with peach and pear trees, data from apple suggest that the long-lasting morphological adaptations in the vascular system, which likely cause reduced sap flow, triggers the ability of apple trees to survive phytoplasma infection. Some phytohormone-mediated defences might support the tolerance.

Species-Specific and Distance-Dependent Dispersive Behaviour of Forisomes in Different Legume Species.

Forisomes are giant fusiform protein complexes composed of sieve element occlusion (SEO) protein monomers, exclusively found in sieve elements (SEs) of legumes. Forisomes block the phloem mass flow by a Ca2+-induced conformational change (swelling and rounding). We studied the forisome reactivity in four different legume species-Medicago sativa, Pisum sativum, Trifolium pratense and Vicia faba. Depending on the species, we found direct relationships between SE diameter, forisome surface area and distance from the leaf tip, all indicative of a developmentally tuned regulation of SE diameter and forisome size. Heat-induced forisome dispersion occurred later with increasing distance from the stimulus site. T. pratense and V. faba dispersion occurred faster for forisomes with a smaller surface area. Near the stimulus site, electro potential waves (EPWs)-overlapping action (APs), and variation potentials (VPs)-were linked with high full-dispersion rates of forisomes. Distance-associated reduction of forisome reactivity was assigned to the disintegration of EPWs into APs, VPs and system potentials (SPs). Overall, APs and SPs alone were unable to induce forisome dispersion and only VPs above a critical threshold were capable of inducing forisome reactions.

The Physiological and Biochemical Effects on Napier Grass Plants Following Napier Grass Stunt Phytoplasma Infection.

Napier grass stunt (NGS) phytoplasma, a phloem-limited bacterium, infects Napier grass leading to severe yield losses in East Africa. The infected plants are strongly inhibited in growth and biomass production. In this study, phytoplasma-induced morphological changes of the vascular system and physiological changes were analyzed and compared with uninfected plants. The study showed that the phytoplasmas are more abundant in source leaves and range from 103 bacteria/µg total DNA in infected roots to 106 in mature Napier grass leaves. Using microscopical, biochemical, and physiological tools, we demonstrated that the ultrastructure of the phloem and sieve elements is severely altered in the infected plants, which results in the reduction of both the mass flow and the translocation of photoassimilates in the infected leaves. The reduced transport rate inhibits the photochemistry of photosystem II in the infected plants, which is accompanied by loss of chloroplastic pigments in response to the phytoplasma infection stress eventually resulting in yellowing of diseased plants. The phytoplasma infection stress also causes imbalances in the levels of defense-related antioxidants, glutathione, ascorbic acid, reactive oxygen species (ROS), and-in particular-hydrogen peroxide. This study shows that the infection of NGS phytoplasma in the phloem of Napier grass has an impact on the primary metabolism and activates a ROS-dependent defense response.

An Endophytic Trichoderma Strain Promotes Growth of Its Hosts and Defends Against Pathogen Attack.

Plants host numerous endophytic microbes which promote plant performance, in particular under stress. A new endophytic fungus was isolated from the leaves of a deciduous wood tree Leucas aspera. Morphological inspection and multilocus phylogeny identified the fungus as a new Trichoderma strain. If applied to Arabidopsis thaliana and Nicotiana attenuata, it mainly colonizes their roots and strongly promotes initial growth of the plants on soil. The fungus grows on high NaCl or mannitol concentrations, and shows predatory capability on the pathogenic fungus Alternaria brassicicola. Colonized Arabidopsis plants tolerate higher salt stress and show lower A. brassicicola spread in roots and shoots, while arbuscular mycorrhiza formation in N. attenuata is not affected by the Trichoderma strain. These beneficial features of the novel Trichoderma strain are important prerequisites for agricultural applications.

Measurement of Electropotential Waves in Intact Phloem Sieve Elements Using Microelectrodes.

Electropotential waves (EPW) are involved in plant responses to both abiotic and biotic stresses. Three different types of EPWs have been identified: action potential, variation potential, and system potential, all of which have been indicated to participate in phloem-based communication between plant organs. In this chapter we describe in detail how to measure EPWs in plants, including how to access the phloem, and how to insert microelectrodes. Such experiments can be used, for example, to study the local and systemic signaling in response to diverse stimuli like microbial threat or herbivore attack.

Measurement of Electropotential Waves in Intact Sieve Elements Using Aphids as Bioelectrodes.

Electropotential waves (EPWs) are thought to transmit sudden and profound physiological changes between plant organs. The recording of EPWs can be performed via extracellular or intracellular probes. Both approaches have advantages and disadvantages. Since the phloem is responsible for long distance transport of the most forms of EPWs, the direct measurement in sieve elements is preferable. The conductance using glass microelectrodes inserted into free lying sieve elements is described in Chapter 34 . In this chapter the measurement of EPWs by using aphids as bioelectrodes is described in detail.The electrical penetration graph technique (EPG) takes advantage of the flexible mouthparts (stylet) of aphids, which specifically penetrate into sieve elements. The use of aphids as bioelectrodes enables multiple electrode recordings and long-distance observations of EPWs. Importantly, this method allows for noninvasive, intracellular measurements.

Beneficial and Pathogenic Arabidopsis Root-Interacting Fungi Differently Affect Auxin Levels and Responsive Genes During Early Infection.

Auxin (indole-3-acetic acid, IAA) is an important phytohormone involved in root growth and development. Root-interacting beneficial and pathogenic fungi utilize auxin and its target genes to manipulate the performance of their hosts for their own needs. In order to follow and visualize auxin effects in fungi-colonized Arabidopsis roots, we used the dual auxin reporter construct DR5::EGFP-DR5v2::tdTomato and fluorescence microscopy as well as LC-MS-based phytohormone analyses. We demonstrate that the beneficial endophytic fungi Piriformospora indica and Mortierella hyalina produce and accumulate IAA in their mycelia, in contrast to the phytopathogenic biotrophic fungus Verticillium dahliae and the necrotrophic fungus Alternaria brassicicola. Within 3 h after exposure of Arabidopsis roots to the pathogens, the signals of the auxin-responsive reporter genes disappeared. When exposed to P. indica, significantly higher auxin levels and stimulated expression of auxin-responsive reporter genes were detected both in lateral root primordia and the root elongation zone within 1 day. Elevated auxin levels were also present in the M. hyalina/Arabidopsis root interaction, but no downstream effects on auxin-responsive reporter genes were observed. However, the jasmonate level was strongly increased in the colonized roots. We propose that the lack of stimulated root growth upon infection with M. hyalina is not caused by the absence of auxin, but an inhibitory effect mediated by high jasmonate content.

The Beneficial Root-Colonizing Fungus Mortierella hyalina Promotes the Aerial Growth of Arabidopsis and Activates Calcium-Dependent Responses That Restrict Alternaria brassicae-Induced Disease Development in Roots.

The endophytic fungus Mortierella hyalina colonizes the roots of Arabidopsis thaliana and stimulates growth and biomass production of the aerial parts but not of roots. An exudate fraction from the fungus induces rapid and transient cytoplasmic Ca2+elevation in the roots. The Ca2+ response does not require the well-characterized (co)receptors BAK1, CERK1, and FLS2 for pathogen-associated molecular patterns, and the Ca2+ channels GLR-2.4, GLR-2.5, and GLR-3.3 or the vacuolar TWO PORE CHANNEL1, which might be involved in cytoplasmic Ca2+ elevation. We isolated an ethyl-methane-sulfonate-induced Arabidopsis mutant that is impaired in this Ca2+ response. The roots of the mutant are impaired in M. hyalina-mediated suppression of immune responses after Alternaria brassicae infection, i.e., jasmonate accumulation, generation of reactive oxygen species, as well as the activation of jasmonate-related defense genes. Furthermore, they are more colonized by M. hyalina than wild-type roots. We propose that the mutant gene product is involved in a Ca2+-dependent signaling pathway activated by M. hyalina to suppress immune responses in Arabidopsis roots.

Collection of Phloem Sap in Phytoplasma-Infected Plants.

Phytoplasmas colonize specifically the phloem sieve elements (SEs) of plants and influence effectively the plant physiology. To study and understand the interaction of phytoplasmas and host plants an access to the cellular, microscale volume of SEs is demanded. Different methods are suitable to collect phloem sap of phytoplasma-infected plants. The two most common methods are the EDTA-facilitated exudation and the stylectomy. For the EDTA-facilitated method, the cut end of a leaf is placed into an EDTA solution. The EDTA prevents and avoids the Ca2+ dependent (re-) occlusion of SEs by binding Ca2+ ions and the mass flow of SEs is restarted which results in an outflow of the SE content into the EDTA bathing solution. The advantage is on the one hand a simple application and secondly, feasible for all plant species.The stylectomy method requires piercing-sucking insects like any aphids. During phloem-sap ingestion, the stylet is severed by a microcautery device or a laser from the insect body. Due to the high turgor pressure of the SEs the phloem sap is forced out through the remaining stylet and can be collected with a glass capillary, for example. The stylectomy delivers pure phloem sap, however, the collected volumes are in the range of nano liters and the temporal and staff costs are tremendous. A third method is the spontaneous exudation in phytoplasma-infected apple trees providing only in springtime large volumes of vascular sap after cutting along the bark. For the spontaneous exudation the proportion of phloem sap is unclear. Thus, this third method still needs a closer examination in prospective surveys.