Competency of Rhizobial Inoculation in Sustainable Agricultural Production and Biocontrol of Plant Diseases

The rate of growth of the global population poses a risk to food security, demanding an increase in food production. Much of the world's cultivable soils also do not have ideal farming conditions such as soil health and fertility problem and increased pest attacks, which are challenges of food production. In this perspective, there is a need to increase agricultural production using a more economically and environmentally sustainable approach. As practices of agricultural production and improvement, rhizobial inoculants represent a practically effective, ecologically safe, and economically alternative means of realizing maximum agricultural production. This review addressed how rhizobial inoculation advances agricultural production through improving plant growth, nutrient availability and uptake, and yields by enhancing bio-fixation of atmospheric nitrogen and solubilization of soil nutrients. Besides, rhizobial inoculants offer biocontrol of plant diseases by providing resistance against disease-causing pathogens or suppression of diseases. Mechanisms involved in biocontrol of plant diseases include competition for infection sites and nutrients, activation of induced systemic resistance, and production of substances such as growth hormones, antibiotics, enzymes, siderophores, hydrogen cyanide, and exo-polysaccharides. Consequently, this approach is promising as sustainable agricultural practices have yet to supplement or replace chemical fertilizers, serving as a basis for future research on sustainable agricultural production. Despite the multifunctional benefits of rhizobial inoculation, there is a variation in the implementation of this practice by farmers. Therefore, researchers should work on eradicating farmers' constraints in using rhizobia, and future studies should be concentrated toward the methods of improving inoculant quality and promotion of the technology.

Systemic Optimization of Legume Nodulation: A Shoot-Derived Regulator, miR2111

Long-distance signaling between the shoot and roots of land plants plays a crucial role in ensuring their growth and development in a fluctuating environment, such as with soil nutrient deficiencies. MicroRNAs (miRNAs) are considered to contribute to such environmental adaptation via long-distance signaling since several miRNAs are transported between the shoot and roots in response to various soil nutrient changes. Leguminous plants adopt a shoot-mediated long-distance signaling system to maintain their mutualism with symbiotic nitrogen-fixing rhizobia by optimizing the number of symbiotic organs and root nodules. Recently, the involvement and importance of shoot-derived miR2111 in regulating nodule numbers have become evident. Shoot-derived miR2111 can systemically enhance rhizobial infection, and its accumulation is quickly suppressed in response to rhizobial inoculation and high-concentration nitrate application. In this mini-review, we briefly summarize the recent progress on the systemic optimization of nodulation in response to external environments, with a focus on systemic regulation via miR2111.

Improving the Fertigation of Soilless Urban Vertical Agriculture Through the Combination of Struvite and Rhizobia Inoculation in Phaseolus vulgaris

Soilless crop production is a viable way to promote vertical agriculture in urban areas, but it relies extensively on the use of mineral fertilizer. Thus, the benefits of fresher, local food and avoiding the transportation and packaging associated with food import could be counteracted by an increase in nutrient-rich wastewater, which could contribute to freshwater and marine eutrophication. The present study aimed to explore the use of mineral fertilizer substitutes in soilless agriculture. Phaseolus vulgaris (common bean) was fertilized with a combination of slow-releasing fertilizer struvite (a source of N, P, and Mg), which is a byproduct of wastewater treatment plants, and inoculation with Rhizobium (a N2-fixing soil bacteria). The experiment included three bean-production lines: (A) 2 g/plant of struvite and rhizobial inoculation; (B) 5 g/plant of struvite and rhizobial inoculation, both irrigated with a Mg-, P-, and N-free nutrient solution; and (C) a control treatment that consisted of irrigation with a full nutrient solution and no inoculation. Plant growth, development, yields, and nutrient contents were determined at 35, 62, and 84 days after transplanting as well as biological N2 fixation, which was determined using the 15N natural abundance method. Treatments A and B resulted in lower total yields per plant than the control C treatment (e.g., 59.35 ± 26.4 g plant–1 for A, 74.2 ± 23.0 g plant–1 for B, and 147.71 ± 45.3 g plant–1 for C). For A and B, the nodulation and N2 fixation capacities appeared to increase with the amount of initially available struvite, but, over time, deficient levels of Mg were reached as well as nearly deficient levels of P, which could explain the lower yields. Nevertheless, we conclude that the combination of struvite and N2-fixing bacteria covered the N needs of plants throughout the growth cycle. However, further studies are needed to determine the optimal struvite quantities for vertical agriculture systems that can meet the P and Mg requirements throughout the lifetime of the plants.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece

Phaseolus vulgaris (L.), commonly known as bean or common bean, is considered a promiscuous legume host since it forms nodules with diverse rhizobial species and symbiovars. Most of the common bean nodulating rhizobia are mainly affiliated to the genus Rhizobium, though strains belonging to Ensifer, Pararhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia have also been reported. This is the first report on the characterization of bean-nodulating rhizobia at the species and symbiovar level in Greece. The goals of this research were to isolate and characterize rhizobia nodulating local common bean genotypes grown in five different edaphoclimatic regions of Greece with no rhizobial inoculation history. The genetic diversity of the rhizobial isolates was assessed by BOX-PCR and the phylogenetic affiliation was assessed by multilocus sequence analysis (MLSA) of housekeeping and symbiosis-related genes. A total of fifty fast-growing rhizobial strains were isolated and representative isolates with distinct BOX-PCR fingerpriniting patterns were subjected to phylogenetic analysis. The strains were closely related to R. anhuiense, R. azibense, R. hidalgonense, R. sophoriradicis, and to a putative new genospecies which is provisionally named as Rhizobium sp. I. Most strains belonged to symbiovar phaseoli carrying the α-, γ-a and γ-b alleles of nodC gene, while some of them belonged to symbiovar gallicum. To the best of our knowledge, it is the first time that strains assigned to R. sophoriradicis and harbored the γ-b allele were found in European soils. All strains were able to re-nodulate their original host, indicating that they are true microsymbionts of common bean.

Applying Agronomic Principles of Rhizobial Inoculation to the Conservation of a Keystone Legume Species in a High Mountain Ecosystem on an Oceanic Island

The Teide broom, Spartocytisus supranubius, is an endemism of the Canary Islands (Spain) and the dominant legume of the Tenerife high-mountain ecosystem in Teide National Park (N.P.). Biotic and abiotic stresses are causing a progressive deterioration and decline of the population of this keystone legume. Since its symbiosis with rhizobia is the main nitrogen (N) input into these soils, diminishing the biological nitrogen fixation could compromise the maintenance of this alpine ecosystem. Symbiotically efficient nitrogen-fixing rhizobia have been widely and successfully used as inoculants for agronomic purposes. However, only rarely has rhizobial inoculation been used for legume species conservation in natural ecosystems. In this study, we assessed three Bradyrhizobium sp. strains as inoculants for S. supranubius on seedlings grown in a greenhouse experiment and on juvenile individuals (2-years-old) transplanted on a field trial in the N.P. Plant growth as well as symbiotic and plant physiological parameters were measured to evaluate the effect of rhizobia inoculation. Our results show that broom plants responded positively to the inoculation both in the greenhouse and field trials. The SSUT18 inoculated plants had significantly higher number and weight of nodules, greater sizes (biovolume) and biomass and also showed the highest N which, being not significant in our experimental conditions, it still contributed to more N per planted hectare than control plants, which could be important for the ecosystem maintenance in these N-poor soils. Positive effects of inoculation were also detected on the plant survival rate and water content. The bradyrhizobial inoculation, by accelerating the plant growth can shorten the greenhouse period and by producing more robust juvenile plants, they could help them to cope better with stresses in its natural habitat. Therefore, inoculation with selected rhizobia is a successful strategy to be integrated into conservation campaigns for this threatened legume species.

Multigene editing reveals that MtCEP1/2/12 redundantly control lateral root and nodule number in Medicago truncatula

The multimember CEP (C-terminally Encoded Peptide) gene family is a complex group that is involved in various physiological activities in plants. Previous studies demonstrated that MtCEP1 and MtCEP7 control lateral root formation or nodulation, but these studies were based only on gain of function or artificial miRNA (amiRNA)/RNAi approaches, never knockout mutants. Moreover, an efficient multigene editing toolkit is not currently available for Medicago truncatula. Our quantitative reverse transcription–PCR data showed that MtCEP1, 2, 4, 5, 6, 7, 8, 9, 12, and 13 were up-regulated under nitrogen starvation conditions and that MtCEP1, 2, 7, 9, and 12 were induced by rhizobial inoculation. Treatment with synthetic MtCEP peptides of MtCEP1, 2, 4, 5, 6, 8, and 12 repressed lateral root emergence and promoted nodulation in the R108 wild type but not in the cra2 mutant. We optimized CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9] genome editing system for M. truncatula, and thus created single mutants of MtCEP1, 2, 4, 6, and 12 and the double mutants Mtcep1/2C and Mtcep5/8C; however, these mutants did not exhibit significant differences from R108. Furthermore, a triple mutant Mtcep1/2/12C and a quintuple mutant Mtcep1/2/5/8/12C were generated and exhibited more lateral roots and fewer nodules than R108. Overall, MtCEP1, 2, and 12 were confirmed to be redundantly important in the control of lateral root number and nodulation. Moreover, the CRISPR/Cas9-based multigene editing protocol provides an additional tool for research on the model legume M. truncatula, which is highly efficient at multigene mutant generation.

EFFECT OF THE USE OF VERMICOMPOST AND RHIZOBIAL INOCULATION ON SOME SOIL CHARACTERISTICS, GROWTH AND YIELD OF MUNG BEAN Vigni radiate L.

A field experiment was conducted in silty loam soil to study the effect of vermicompost fertilizer and inoculums Rhizobium legumenosarum on growth and yield of mung bean (Vigni radiate L.), and some soil properties after planting. The experiment consists of nine treatments as follows :T1: control , T2: full recommended mineral fertilizer, T3: vermicompost 8 t.ha-1, T4: vermicompost 16 t.ha-1, T5: vermicompost 8 t.ha-1+ rhizobia, T6: vermicompost16 t.ha-1+ rhizobia, T7: vermicompost 2 t.ha-1+ ½ recommended mineral fertilizer, T8: rhizobia + ½ recommended mineral fertilizer, T9: vermicompost 8 t.ha-1 + rhizobia +½ recommended mineral fertilizer. The experiment was conducted according to RCBD design with three replications. The results were indicated that the use of vermicompost 16 t. ha-1 with the rhizobia inoculation (T6) has improved some soil properties, as this treatment reduced the pH and EC of the soil to 7.18 and 2.20 dsm-1. While CEC and O.M increased to 47.9 Cmole Kg-1 and 2.96%, respectively. Whereas, the treatment T9 was superior in most of the traits, including plant height (70.1 cm), Root weight (5.8 g plant-1) and a number of active and total root nodes (41.3 and 36.6 nodes plant -1). The treatment T9 also gave the best characteristics of the yield components, including the number of pods plant-1, weight of pods plant-1, weight 1000 seeds (g), total yield and was 51.0 pods plant-1, 92.4 g plant -1,49.8 g.plant-1, 1216.95 kg.ha-1 respectively.