Inoculation of legume seed is an efficient and convenient way of introducing effective rhizobia to soil and subsequently the rhizosphere of legumes. However, its full potential is yet to be realised. Following widespread crop failures, the manufacture of high quality inoculants revolutionised legume technology in Australia in the 1960s. Many improvements to inoculants and the advent of an inoculant control service ensured that quality was optimised and maintained. Minimum standards for the number of rhizobia per seed were set after consideration of several factors including seed size and loss of viability during inoculation. Despite manufacturers' recommendations for storage and application of inoculants, there is a distinct lack of control over the inoculation process; hence the full potential of high quality products may not always be achieved. The efficacy of inoculation varies depending on several factors, all of which affect the number of viable rhizobia available for infection of legume roots. Increased numbers of viable rhizobia per seed by application of inoculant above the commercially recommended rate, results in a continued linear increase in nodulation and yield. Several studies have reported yield increases of up to 25%. However, applying higher quantities of inoculant is uneconomical and technically difficult. Alternatively, higher numbers of viable rhizobia per seed may be achieved by improving survival during seed inoculation. Despite recognition of the factors affecting survival of rhizobia on seed and a substantial demand for commercially pre-inoculated legume seed, poor survival is still a major concern. Desiccation, temperature and seed coat toxicity all influence survival of rhizobia on seed. Their adverse effects may be ameliorated by selecting tolerant rhizobial strains and legume seed cultivars with low toxicity or artificially, by the use of additives in the seed coating. The accumulation of the desiccant protectant trehalose in strains of rhizobia, may result in better survival under desiccation stress. Similarly, the accumulation of exopolysaccharide (EPS) may act as a barrier reducing excessive water loss. Polymeric adhesives such as gum arabic, methyl cellulose and polyvinyl pyrollidone (PVP) have improved survival. However, studies of additives used in inoculation have been ad hoc and little of their mode of action is understood. A better understanding of the mechanisms involved in the protection of rhizobia from adverse conditions will assist in defining the optimum conditions for seed inoculation and storage to ensure a higher quality product for farmers at the time of sowing.

Legume seed inoculation technology—a review

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 109 cfu ml−1 of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.

Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations

Microbial inoculants are becoming more available as sustainable alternatives to fertilizers and other agrichemicals in broad-acre cropping. However, with the exception of legume inoculants little is understood about effective delivery and survival of the inoculum. Legume inoculants are applied to both seed and soil but seed inoculation is the most economical technique. Large quantities of pasture seed in Australia are inoculated by commercial seed coating companies, but the long-term survival of seed-applied inoculum is variable and monitoring of viability requires specialist microbiology skills and facilities. The aim of our research was to define optimum storage conditions for survival of rhizobia on legume seed and evaluate water activity as a means of monitoring shelf-life. The relationship between survival and water activity varied according to seed species, inoculum preparation, coating ingredients, initial water activity and time suggesting that storage conditions would need to be defined for each different combination. Although drying seeds after coating significantly reduced viable numbers of rhizobia, survival of rhizobia on dried commercially coated lucerne seed after 11 weeks was less variable than seeds that had not been dried. The highest numbers were maintained when seeds remained dry with water activities of between 0.47 and 0.38. The quality of inoculated seed could be improved by reducing the death rate of inoculum during preparation and providing optimum storage conditions for long-term survival.

https://www.mdpi.com/2077-0472/2/1/38/pdf?y=1

Conditions Affecting Shelf-Life of Inoculated Legume Seed

Preinoculation of seed is a convenient alternative method to inoculating seed on-farm. With preinoculation, a range of plant-growth and protection agents, polymer adhesives, colour pigments or dyes, and powder materials may be incorporated into an inoculant adhesive-slurry prior to seed coating. However, our recent point-of-sale surveys support findings of previous studies that survival of rhizobia on preinoculated seed is variable and can be poor. We focussed our research, both in the laboratory and at commercial facilities, on some of the factors that may contribute to poor survival of rhizobia on preinoculated seed. We found that rhizobial survival was affected by water quality; filtration improved cell survival but was not equal to distilled water. We also found that polymers affected cell survival differently for each rhizobial strain, and that slowing the desiccation rate reduced the cell rate of decline. Although fewer in cell number, older inoculant afforded more protection for survival of rhizobial cells. There is a need to test each ingredient and stage in the seed-coating process for compatibility to determine the best practices to promote rhizobial survival on seed. Failure to act on these factors prolongs the status quo of the findings from recent retail surveys.

Some factors that contribute to poor survival of rhizobia on preinoculated legume seed

The study consisted of a comparison of two-peat materials (Brazil and Canada) containing different C-total content. The peats were inoculated with strains of Herbaspirillum seropedicae ZAE 94, Burkholderia sp. M130, and Azospirillum brasilense Sp109, and monitored during a period of six months in relation to variation on humidity and survival of bacteria in the inoculant. The quantification of viable cells in the inoculant was measured by the Most Probable Number (MPN) method. The rice seeds were pelleted with the respective inoculants, grown in pots containing soil and maintained outside a greenhouse. The dry mass accumulation, N percent, N-total and yield were determined during the plant cycle. The humidity content varied with storage period. The survival measurement showed that only Burkholderia sp. M130 maintained the number of viable cells around 10 8 g -1 of peat, while there was a reduction in population of other strains. An increase of yield and total N of 13 and 19.4%, respectively in comparison to treatment fertilized with 40 kg N ha -1 was observed for variety IAC4440, inoculated with strain ZAE94. There was no difference in yield of the IR42 inoculated with either ZAE94 or M130, as compared to N control treatment. No significant difference in development of both rice varieties was observed for both peat used. The results suggest that peat can be used as a carrier for production of an inoculant based on diazotrophic bacteria, since it allowed maintenance of a bacterial population up to 10 8 cells g -1 peat during the storage period of up to 100 days. The results obtained encourage the practice of inoculation of non-leguminous plants.

Seleção de inoculantes à base de turfa contendo bactérias diazotróficas em duas variedades de arroz

Morphological changes that take place in peat cultures of several species of rhizobia were examined. These changes seemed to be associated with enhanced survival of cells in peat and after inoculation onto plastic beads, which were used as a model system for seeds. Cell wall changes, in which the periplasmic space appeared to be occluded with electron-dense material, were observed in Rhizobium sp. strain SU343 and Bradyrhizobium lupini WU425 cells after 7 and 14 days in peat, respectively. Nutrient limitation and low O2 concentration in peat are suggested to be factors involved in the induction of the morphological changes. Polyhydroxybutyrate reserves, which were present in broth-cultured cells of both species of rhizobia, were mobilized after transfer into peat but did not appear to influence survival after inoculation onto beads. Enhanced expression of an iron-manganese superoxide dismutase was also observed after the cells were transferred into peat. We conclude that cell wall thickening in rhizobia after transfer from broth cultures into peat is an adaptive response for long-term survival under nutrient-limited conditions in peat. Cells with thickened walls may also be more resistant to other types of stress, such as that encountered on a seed surface.

Morphological changes of rhizobia in peat cultures.


 Crape myrtle (Lagerstroemia indica) is a globally used ornamental woody plant and is the representative species of Lagerstroemia. However, studies on the evolution and genomic breeding of L. indica were hindered by the lack of a reference genome. Here, we assembled the first high-quality genome of L. indica using PacBio combined with Hi-C scaffolding to anchor the 329.14 Mb genome assembly into 24 pseudochromosomes. We detected a previously undescribed independent WGT event occurring 35.5 MYA in L. indica following its divergence from P. granatum. After resequencing 73 accessions of Lagerstroemia, the main parents of modern crape myrtle cultivars were found to be L. indica and L. fauriei. During the process of domestication, genetic diversity tended to decrease in many plants, but this was not observed in L. indica. We constructed a high-density genetic linkage map with an average map distance of 0.33 cM. Furthermore, we synthesized the results of QTL mapping using genetic mapping and BSA localized the main-effect interval determining internode length (IL) to chr1, which contains CDL15, CRG98, and GID1b1 associated with the phytohormone pathways. Analysis of gene expression of the red, purple and white flower colour flavonoid pathways revealed that differential expression of multiple genes determined the flower colour of L. indica, with white flowers having the lowest gene expression. In addition, BSA of purple- and green-leaved individuals of populations of L. indica was performed, and the leaf colour loci were mapped to chr12 and chr17. Within these intervals, we identified MYB35, NCED, and KAS1. Our genome assembly provided a foundation for investigating the evolution, population structure and differentiation of Myrtaceae species and accelerated the molecular breeding of L. indica.

Genome assembly and resequencing analyses provide new insights into the evolution and ornamental traits of crape myrtle

Polyhydroxybutyrate (PHB) accumulates in large amounts in free-living and bacteroid cells of many species of rhizobia, but the significance of this reserve material for nitrogen fixation is still unclear. PHB has been suggested to provide reserves of carbon and reductant that are important in sustaining respiratory activities to protect nitrogenase from damage by and in extending nitrogen fixation into the pod-filling stage (Romanov et al. 1980, Bergersen et al. 1991). On the other hand, there is evidence that suggests that, at least in some species of bacteroids, PHB reserves are not important for nitrogen fixation, and may actually reduce its capacity. Bacteroids in some symbioses (eg, chickpea, alfalfa) do not accumulate PHB, and nodules formed with Rhizobium mutants unable to synthesise PHB exhibit increased nitrogen-fixing activity (Povolo et al. 1994, Cevallos et al. 1996). Indeed, seeds from Phaseolus vulgaris plants nodulated by one such R. etli mutant contained significantly more nitrogen than seeds from plants nodulated with the wild type strain (Cevallos et al. 1996). Thus, questions remain as to whether carbon is allocated actively to PHB synthesis, or whether PHB synthesis is the result of an overflow pathway that operates because oxidation of carbon in the tricarboxylic acid cycle of bacteroids is constrained (Walshaw et al. 1997). To increase our understanding of the role of PHB symbiotic nitrogen fixation, we have been studying factors that influence the accumulation and mobilisation of this reserve material in several species of rhizobia. These studies are also aimed at determining whether accumulated PHB reserves enhance the survival of rhizobia during storage in peat cultures and after inoculation on to seeds.

Polyhydroxybutyrate in Nitrogen-Fixing Symbioses

Glaucium Mill. (horned poppy), belonging to the family Papaveraceae, is represented by a total of 25 species worldwide, and especially distributed throughout Western, Northern and Eastern Asia, Europe, Northern Africa, and Australia. As a country, Iran harbors relatively more species of the genus Glaucium (11-13 species) and hence, this country is considered as the hot spot of the genus. As a result, we conducted a molecular analysis of the data for this genus due to the relevance of these species of plants. We employed 75 plants from seven species and seven provinces that were randomly picked for this investigation. Five primers were used to amplify genomic DNA, yielding 78 bands, 73 of which were polymorphic (97.78%). ISSR primers have a great capability to recognise polymorphic loci among Glaucium species, as evidenced by the high average PIC and MI values obtained. The genetic similarity of seven samples was calculated to be between 0.77 and 0.92. Glaucium corniculatum var. corniculatum and Glaucium elegans var. elegans showed the lowest similarity, while Glaucium oxylobum and Glaucium grandiflorum had the highest similarity, according to Inter-Simple sequence repeats (ISSR) markers analysis. The following are the study’s goals: 1) Is it possible to identify Glaucium species using ISSR markers? 2) In Iran, how are these taxa genetically structured? 3) what is the inter-species relationship? According to this study, ISSR markers can be utilized to distinguish species.

Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers: A high value medicinal plant

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Papers

Morphological changes of rhizobia in peat cultures.

Genome assembly and resequencing analyses provide new insights into the evolution and ornamental traits of crape myrtle

Polyhydroxybutyrate in Nitrogen-Fixing Symbioses

Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers: A high value medicinal plant