A simple method to evaluate the number of bradyrhizobia on soybean seeds and its implication on inoculant quality control

Penna, Claudio; Massa, Rosana; Olivieri, F. P.; Gutkind, Gabriel; Cassán, Fabricio

doi:10.1186/2191-0855-1-21

Cited by 18 publications

(12 citation statements)

References 36 publications

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“…At 100% RH, the number of CFU was not affected negatively in sand nor on filters, however, when placed on sterile alfalfa seeds, CFU did decline to 0.27%. This can be explained by a toxic effect of the seed-surface, which has been described previously (Salema et al 1982 Death of rhizobia on inoculated seed, Smith 1992, Vriezen et al 2006, Penna et al 2011). We chose to further explore the responses of S. meliloti 1021 to desiccation using nitrocellulose filters, since the error on filter is lower compared to the use of a sand matrix, and no negative matrix effect is seen similar to drying on alfalfa seeds.…”

Section: Resultsmentioning

confidence: 68%

Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti 1021

2012

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Sinorhizobium meliloti is a microorganism commercially used in the production of e.g. Medicago sativa seed inocula. Many inocula are powder-based and production includes a drying step. Although S. meliloti survives drying well, the quality of the inocula is reduced during this process. In this study we determined survival during desiccation of the commercial strains 102F84 and 102F85 as well as the model strain USDA1021.The survival of S. meliloti 1021 was estimated during nine weeks at 22% relative humidity. We found that after an initial rapid decline of colony forming units, the decline slowed to a steady 10-fold reduction in colony forming units every 22 days. In spite of the reduction in colony forming units, the fraction of the population identified as viable (42-54%) based on the Baclight live/dead stain did not change significantly over time. This change in the ability of viable cells to form colonies shows (i) an underestimation of the survival of rhizobial cells using plating methods, and that (ii) in a part of the population desiccation induces a Viable But Non Culturable (VBNC)-like state, which has not been reported before. Resuscitation attempts did not lead to a higher recovery of colony forming units indicating the VBNC state is stable under the conditions tested. This observation has important consequences for the use of rhizobia. Finding methods to resuscitate this fraction may increase the quality of powder-based seed inocula.

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Section: Resultsmentioning

confidence: 68%

Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti 1021

2012

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“…Following the current methodology used in South America for cellular recovery of Bradyrhizobium spp. from soybean seeds [26][27][28][29] , A. brasilense strains Ab-V5 and Ab-V6 were single inoculated into maize seeds and submitted to the cell recovery process. Zero or near to zero cells were recovered, being classified in the category of "very low" (Table 1), highlighting that the methodology was not suitable to evaluate the recovery of viable cells of A. brasilense from inoculated maize seeds.…”

Section: Resultsmentioning

confidence: 99%

“…Based on these results, the following conditions were chosen for A. brasilense recovery: seeds previously hydrated in distilled sterile water for 2 h, inoculum diluted in distilled sterile water prior to seed inoculation (1:2, v:v), recovery step to the dilution 10 0 in water with Tween 80 (0.4 mL L -1 ), and shaking for 30 min. The subsequent steps, to obtain the following dilutions (10 -1 , 10 -2 , 10 -3 and 10 -4 ), plating of the samples and counting of CFU followed the current methodology for recovery of Bradyrhizobium from soybean seeds [26][27][28][29] . The steps can be visualized in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Method for Recovering and Counting Viable Cells from Maize Seeds Inoculated with Azospirillum brasilense

Santos¹,

Soja²,

Rodrigues³

et al. 2020

J. Pure Appl. Microbiol.

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The inoculation of seeds with nitrogen-fixing and plant-growth promoting bacteria is a well-established agricultural practice that has been increasingly adopted worldwide, decreasing costs and environmental impacts of food production. Most of the globally commercialized inoculants are for the soybean crop, and a method for recovery of Bradyrhizobium cells from inoculated soybean seeds for subsequent counting has been adopted by several laboratories of South America, especially to investigate the bacterial survival on seeds treated with pesticides. However, the use of inoculants containing Azospirillum brasilense in cereal crops has exponentially increased, requiring investigation about the recovery and counting of cells from inoculated seeds. We first verified that the method used for recovery and counting of viable cells of Bradyrhizobium from soybean seeds was not applicable for maize seeds inoculated with A. brasilense. We then modified several steps of the method, aiming at succeeding in recovering Azospirillum viable cells. The main limitation was identified in the nature of the seed tegument, dry and poor in nutrients, resulting in A. brasilense cell aggregation. Pre-hydration of seeds for 2 h in sterile distilled water, followed by shaking for 30 min in sterile distilled water with Tween 80 allowed proper counting of A. brasilense cells recovered from maize seeds. The method was successfully applied to count Azospirillum cells recovered from pre-inoculated maize seeds, and to estimate the impact of seed treatment with pesticides on cell survival.

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“…The recovery of viable Bradyrhizobium cells from inoculated seeds and counting followed the methodology previously described (Araujo et al, 2017;Penna, Massa, Olivieri, Gutkimd, & Cassán, 2011;Santos et al, 2020), also included in the Brazilian legislation (Mapa, 2010). Five treatments were evaluated, and they are described in detail in the item of field experiments.…”

Section: Concentration Purity and Strain Identity In The Inoculant mentioning

confidence: 99%

Seed pre‐inoculation with Bradyrhizobium as time‐optimizing option for large‐scale soybean cropping systems

et al. 2020

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Nitrogen is a key nutrient for soybean cropping and can be fully supplied by the biological nitrogen fixation (BNF) process. Inoculation with elite Bradyrhizobium strains greatly improves the supply of N to soybean; however, inoculation of large areas in short-sowing windows delays the sowing process, demanding the development of technologies for pre-inoculation. Here we report the evaluation of a liquid formulation containing cell protectors that proved, in four field experiments located in different edaphoclimatic conditions of Brazil, symbiotic performance comparable to the peat-based inoculant, traditionally considered as the best carrier. The liquid inoculant was also effective when applied to seeds not treated with pesticides 15 days before sowing, providing efficient BNF and timeflexibility to the farmers. Benefits of the liquid inoculant in improving grain yield were confirmed in two areas cropped for the first time and devoid of Bradyrhizobium, with an average increase of 89%, and also in two areas traditionally cropped with inoculated soybean, with an average increase of 6.8%, both in comparison to the non-inoculated control without N-fertilizers (NI). It is worth mentioning that, also in comparison to the NI control, the addition of 200 kg N ha −1 impacted grain yield in +54% and −1,8%, in new and traditional areas, respectively, indicating that BNF was more effective than the application of N-fertilizer. Large increases in total N content in grains were observed as well in response to the liquid inoculant, on average 47% and 27%, when compared to the NI and NI + N controls, respectively.

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A simple method to evaluate the number of bradyrhizobia on soybean seeds and its implication on inoculant quality control

Cited by 18 publications

References 36 publications

Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti 1021

Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti 1021

Method for Recovering and Counting Viable Cells from Maize Seeds Inoculated with Azospirillum brasilense

Seed pre‐inoculation with Bradyrhizobium as time‐optimizing option for large‐scale soybean cropping systems

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