F1000Research 2024, 12:781 Last updated: 23 JUL 2024 RESEARCH ARTICLE Effects of priming duration and concentration of metabolites from rhizosphere bacteria on the germinability of cowpea, soybean, sesame, and okra seeds [version 4; peer review: 2 approved with reservations, 1 not approved] Previous Title 'Effects of steeping duration and concentration of metabolites from rhizosphere bacteria on germinability of cowpea (Vigna unguiculata), soybean (Glycine max), sesame (Sesamum indicum) and okra ( Abelmoschus esculentus)' Oghenerobor Akpor , Ayotunde Ajinde, Tolulope Ogunnusi Department of Biological Sciences, Afe Babalola University, Ado Ekiti, Ekiti, 360102, Nigeria v4 First published: 05 Jul 2023, 12:781 https://doi.org/10.12688/f1000research.137322.1 Open Peer Review Second version: 09 Jan 2024, 12:781 https://doi.org/10.12688/f1000research.137322.2 Approval Status Third version: 03 Jun 2024, 12:781 https://doi.org/10.12688/f1000research.137322.3 1 Latest published: 23 Jul 2024, 12:781 https://doi.org/10.12688/f1000research.137322.4 2 3 version 4 (revision) Abstract Seed priming enhances germination and growth, which are important determinants of crop yield. This study was carried out to assess the effect of priming duration and metabolite concentration on the priming of five (5) different crops, using the metabolites of five (5) bacterial isolates. The crop seeds were treated in the cold-extracted metabolites of the five isolates at five (5) different priming durations (1, 2, 3, 4, and 5 h) and then in five metabolite concentrations (200, 400, 600, 800, and 1000 mg/L) of the five extracted metabolites at the optimal priming duration determined in the first experiment. Characterization of the cold-extracted metabolites was also carried out using gas-chromatography-mass spectrometry (GC-MS). Results revealed that priming cowpea and soybean for longer durations (< 3 h) could hinder their growth and development. Lower concentrations were observed to be optimal for cowpea and soybean, but for sesame and okra, there was no detectable pattern with metabolite concentration. The GC-MS revealed the presence of some molecules (e.g. hexadecanoic acid) that have shown plant growth promotion potential in other studies. This study showed that seeds with large endosperm, such as, cowpea and soybean, are more prone to the deleterious effects of treatment for longer durations. Further experiments should be carried out to isolate and purify the bioactive 23 Jul 2024 version 3 (revision) view 03 Jun 2024 version 2 (revision) view 09 Jan 2024 version 1 05 Jul 2023 view 1. Debasis Mitra view , Raiganj University, Raiganj, India International Rice Research Institute, Manila, Philippines 2. Hillary Righini, University of Bologna, Bologna, Italy Page 1 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 moieties for further studies and onward application. Keywords Germinability, rhizobacteria, germinability, seed vigor 3. Sowmyalakshmi Subramanian , McGill University, Montreal, Canada Any reports and responses or comments on the article can be found at the end of the article. This article is included in the Agriculture, Food and Nutrition gateway. Corresponding author: Oghenerobor Akpor (akporob@abuad.edu.ng) Author roles: Akpor O: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing; Ajinde A: Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Original Draft Preparation; Ogunnusi T: Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2024 Akpor O et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite this article: Akpor O, Ajinde A and Ogunnusi T. Effects of priming duration and concentration of metabolites from rhizosphere bacteria on the germinability of cowpea, soybean, sesame, and okra seeds [version 4; peer review: 2 approved with reservations, 1 not approved] F1000Research 2024, 12:781 https://doi.org/10.12688/f1000research.137322.4 First published: 05 Jul 2023, 12:781 https://doi.org/10.12688/f1000research.137322.1 Page 2 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 REVISED Amendments from Version 3 The p-values of post hoc comparisons have been included in the supplementary files. Any further responses from the reviewers can be found at the end of the article Introduction Seeds are critical inputs in agricultural production. Hence, it is expedient to plant only seeds that can produce the best agronomic performance. To this end, seed priming has been proposed and used. The beneficial effects of seed priming on a variety of crops have been confirmed.1–4 Although priming, as a plant-growth enhancement technique, can be applied at many stages in the developmental cycle of a plant, seed treatment is popular due to its practicality and simplicity. Priming usually involves soaking seeds in a solution to kickstart various pre-germinative activities,5,6 and it typically requires re-drying the seed before planting.7 Several priming methods are used to enhance the agro-morphic parameters of crop seeds. Hydropriming involves priming seeds in water8; osmopriming entails the use of an osmoticum9; halopriming involves soaking in salt solutions10; solid-matrix priming involves priming on a solid material11; and hormonal priming, which requires the use of plant growth regulators such as abscisic acid (ABA),12,13 gibberellic acids (GAs)5 or salicylic acid (SA).14–16 Biopriming, which involves using microbial products, is a relatively new priming strategy that offers the advantage of environmental friendliness and may also be less expensive than most of the priming methods available. Seed priming can enhance germination, reduce germination time, and improve seedling vigor.17 Priming also increases the resistance of seeds to environmental stress.18 Although the exact mechanism of priming is not well understood, it is understood to involve specific physiological and biochemical reactions.19,20 Optimal priming duration is plant-specific due to seed structures that are unique morphologically and physiologically. Since bacteria secret substances that can promote plant growth,21,22 the determination of their effective concentration is necessary for wholesome application. Optimal concentrations of chemicals used in priming have been determined.23–25 However, while there is ample research on other priming methods, microbial priming is less well-researched, and their optimization is even less so. Hence, we set out to understand the impact of priming duration and metabolite concentration on the growth promotion activity of secondary metabolites on selected crops (cowpea, soybean, sesame, and okra) visa-vis some agro-morphic parameters by sowing seeds soaked in metabolites of some previously-isolated bacteria immediately without drying. The crops used for priming in this study are of significant economic importance in tropical regions where they are a vital source of dietary requirements. Therefore, it is necessary to boost their production, and seed priming is a veritable tool for achieving this end. Methods Isolation Seventy-five (75) bacterial strains were isolated from rhizospheres within Afe Babalola University (Ado-Ekiti, Nigeria) using the pour-plate technique, as reported by Sanders.26 The five (5) strains used in this study were chosen based on their germinability enhancement potential observed from a previous in planta experiment using seeds (data not presented). Pure cultures of the five (5) strains were stored on nutrient agar slants at 4 °C
2 °C until needed. Molecular identification Identification of the 15 rhizobacterial strains was done using the 16s rRNA gene sequencing technique. The CTAB protocol was used in DNA extraction.27 Subsequently, the V3-V4 hypervariable regions of the 16s rRNA gene were amplified using 27F 50 AGAGTTTGATCMTGGCTCAG 30 and 1525R 50 AAGGAGGTGATCCAGCC 30 primers and the PCR conditions (profile) comprised an initial denaturation at 94 °C for 5 min; followed by 30 cycles consisting of 94 °C for 30 s, annealing at 50 °C for 60 s, and extension at 72 °C for 1 min 30 s; and a final extension at 72 °C for 10 mins, in a GeneAmp 9700 PCR System Thermalcycler (Applied Biosystem Inc., USA). The integrity check of the amplified products was carried out using 1% Agarose gel and purified using 95% ethanol and 3M of sodium acetate. The purified PCR products were sequenced using a Genetic Analyzer 3130xl sequencer (Applied Biosystem Inc., USA) according to the manufacturer’s instructions. The sequences were compared to those in a database for identification via the NCBI site (BLAST). Then, the 16S rRNA sequences were aligned using the ClustalW program, and the neighbor-joining phylogenetic tree was constructed using MEGA 11.28 The isolates were identified as OP830504 – Serratia liquefaciens Page 3 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 AYO-O; OP830503 – S. liquefaciens AYO-N; OP830491 – Providencia rettgeri AYO-B; OP830498 – P. rettgeri AYO-I; and OP830501 – Bacillus cereus AYO-L. Test seeds The seeds used for the study were cowpea (Vigna unguiculata), soybean (Glycine max), sorghum (Sorghum bicolor), sesame (Sesamum indicum), and okra (Abelmoschus esculentus). All the seeds were sourced from a local seed dealer in Ado-Ekiti (Ekiti State, Nigeria). First, a test was carried out on seeds in order to assess their viability. For this, over (500) seeds were released into a beaker containing 500 mL of sterile, distilled water, and floated (non-viable) seeds were collected and discarded, and the water decanted. From the remaining seeds, 50 seeds were sown in four replicates on 3.5 grams of absorbent cotton wool placed within transparent plastic containers measuring 100 mm by 40 mm by 20 mm. Incubation was under fluorescent light with daily watering for five (5) days under laboratory conditions. A final germination percentage of 80% was indicative of good germination potential (seed quality) for all the seeds, except okra at 60%. Test metabolites The metabolites were extracted from the cultures of the five strains: A = OP830504 – Serratia liquefaciens AYO-O; B = OP830503 – S. liquefaciens AYO-N; C = OP830491 – Providencia rettgeri AYO-B; D = OP830498 – P. rettgeri AYO-I; and E = OP830501 – Bacillus cereus AYO-L. The cold extraction method reported by Ref. 29 was adopted. The 48-hour broth cultures of the isolates grown for 48 h at 25 °C
2 °C were centrifuged at 5000 rpm for 15 min to obtain the cell-free supernatants, which were acidified to a pH of 2 by the addition of 1M HCl. Following acidification, an equal volume of methanol:ethylacetate (1:2) mixture was added and incubated at 4 °C
2 °C for 24 h. After incubation, the mixtures were transferred to a separating funnel to separate the solvent from the broth and precipitate the metabolite. The precipitated metabolite was then dried by placing the beakers containing the separated solvents in a water bath at a temperature of 80 °C. The dried metabolite was then quantified and stored in clean universal bottles at 4 °C
2 °C until needed. Metabolite characterization Metabolite identification was carried out using a gas chromatograph (GC) connected to a mass spectrometer (Varian 3800/4000; Agilent Technologies, USA). The equipment has a splitter split/splitless HP5 (30 mm  0.25 mm) silicabased, cross-linked column with nitrogen as a gas carrier. The injector and detector temperatures were set at 300 °C. The GC temperature regimen started at 50 °C and increased to 100 °C with a ramp rate of 10 °C/min and held at 100 °C for 2 minutes and was increased to 250 °C at the rate of 5 °C/min for 2 mins. It was finally raised to 300 °C with a ramp rate of 3 °C/min for 15 min. A sample volume of 1 uL was used and the carrier gas at a rate of 1 mL/min. The MS was scanned from 30–400 amu at 1.562 u/s and operated in EI mode at 70 eV. The mass spectral data were compared with those of the National Institute of Standards and Technology (NIST) and Wiley libraries. Only the mass spectral data of compounds with at least 90% matching accuracy were reported. Germinability studies Germinability experiments were carried out to investigate the effects of priming duration and metabolite concentration on the seeds. The investigation of the effect of priming duration on the germinability of the seeds was carried out for 1, 2, 3, 4, or 5 h. Viable, surface-sterilized seeds of the four seeds were treated in a 1000 mg/L (water dilution) of the five metabolites. Every one hour, for a 5-h duration, seven (7) seeds were withdrawn and planted in transparent plastic cups in six (6) replicates and incubated for 8 days with daily watering. At the expiration of incubation, final germination percentage, mean germination time, germination index and vigor index were estimated as follows: • Final germination percentage (FGP) = total number of germinated seeds/total number of seeds sown100%30 • Mean germination time (MGT) = ∑fxf31 Where f is the number of seeds germinated on day x • Germination index (GIX) = 8N1+7N2+6N3+ … +1N832 Page 4 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Where N1, N2, N3 … N8 represent the number of seeds that germinated on the first, second, and third until the 8th day, and 8, 9, 7 … 1 are the weights given to the number of germinated seeds on the first, second, and third day up to the 8th day. • Vigor index (VIX) = FGP  average plant height33 For effect of metabolite concentration, 200 mg/L, 400 mg/L, 600 mg/L, 800 mg/L, and 1000 mg/L (water dilutions) were used for the study. The seeds were treated in the respective metabolite concentrations and allowed to stand for the optimal priming time obtained in the first experiment before planting and incubation using six (6) replicates. At the expiration of the 8-day incubation period, final germination percentage, mean germination time, germination index, and vigor index were estimated as described earlier. Results Effect of priming duration in metabolites Generally, the final germination percentage of the cowpea seeds showed significantly highest and lowest values in seeds treated for 2 and 5 h, 1 and 5 h, 1, 2, and 3 and 4 h in the metabolites from Isolates K, L and M, respectively. There was no significant difference in final germination percentage at the different priming durations for seeds treated in the metabolite from Isolate N. In addition, seeds treated with the metabolite from Isolate O showed significantly lowest final germination percentage at 3 and 5 h. With respect to mean germination time, significantly lowest values were recorded for seeds primed for 1 h (metabolites from Isolates K and M), 4 and 5 h (metabolite from isolate L), 1, 4, and 5 h (metabolites from Isolate N), and 1, 2, and 4 (metabolite from Isolate O). Germination index showed significantly lowest values at 3 h (metabolite from Isolate L), 4 and 5 h (metabolite from Isolate M), 1-3 h (metabolite from Isolate N), and 3 and 5 h (metabolite from Isolate O). For vigor index, significantly lowest values were observed at 2, 4 and 5 h (metabolite from Isolate M), 1, 3, and 5 h (metabolite from Isolate N), and 4 and 5 h (metabolite from Isolate O). Also, germination and vigor indices of the seeds showed significantly lowest values in setups treated for 5 h (metabolites K and L) (Table 1). Probability values are shown in supplementary file 1. In the case of the soybean seeds, the final germination percentage of the seeds showed the highest values at 2 h priming duration when treated in the respective metabolites, apart from those treated in Isolate O, where 1 h priming duration was observed to show the highest value. Furthermore, significantly lowest mean germination times were recorded in seeds primed for 1, 2, and 3 h, 1 and 2 h, 1, 4, and 5 h in the metabolites from isolates L, M and N, respectively, and 2 h in the metabolites from Isolate K, and 1, 3, and 4 h in the metabolite from Isolate O. However, significantly highest germination index was observed in the seeds treated for 1 and 2 h in Metabolite K, 2 h in Metabolite L, 1-3 h in Metabolite M, 2 and 3 h in Metabolite N, and 1 h in Metabolite O. Also, for seedling vigor index, significantly highest values were observed at 2 h (Metabolites K and L), 1, 3, 4, and 5 h (Metabolite M), 2 and 3 h (Metabolite N), and 1 h (Metabolite O) (Table 2). Probability values are shown in supplementary file 2. For the sesame seeds, remarkably high final germination values (> 78%) were observed in the respective treatments, regardless of the priming duration. However, a significantly lowest mean germination time was observed for seeds treated for 1 h (metabolites from Isolate O), 2 h (metabolites from Isolates L and M) and 1, 2, 3, and 5 h (metabolites from Isolate K). Also, significantly highest germination index was observed for seeds treated for 2 and 4 h (metabolite from Isolate K), 2 h (metabolites from Isolates L and M) and 1 h (metabolites from Isolates N and O). In the case of the seedling vigor index, seeds treated for 2 and 3 h (metabolites from Isolates K and O), 1 h (metabolite from Isolates L and M) and 3 h (metabolite from Isolate N) showed the significantly highest values (Table 3). Probability values are shown in supplementary file 3. When the okra seeds were treated in the different metabolites for varying durations, final germination percentage of the okra seeds showed significantly highest values in seeds treated for 3 h (Metabolite K), 4 h (Metabolite L) and 1 and 5 h (Metabolite N). There was no significant difference in final germination percentage between seeds treated with the metabolite from Isolate M at the different priming durations. Also, mean germination time showed no significant difference at the different priming durations for seeds treated with the metabolites from Isolates K, M and O. However, significantly lowest mean germination times were observed for seeds treated for 2 and 5 h and 3-5 h with the metabolites from Isolates L and N, respectively. Furthermore, seeds primed for 3 h showed significantly highest germination and vigor indices when treated with the metabolites from Isolates K respectively. Significantly highest germination index values were observed for seeds treated for 3 h (Metabolite K), 2-4 h (metabolite L), 1, 3, and 4 h (metabolite M), 1, 3, 4, and 5 h (Isolate N), and 1-4 h (Isolate O). Finally, for seedling vigor index, significantly highest values were observed at 3 h (Metabolite K), 3 and 4 h (Metabolite L), 3 and 4 h (Metabolite M), 1, 4, and 5 (Metabolite N), and 3 and 4 h (Metabolite O) (Table 4). Probability values are shown in supplementary file 4. Page 5 of 34 Table 1. Germinability of the cowpea seeds at the different priming durations in the respective metabolites. Parameter Time Isolate K Isolate L Isolate M Isolate N Isolate O FPG 1h 71.43a (
0.00) 78.57a (
7.82) 85.71a (
0.00) 64.29a (
7.82) 78.57a (
7.82) 2h 85.71b (
15.65) 64.29b (
7.82) 71.43ac (
15.65) 78.57a (
7.82) 78.57a (
7.82) 3h 71.43a (
0.00) 42.86c (
15.65) 71.43ac (
31.30) 71.43a (
0.00) 50.00b (
23.47) MGT GIX 4h 71.43 (
0.00) 57.14 (
0.00) 50.00 (
7.82) 78.57 (
23.47) 78.57a (
7.82) 5h 54.29c (
15.65) 21.43d (
7.82) 64.29bc (
7.82) 78.57a (
7.82) 64.29ab (
23.47) a b 5.14 (
0.05) 5.45 (
0.25) 5.04 (
0.04) 5.14 (
0.06) 5.24a (
0.06) 2h 5.51b (
0.15) 5.37ab (
0.01) 5.34b (
0.02) 5.36b (
0.08) 5.25a (
0.17) 5.32 (
0.04) 5.23 (
0.00) 5.39 (
0.06) 5.40 (
0.17) 5.40b (
0.16) 4h 5.43b (
0.08) 5.11c (
0.00) 5.41b (
0.10) 5.21a (
0.11) 5.25a (
0.07) (
0.08) 5h 5.38 1h 129.50a (
3.83) a 5.25 bc (
0.27) 120.00a (
29.58) a b a 3h bc b a a 1h c a b c b 5.57 (
0.07) 5.24 (
0.06) 5.45b (
0.15) 164.50a (
3.83) 116.00ab (
18.62) 133.50a (
8.22) b a ab 133.00a (
0.00) 2h 125.00 (
33.96) 101.50 (
11.50) 115.50 (
26.84) 124.00 3h 116.00a (
3.29) 73.50b (
26.84) 112.50b (
53.13) 110.00b (
10.95) 79.00b (
44.91) 4h 109.00a (
4.38) 105.00a (
0.00) 77.00c (
7.67) 133.50a (
31.22) 131.00a (
9.86) b c bc 5h 84.40 (
19.72) 35.00 (
7.67) 89.50 1h 818.88a (
69.86) 862.35a (
163.31) 1578.37a (
104.63) 2h b 1212.14 (
240.22) 575.61 (
130.67) 697.76 3h 788.78a (
32.42) 323.67c (
239.21) 888.57c (
625.97) a b b (
16.98) bc (
202.77) b (
18.62) a 133.50 (
8.22) 93.00b (
25.20) 783.78a (
215.62) 943.06ab (
2.46) b 1390.10 (
258.55) 1064.49a (
77.35) 700.51a (
100.04) 608.47b (
535.09) b 4h 877.55 (
125.19) 507.35 (
66.62) 343.98 (
156.16) 1305.20 (
800.23) 556.43c (
266.37) 5h 434.12c (
247.37) 37.55d (
29.51) 353.88b (
68.41) 999.18ab (
229.37) 466.53c (
347.41) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 6 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX a Table 2. Germinability of the soybean seeds at the different priming durations in the respective metabolites. Parameter FPG MGT GIX 1h Isolate K a 64.29 (
7.82) a Isolate L 50.00 ad (
7.82) ac 57.14 (
15.65) Isolate O a 85.71a (
0.00) b 50.00 (
7.82) 71.43 (
15.65) 64.29 (
7.82) 71.43 (
0.00) 78.57 (
7.82) 57.14bc (
0.00) 3h 42.86b (
15.65) 42.86a (
0.00) 71.43b (
0.00) 71.43b (
15.65) 50.00b (
7.82) (
15.65) 4h 57.14 5h 57.14ab (
0.00) a 57.14 bd (
0.00) 50.00ad (
7.82) a 50.00 (
7.82) 57.14 (
0.00) 50.00b (
7.82) 64.29bc (
7.82) 50.00a (
7.82) 64.29c (
7.82) 5.37 (
0.01) 5.34 (
0.02) 5.31 (
0.14) 5.50 (
0.25) 5.51ab (
0.21) 2h 5.27b (
0.05) 5.44ac (
0.06) 5.37a (
0.10) 5.69b (
0.10) 5.61a (
0.12) 5.72 (
0.15) 5.34b (
0.12) 5.65b (
0.04) 5.55b (
0.06) 5.61ab (
0.12) 5.52ab (
0.23) 5.50c (
0.20) 5.60b (
0.01) 5.50a (
0.00) 5.54a (
0.08) 5.67 (
0.06) 5.41 4h 5.48d (
0.02) 5h 5.50e (
0.00) ab (
11.50) 1h 101.50 2h 119.00b (
23.00) c (
0.10) a b ac 80.50 (
11.50) 95.00 98.00b (
15.34) 112.50a (
7.12) c (
33.96) ac 3h 57.00 (
23.00) 66.50 (
3.83) 101.00 4h 84.50a (
23.55) 75.50ac (
2.74) 70.50b (
8.22) a ac 5h 84.00 (
0.00) 71.50 1h 490.00a (
60.14) 326.63a (
28.50) (
1.64) c a 71.50 (
0.55) 124.50a (
16.98) 99.00b (
4.38) 78.00bc (
6.57) 91.00 bc (
27.39) 78.00ac (
6.57) 71.00b (
1.10) 88.50 (
11.50) 73.50 (
11.50) 92.00cd (
15.34) 607.45a (
339.48) 241.73ad (
85.74) 1027.96a (
329.30) 593.67 (
120.95) 845.41 (
39.68) 897.14 (
367.98) 494.69b (
66.17) 3h 243.67a (
223.11) 252.24a (
91.88) 805.10ab (
60.36) 711.63bc (
437.06) 418.88b (
196.62) 4h 474.59a (
374.35) 305.71a (
75.56) 422.45a (
135.48) 556.73c (
15.20) 243.67c (
44.26) 346.94 (
76.01) a 279.08 (
85.06) b a 81.00bd (
18.62) 926.73 (
416.05) a b (
12.05) b 2h 5h b a 5.53 (
0.15) 3h ac a a 1h c a b Isolate N 2h ab b Isolate M a 440.00 (
110.89) b d 232.96 (
115.02) 494.59b (
132.24) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 7 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX Time Table 3. Germinability of the sesame seeds at the different priming durations in the respective metabolites. Parameter Time Isolate K Isolate L Isolate M Isolate N Isolate O FPG 1h 92.86a (
7.82) 100.00a (
0.00) 100.00a (
0.00) 92.86a (
7.82) 100.00a (
0.00) 2h 100.00a (
0.00) 100.00a (
0.00) 100.00a (
0.00) 100.00b (
0.00) 100.00a (
0.00) 3h 92.86a (
7.82) 85.71b (
0.00) 92.86b (
7.82) 100.00b (
0.00) 92.86b (
7.82) 4h a 100.00 (
0.00) 5h 92.86a (
7.82) MGT GIX 78.57 (
7.82) a 100.00 (
0.00) 100.00 (
0.00) 92.86b (
7.82) 100.00a (
0.00) 92.86b (
7.82) 100.00b (
0.00) 100.00a (
0.00) 1h 5.33 (
0.14) 5.42 (
0.00) 5.30 (
0.04) 5.00 (
0.00) 5.16a (
0.04) 2h 5.27ab (
0.08) 5.20b (
0.00) 5.13b (
0.15) 5.42a (
0.00) 5.38b (
0.04) a a 5.25 (
0.02) 5.27 (
0.05) 5.37 (
0.06) 5.34 (
0.00) 5.37b (
0.04) 4h 5.34b (
0.00) 5.35d (
0.04) 5.34a (
0.08) 5.34a (
0.00) 5.53c (
0.03) (
0.01) 5h 5.33 1h 148.50a (
0.55) b d a a 3h ab c a b 5.34 (
0.08) 5.50 (
0.00) 5.42 (
0.00) 5.38b (
0.04) 154.00a (
0.00) 164.50a (
3.83) 182.00a (
15.34) 178.50a (
3.83) 168.00 (
7.67) 175.00 (
0.00) 182.00 (
15.34) 154.00 (
0.00) 157.50b (
3.83) 3h 157.50a (
11.50) 143.50c (
3.83) 147.00c (
7.67) 161.00b (
0.00) 147.00c (
15.34) 4h 161.00b (
0.00) 126.00d (
15.34) 161.00a (
7.67) 161.00b (
0.00) 133.50d (
8.22) 150.50 (
11.50) 161.00 (
7.67) 136.50 (
11.50) 154.00 (
0.00) 157.50b (
3.83) 1h 528.67a (
111.00) 800.00a (
178.40) 952.86a (
7.82) 666.53a (
93.00) 756.43a (
71.20) b c b 5h b a b a 2h a b c a a b bd 2h 711.43 (
12.52) 660.00 (
115.80) 887.86 (
2.35) 506.43 3h 678.16b (
33.31) 615.31b (
2.01) 721.63b (
42.48) 829.29c (
97.81) a c c (
44.60) b 877.86b (
28.95) 791.43ab (
78.25) 4h 592.86 (
31.30) 339.18 (
22.80) 582.86 (
50.08) 553.57 (
44.60) 447.55c (
138.83) 5h 511.73a (
95.12) 455.71d (
0.00) 461.73d (
106.08) 450.71d (
22.69) 496.43c (
44.60) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 8 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX ab c Table 4. Germinability of the okra seeds at the different priming durations in the respective metabolites. Parameter FPG MGT GIX 1h Isolate K a 28.57 (
15.65) b Isolate L 57.14 ad ab (
15.65) Isolate M Isolate N a 85.71 (
0.00) 71.43ab (
15.65) b 85.71 (
0.00) 2h 71.43 (
0.00) 71.43 (
0.00) 78.57 (
7.82) 64.29 (
7.82) 71.43ab (
15.65) 3h 85.71c (
0.00) 85.71bc (
0.00) 78.57a (
7.82) 71.43bc (
0.00) 85.71a (
15.65) b 4h 64.29 (
7.82) 92.86 (
7.82) 78.57 (
7.82) 64.29 (
23.47) 78.57ab (
7.82) 5h 71.43b (
15.65) 50.00d (
23.47) 78.57a (
23.47) 78.57ac (
7.82) 64.29b (
7.82) a c a Isolate O a 5.41 (
0.10) 5.28 (
0.01) 5.30 (
0.08) 5.47 (
0.20) 5.22a (
0.24) 2h 5.33a (
0.16) 5.14bd (
0.05) 5.39a (
0.33) 5.91b (
0.06) 5.29a (
0.23) 5.29 (
0.07) 5.36 (
0.02) 5.30 (
0.13) 5.35 (
0.04) 5.28a (
0.30) 4h 5.33a (
0.11) 5.39a (
0.06) 5.19a (
0.01) 5.27c (
0.05) 5.20a (
0.05) 5h 5.28a (
0.31) 5.19cd (
0.21) 5.38a (
0.17) 5.34c (
0.02) 5.35a (
0.26) 45.50 (
26.84) 93.00 (
24.10) 140.50 (
7.12) 124.00 (
18.62) 120.00ab (
8.76) 2h 114.00b (
13.15) 129.50b (
3.83) 121.00bc (
13.15) 71.00b (
5.48) 122.50ab (
42.17) b a c 1h c a a a 3h a a a b 1h a ac a abd 3h 141.00 (
6.57) 133.00 (
1.10) 127.50 4h 102.50b (
4.93) 144.50b (
18.07) 137.00ab (
12.05) b 113.00 (
1.10) 139.00ab (
1.10) 108.50a (
42.17) 137.00a (
18.62) 114.50 (
2.74) 84.00 (
30.67) 120.00 (
24.10) 126.50 (
14.79) 102.00b (
3.29) 1h 108.27a (
92.67) 335.31a (
154.70) 580.41ac (
45.61) 743.27a (
73.77) 488.98ac (
140.40) 539.29 (
126.87) 303.88 (
9.17) 522.65ac (
230.94) 869.39c (
8.05) 836.33c (
12.07) 643.88ab (
133.02) 511.22c (
4.47) 818.88b (
292.31) 537.45b (
112.12) 754.08c (
109.77) 713.67b (
61.93) 553.47ac (
336.68) 710.20ab (
140.84) 495.92 3h 4h 5h d (
36.89) 410.82 (
171.02) a 250.41 (
222.00) 488.47 ad a 557.65 (
73.22) 2h b cd a 5h bd a (
1.64) a cd (
104.96) 559.90 b ac (
100.27) 392.55c (
100.94) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 9 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX Time F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Effect of metabolite concentration At the respective concentrations of the different metabolites, significantly lowest final germination percentages were observed in the cowpea seeds treated with 800 mg/L of the metabolites from Isolates K, L, and N. The final germination percentage of the cowpea seeds treated with metabolites from isolates M and O did not differ significantly at the respective concentrations. Also, significantly lowest mean germination times were observed for seeds treated with metabolite concentrations of 200, 600, and 800 mg/L (metabolite from Isolate K), 400 and 800 mg/L (metabolite from Isolate L), and 400 mg/L (metabolite from Isolate M), 200 – 800 mg/L (Metabolite N), and 200-600 mg/L (Metabolite O). Significantly highest germination index values were observed in the seeds treated with metabolite concentrations of 200 and 600 mg/L (Metabolite K), 600 mg/L (Metabolite L), 200, 400, 600, and 1000 mg/L (Metabolite N), and 200, 600, and 800 mg/L (Metabolite O) (Table 5). In the case of seedling vigor index, significantly highest values were observed at concentrations of 600 mg/L (Metabolite K), 200, 600, and 1000 mg/L (Metabolite L), 600 and 1000 mg/L (Metabolite M), 400, 600, and 1000 mg/L (Metabolite N), and 200-600 mg/L (Metabolite O) (Table 5). Probability values are shown in supplementary file 5. For the soybean seeds, significantly lowest final germination percentages were observed in treatment with metabolite concentrations of 200, 600, 800, and 1000 mg/L (metabolite from Isolate K), 800 mg/L (metabolite from Isolate M), and 600 mg/L (metabolite from Isolate N), and 1000 mg/L (metabolite from Isolate O). In the case of mean germination time, significantly lowest values were recorded in the seeds that were treated with 200, 400, and 800 mg/L (metabolite from Isolate K), 200, 400, 600, and 1000 mg/L (metabolite from Isolate L), 200, 600, and 1000 mg/L (metabolite from Isolate M), 200, 400, and 800 mg/L (metabolite from Isolate N), 200, 400, and 600 mg/L (metabolite from Isolate M). Generally, highest germination index values were observed in the seeds that were treated with 400 mg/L, 600 and 1000 mg/L, 400 and 800 mg/L, and 400 and 800 mg/L of metabolites from Isolates K, M, N, and O, respectively. For the seeds treated with metabolites from Isolates K, M, N, and O, significantly highest seedling vigor index values were observed in the seeds treated with 400 mg/L, 600 mg/L, 400 mg/L, and 600 mg/L, respectively (Table 6). Probability values are shown in supplementary file 6. In the case of the sesame seeds, remarkably high final germination (> 90%) was observed at all priming duration for all priming durations. Significantly lowest mean germination time was recorded for seeds treated with 600 mg/L (metabolites from isolates K and L), 1000 mg/L (metabolites from isolates M and O) and 200, 400, and 600 mg/L (metabolite from isolate N). Generally, significantly highest germination index was observed in seeds treated with 600 mg/L of metabolite from isolate K, 600 mg/L, 800 mg/L, and 1000 mg/L of metabolites from isolate L, 1000 mg/L of metabolites from isolate M, 200-600 mg/L of metabolites from isolate N, and 1000 mg/L of metabolites from isolate O. For seedling vigor index, significantly highest values were observed at 400 and 600 mg/L for metabolite K, 600 and 800 mg/L for metabolite L, 200 and 1000 mg/L for metabolite M, 200 mg/L for metabolite N, and 1000 mg/L for metabolite O (Table 7). Probability values are shown in supplementary file 7. Furthermore, the final germination percentage of the okra seeds showed significantly lowest values in setups that were treated with 800 mg/L of metabolites from Isolates K and N, 200, 800, and 1000 mg/L of metabolite from Isolate L, 800 and 1000 mg/L of metabolite from Isolate M, and 200 mg/L of metabolite from Isolate O. In the case of mean germination time, seeds treated with the metabolites from Isolates M and N showed no significance difference at the different concentrations, while treatment in 800 mg/L and 200, 400, and 1000 mg/L of the metabolites from Isolates K and O showed significantly highest values, respectively. With respect to germination index, significantly highest values were recorded in the seeds treated with 400, 600, and 1000 mg/L (metabolite from Isolate K), 600 mg/L (metabolite from Isolate L), 200-800 mg/L (metabolite from Isolate M), and 800 and 1000 mg/L (metabolites from Isolates N and O). For vigor index, the seeds treated in the metabolites at 400, 600, and 1000 mg/L (Metabolite K), 600 mg/L (Metabolite L), 200-600 mg/L (Metabolites M and N), and 800-1000 mg/L (Metabolite K) (Table 8). Probability values are shown in supplementary file 8. Detected compounds in the metabolites In the metabolite from S. liquefaciens AYO-O, the major compounds that were detected included methyl lactate (10.40%), 9,12-octadecadienoic acid (Z,Z)- (17.50%), n-hexadecanoic acid (13.38%), phytol (5.96%), oleic acid (11.48%) and 9,12-octadecadienoic acid (Z,Z)- (17.01%). Also, for the metabolite from P. rettgeri (OP830491), n-hexadecanoic acid (14.13%), octadecane (7.90%), phytol (9.31%), 11,14,17-eicosatrienoic acid, methyl ester (5.96%), lupeol (7.74%), stigmasterol (15.00%) and β-sitosterol (12.19%) were the most dominant compounds (Table 9). In addition, the metabolites from S. liquefaciens AYO-N revealed the presence of tetradecanoic acid (8.85%), phytol (29.23%), and 11,14,17-eicosatrienoic acid, methyl ester (25.17%) as the most dominant species. In the case of the Page 10 of 34 Table 5. Germinability of the cowpea seeds at the different concentrations of the respective metabolites. Parameter Concentration Isolate K Isolate L Isolate M Isolate N Isolate O FPG 200 mg/L 71.43ab (
0.00) 64.29a (
23.47) 50.00a (
7.82) 64.29a (
23.47) 64.29a (
7.82) 400 mg/L 57.14ac (
31.30) 42.86b (
0.00) 42.86a (
0.00) 85.71b (
0.00) 57.14a (
46.95) 600 mg/L 85.71b (
0.00) 85.71c (
0.00) 57.14a (
31.30) 85.71b (
15.65) 78.57a (
7.82) MGT GIX 800 mg/L 50.00 (
7.82) 14.29 (
0.00) 50.00 (
7.82) 42.86 (
0.00) 64.29a (
7.82) 1000 mg/L 71.43abc (
15.65) 64.29a (
23.47) 57.14a (
0.00) 78.57ab (
23.47) 71.43a (
15.65) a d 5.32 (
0.25) 5.67 (
0.09) 5.61 (
0.12) 5.45 (
0.05) 5.37ab (
0.01) 400 mg/L 5.62b (
0.12) 5.38bc (
0.07) 5.25b (
0.27) 5.47ab (
0.11) 5.28a (
0.31) b a a 200 mg/L ab a a a a ab 600 mg/L 5.39 (
0.09) 5.47 (
0.08) 5.54 (
0.04) 5.47 800 mg/L 5.52ab (
0.23) 5.25c (
0.27) 5.69a (
0.04) 5.41a (
0.10) b ab 1000 mg/L 5.64 (
0.35) 5.51 200 mg/L 116.50a (
18.07) 84.00a (
35.05) b (
0.01) 5.52bc (
0.07) 5.55 (
0.06) 5.56 (
0.07) 5.75d (
0.16) 67.50a (
4.93) 98.00a (
38.34) 101.50ab (
11.50) 79.00 (
47.10) 67.00 (
3.29) 73.50 (
11.50) 127.00 (
7.67) 84.50a (
61.89) 600 mg/L 133.50a (
7.12) 127.00b (
6.57) 81.00a (
42.72) 125.50a (
26.84) 120.50b (
6.02) 800 mg/L 71.00b (
1.10) 24.50c (
3.83) 64.50a (
8.22) 66.50b (
3.83) 92.00ab (
7.67) 88.50 (
3.83) 92.50 (
32.32) 81.00 (
3.29) 109.50 (
27.93) 85.50a (
8.22) 200 mg/L 675.00a (
123.52) 584.69ab (
234.07) 318.98a (
122.73) 741.12a (
481.21) 765.92a (
137.94) (
540.57) 400 mg/L 588.98 600 mg/L 1100.20b (
10.06) c 331.53 (
147.89) 1279.59 (
34.88) 1042.55a (
1097.35) 918.37b (
490.94) 719.18b (
627.31) 1406.33b (
612.78) 1065.82a (
254.30) c a a (
42.92) 308.57 ac a a 1000 mg/L ac a a b 5.40ac (
0.24) 400 mg/L b a a (
0.05) a b a 800 mg/L 320.92 (
27.61) 16.94 (
0.22) 330.41 (
73.55) 302.76 (
66.73) 796.73b (
137.71) 1000 mg/L 525.31ac (
178.40) 744.49ab (
618.37) 411.84ab (
97.03) 1240.41b (
443.99) 863.06b (
236.30) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 11 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX c Table 6. Germinability of the soybean seeds at the different concentrations of the respective metabolites. Parameter FPG MGT GIX 200 mg/L Isolate K 42.86 ab (
15.65) a Isolate L a 42.86 (
31.30) a Isolate M Isolate N a 71.43 (
15.65) a 50.00 (
7.82) 50.00a (
7.82) ad (
7.82) 57.14a (
15.65) 400 mg/L 50.00 (
7.82) 42.86 (
31.30) 64.29 (
7.82) 50.00 600 mg/L 42.86ab (
0.00) 42.86a (
15.65) 85.71b (
0.00) 14.29b (
0.00) b 35.71 (
7.82) 35.71 (
23.47) 42.86 (
15.65) 57.14 (
0.00) 71.43c (
0.00) 1000 mg/L 42.86ab (
15.65) 42.86a (
15.65) 85.71d (
0.00) 42.86cd (
15.65) 35.71d (
7.82) a c ab 200 mg/L 5.43 (
0.08) 5.50 (
0.00) 5.52 400 mg/L 5.41a (
0.10) 5.59ab (
0.10) 5.55a (
0.10) bc (
0.17) 600 mg/L 5.66 800 mg/L 5.52ab (
0.31) 1000 mg/L 5.73c (
0.00) ab (
26.84) 200 mg/L 66.50 400 mg/L 77.00b (
7.67) a 5.59 ab (
0.10) (
0.00) b a 85.71b (
0.00) 800 mg/L a a Isolate O ac 5.59 ab (
0.10) 5.55a (
0.06) bcd 5.35a (
0.04) 5.36a (
0.05) 5.47 (
0.00) 5.75 5.93b (
0.62) 5.55a (
0.06) 5.61ad (
0.00) 5.54b (
0.05) 5.68ab (
0.24) 5.51ab (
0.01) 5.81c (
0.09) 5.59b (
0.15) a (
0.27) 5.39a (
0.08) 63.00 (
46.01) a 101.00 (
23.00) 68.00 (
5.48) 77.50a (
8.22) 57.00a (
39.44) 89.50a (
4.93) 70.50ac (
8.22) 91.00ac (
23.00) 600 mg/L 57.00 (
6.57) 57.50 (
16.98) 127.00 (
0.00) 18.00 (
3.29) 136.50b (
3.83) 800 mg/L 48.50a (
0.55) 50.50a (
44.37) 60.00c (
19.72) 78.00c (
0.00) 102.00c (
3.29) a a 54.00 (
19.72) 58.00 (
29.58) 124.00 (
2.19) 49.00 (
14.24) 50.00d (
15.34) 200 mg/L 237.35a (
216.63) 153.98a (
133.13) 847.55ac (
398.16) 305.10a (
53.88) 439.29a (
98.25) a b b 1000 mg/L b a b a ad b 183.27 (
62.15) 450.61a (
234.07) 400 mg/L 390.10 (
115.47) 355.31 (
351.88) 581.94 600 mg/L 146.94a (
12.74) 238.17a (
160.29) 2450.20b (
1569.39) 25.31c (
17.44) 1251.43b (
29.51) 800 mg/L 154.29a (
32.19) 304.90a (
317.90) 266.12a (
202.99) 319.59a (
68.41) 703.57c (
124.63) 1000 mg/L a 149.18 (
68.19) a 280.82 (
229.82) 1249.59 (
139.39) d cd (
98.59) c 62.45 (
11.18) 180.82d (
50.08) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 12 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX Concentration Table 7. Germinability of the sesame seeds at the different concentrations of the respective metabolites. Parameter Concentration Isolate K Isolate L Isolate M Isolate N Isolate O FPG 200 mg/L 96.43ab (
3.91) 85.71a (
0.00) 100.00a (
0.00) 100.00a (
0.00) 92.86a (
7.82) 400 mg/L 100.00a (
0.00) 85.71a (
0.00) 100.00a (
0.00) 100.00a (
0.00) 92.86a (
7.82) 600 mg/L 100.00a (
0.00) 92.86b (
7.82) 92.86b (
7.82) 100.00a (
0.00) 92.86a (
7.82) MGT GIX 800 mg/L 92.86 (
7.82) 100.00 (
0.00) 92.86 (
7.82) 92.86 (
7.82) 92.86a (
7.82) 1000 mg/L 92.86b (
7.82) 100.00c (
0.00) 100.00a (
0.00) 100.00a (
0.00) 100.00a (
0.00) a c a b a b ac 200 mg/L 5.20 (
0.00) 5.32 (
0.13) 5.30 (
0.04) 5.23 400 mg/L 5.20a (
0.08) 5.18b (
0.03) 5.38b (
0.04) 5.22a (
0.05) b c ab 600 mg/L 5.06 (
0.07) 5.03 (
0.03) 5.33 800 mg/L 5.14c (
0.01) 5.21b (
0.09) 5.37b (
0.06) ac (
0.03) 1000 mg/L 5.17 200 mg/L 168.25a (
7.39) a b (
0.01) c (
0.04) a 5.21ab (
0.06) 5.32b (
0.10) 5.25b (
0.02) bc 5.16 (
0.04) 5.30 138.00a (
10.95) 164.50a (
3.83) 171.50a (
3.83) (
0.04) 161.00a (
15.34) 175.00 (
7.67) 151.00 (
3.29) 157.50 (
3.83) 172.00 (
4.38) 164.50a (
3.83) 600 mg/L 189.00b (
7.67) 178.50c (
11.50) 150.50b (
11.50) 172.00a (
4.38) 161.00a (
7.67) 800 mg/L 168.00a (
15.34) 172.50c (
10.41) 147.00b (
7.67) 150.50b (
3.83) 157.50a (
11.50) 1000 mg/L 164.50 (
11.50) 182.00 (
0.00) 178.50 (
3.83) 164.50 (
3.83) 182.00b (
0.00) 200 mg/L 470.20a (
54.55) 377.14a (
59.02) 542.86ad (
10.95) 529.29a (
10.17) 437.96a (
39.79) b c a d a 5.13c (
0.00) 400 mg/L a a 5.17a (
0.11) 5.22 (
0.05) 5.13 (
0.00) b 5.21ab (
0.02) bc 400 mg/L 603.57 (
151.01) 380.20 (
3.35) 458.57 600 mg/L 682.14b (
66.51) 491.63bc (
137.49) 450.82c (
53.88) a ab 800 mg/L 470.00 (
17.21) 409.29 (
44.60) 1000 mg/L 437.86a (
85.29) 567.86c (
88.42) (
104.85) c c b 478.57 (
28.17) 433.16a (
37.22) 445.71b (
20.34) 455.51a (
45.61) b 407.96 (
25.71) 457.45 (
35.66) 432.76a (
22.02) 525.00d (
30.52) 450.71b (
69.64) 524.29b (
0.00) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 13 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX b Table 8. Germinability of the okra seeds at the different concentrations of the respective metabolites. Parameter FPG MGT GIX 200 mg/L Isolate K a 28.57 (
0.00) b Isolate L a 28.57 (
0.00) a 42.86 (
31.30) Isolate O a 28.57a (
0.00) a 42.86 (
15.65) 42.86 (
15.65) 50.00 (
7.82) 42.86 (
0.00) 42.86 (
15.65) 42.86b (
0.00) 600 mg/L 42.86b (
15.65) 64.29c (
7.82) 42.86a (
15.65) 28.57b (
0.00) 35.71c (
7.82) a a Isolate N 400 mg/L c b Isolate M ab 800 mg/L 7.14 (
7.82) 28.57 (
0.00) 28.57 1000 mg/L 50.00b (
7.82) 35.71a (
7.82) 14.29b (
0.00) a 64.29d (
7.82) 64.29d (
7.82) 71.43e (
0.00) 5.37 (
0.15) 5.37 (
0.15) 5.44 (
0.06) 5.26 (
0.17) 5.50a (
0.00) 400 mg/L 5.37a (
0.15) 5.43ab (
0.08) 5.41a (
0.10) 5.67a (
0.06) 5.50a (
0.00) 5.23 (
0.00) 5.54 (
0.05) 5.50 (
0.00) 5.86 (
0.15) 5.41b (
0.10) 800 mg/L 5.75b (
3.01) 5.37a (
0.15) 5.50a (
0.00) 5.75a (
3.01) 5.32c (
0.04) 1000 mg/L 5.27a (
0.05) 5.44ab (
0.23) 5.50a (
0.00) 5.50a (
0.00) 5.47ab (
0.09) 45.50 (
3.83) 45.50 (
3.83) 66.50 (
49.84) 74.00 (
33.96) 42.00a (
0.00) 400 mg/L 66.50b (
19.17) 77.00b (
15.34) 66.50a (
3.83) 57.00a (
23.00) 63.00b (
0.00) 73.50 (
26.84) 91.50 (
8.22) 63.00 (
23.00) 33.00 (
3.29) 56.00b (
15.34) 800 mg/L 10.50c (
11.50) 45.50a (
3.83) 42.00ab (
0.00) 10.50bc (
11.50) 105.00c (
15.34) 84.00 (
15.34) 53.00 (
4.38) 21.00 (
0.00) 94.50 (
11.50) 106.00c (
6.57) 200 mg/L 83.06a (
9.61) 106.94a (
20.12) 285.10a (
275.65) 217.96a (
124.30) 84.08a (
16.54) 400 mg/L b 181.02 (
105.74) 201.94 (
2.12) 253.16 600 mg/L 212.24b (
126.09) 410.41c (
160.74) 205.51ab (
109.32) 93.06ab (
15.20) 205.71a (
81.82) 800 mg/L 10.82a (
11.85) 66.73a (
14.53) 99.18bc (
21.01) 15.20b (
16.66) 479.39b (
223.78) 1000 mg/L 232.04 (
70.42) b 126.43 ab (
45.94) b b 1000 mg/L b a a a 600 mg/L b c a a 200 mg/L b a a a 600 mg/L a b a 7.14 (
7.82) 200 mg/L a a (
0.00) c c ab (
34.54) 29.69 (
7.49) c 174.08 ab c (
76.68) 621.53 (
274.87) 196.53a (
55.00) 589.80b (
40.24) Values are averages of six replicates. Values in parenthesis represent
standard deviations of means. Same and different superscripts on same column for a particular factor connote no significant difference and significant difference, respectively. K, L, M, N, and O represent Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). FPG = final percent germination, MGT = mean germination time, GIX = germination index, and VIX = vigor index. Page 14 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 VIX Concentration Table 9. Detected compounds in the metabolites. Peak # RT Compound detected Comp. wt% Metabolite from S. liquefaciens (OP830504) RT Compound detected Comp. wt% Metabolite from P. rettgeri (OP830491) 1 2.50 Methyl lactate 10.40 2.54 Methyl lactate 2.28 2 2.99 Cyclohexanol, 5-methyl-2-(1-methylethyl)- 3.93 8.00 Cyclohexanol, 5-methyl-2-(1-methylethyl)- 5.00 3 4.46 Pentanoic acid, 2-methylbutyl ester 1.38 12.48 Pentanoic acid, 2-methylbutyl ester 2.51 4 8.50 9,12-Octadecadienoic acid (Z,Z)- 17.50 14.23 9,12-Octadecadienoic acid (Z,Z)- 2.07 5 9.52 Tetradecanoic acid 1.57 16.60 Tetradecanoic acid 2.55 6 15.15 Dibutyl phthalate 1.39 17.58 Dibutyl phthalate 1.97 7 16.00 9-Octadecenoic acid (Z)-, methyl ester 3.97 25.00 n-Hexadecanoic acid 14.13 8 21.00 3,7,11,15-tetramethyl-2-hexadecen-1-ol 1.29 30.00 3,7,11,15-tetramethyl-2-hexadecen-1-ol 1.52 9 25.00 Octadecane 2.03 31.97 Octadecane 7.90 10 32.00 n-Hexadecanoic acid 13.38 35.51 9-Octadecenoic acid (Z)-, methyl ester 1.69 11 34.50 Phytol 5.96 36.25 Phytol 9.31 12 35.81 Oleic acid 11.48 37.62 Oleic acid 2.98 13 37.52 9,12-Octadecadienoic acid (Z,Z)- 17.01 39.94 9,12-Octadecadienoic acid (Z,Z)- 1.25 14 39.00 11,14,17-Eicosatrienoic acid, methyl ester 3.89 41.00 11,14,17-Eicosatrienoic acid, methyl ester 5.96 15 41.98 Squalene 2.51 41.50 Lupeol 7.74 16 44.50 Stigmasterol 1.83 Stigmasterol 15.00 44.23 β-Sitosterol 12.19 18 44.99 Squalene 2.78 Metabolite from S. liquefaciens (OP830503) Metabolite from P. rettgeri (OP830498) Page 15 of 34 1 7.00 Methyl lactate 1.90 8.02 Methyl lactate 1.20 2 14.23 Cyclohexanol, 5-methyl-2-(1-methylethyl)- 1.86 10.50 Cyclohexanol, 5-methyl-2-(1-methylethyl)- 5.73 3 15.98 Pentanoic acid, 2-methylbutyl ester 1.94 11.43 Pentanoic acid, 2-methylbutyl ester 2.31 4 18.80 9,12-Octadecadienoic acid (Z,Z)- 4.90 13.25 9,12-Octadecadienoic acid (Z,Z)- 6.05 5 20.50 Tetradecanoic acid 8.85 16.50 Tetradecanoic acid 6.26 6 23.11 n-Hexadecanoic acid 2.92 20.81 Dibutyl phthalate 3.61 7 24.06 Dibutyl phthalate 1.55 23.03 9-Octadecenoic acid (Z)-, methyl ester 2.93 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 43.50 17 Table 9. Continued Peak # RT Compound detected Comp. wt% RT Compound detected Comp. wt% 8 26.98 3,7,11,15-tetramethyl-2-hexadecen-1-ol 3.83 26.02 3,7,11,15-tetramethyl-2-hexadecen-1-ol 17.54 9 28.15 Octadecane 3.14 28.50 Octadecane 3.99 10 31.50 9-Octadecenoic acid (Z)-, methyl ester 1.90 32.50 n-Hexadecanoic acid 21.96 11 33.98 Phytol 1.96 35.71 Phytol 3.88 12 35.50 Oleic acid 29.23 37.20 Oleic acid 1.63 13 37.00 9,12-Octadecadienoic acid (Z,Z)- 2.62 37.80 9,12-Octadecadienoic acid (Z,Z)- 12.45 14 39.00 11,14,17-Eicosatrienoic acid, methyl ester 25.17 39.75 11,14,17-Eicosatrienoic acid, methyl ester 2.56 15 40.00 Squalene 4.37 40.75 Squalene 3.04 16 48.49 Stigmasterol 1.94 43.52 Stigmasterol 3.27 17 54.00 Lupeol 1.66 48.21 Stearyltrimethylammonium chloride 1.18 Metabolite from Bacillus cereus (OP830501) 7.61 Methyl lactate 4.06 2.03 2 14.13 Cyclohexanol, 5-methyl-2-(1-methylethyl)- 2.13 1.73 3 15.98 Pentanoic acid, 2-methylbutyl ester 2.67 1.87 4 18.80 9,12-Octadecadienoic acid (Z,Z)- 4.27 5.83 5 20.50 Tetradecanoic acid 15.00 16.24 6 23.11 n-Hexadecanoic acid 3.20 2.51 7 24.00 Dibutyl phthalate 4.03 2.55 8 26.98 3,7,11,15-tetramethyl-2-hexadecen-1-ol 4.04 3.29 9 28.15 Octadecane 2.16 1.82 10 31.50 9-Octadecenoic acid (Z)-, methyl ester 1.07 1.80 11 33.98 Phytol 1.25 1.52 12 35.50 Oleic acid 26.69 27.33 13 37.00 9,12-Octadecadienoic acid (Z,Z)- 5.03 4.10 14 39.00 11,14,17-Eicosatrienoic acid, methyl ester 19.22 20.63 15 40.00 Squalene 3.19 3.00 16 48.49 Stigmasterol 1.06 1.81 17 54.00 Lupeol 0.92 1.60 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Page 16 of 34 1 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 metabolite from P. rettgeri AYO-I, cyclohexanol, 5-methyl-2-(1-methylethyl)- (5.73%), 9,12-octadecadienoic acid (Z,Z)- (6.05%), tetradecanoic acid (6.26%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (17.54%), n-hexadecanoic acid (21.96%), and 9,12-octadecadienoic acid (Z,Z)- (12.45%) were the most dominant moieties (Table 9). Finally, in the metabolite from B. cereus AYO-L, tetradecanoic acid (15.00%), oleic acid (26.69%) and 11,14,17eicosatrienoic acid, and methyl ester (19.22%) were the most dominant species (Table 9). Discussion Seed germination is fundamental to plant development and affects productivity,34,35 and seeds that germinate vigorously produce better yields.36 Generally, for the cowpea seeds, the final germination percentage reached a significantly highest value at shorter priming durations (1, 2, or 3 h), then decreased afterward. However, for Metabolites N and O, all values were statistically the same or nearly so. With the soybean seeds, the final germination percentage increased with increasing priming duration until 1, 2, or 3 h, then decreasing afterward, even though this decrease was sometimes insignificant. Although microbial metabolites can promote germination,37–39 the result for cowpea and soybean could be ascribed to nutrient and electrolyte leakage at prolonged priming duration.40 Long priming durations can result in overimbibition,41 leading to swollen seeds that may not germinate. Microbial deterioration can occur in large endosperm seeds (soybean and cowpea) when primed for longer durations, especially when the seeds are sown immediately without drying, as in this study. The results of this study show that priming soybean and cowpea for shorter periods is sufficient for maximal germination values. Through the application of the traditional priming technique that involves re-drying the seeds, higher optimal priming durations of 6 hours (cowpea), 8 and 18 hours (soybean) were reported.42,43,44 In this study, seeds were planted directly without re-drying. Priming duration did not seem to significantly impact the final germination pattern of the sesame seeds, as high values were distributed without order at the different priming durations, and there was no significant difference between them. The sesame seeds did not become over-bloated as in cowpea and soybean. Sesame seeds are comparatively impervious and have a small endosperm. A high optimal priming duration (12 hours) was recorded by Tizazu et al.45 using the traditional priming method. There was also no order to the distribution of FGP values for okra, indicating that the priming duration did not significantly influence germination. This uneven germination pattern could be due to the hard seed coat of okra,46 which perhaps limited imbibition. This hard seed coat likely resulted in high maximal priming durations of 12 h47 and 48 h48 for okra. Insufficient imbibition can lead to germination delay,49 a situation that could have happened with a hardy seed such as okra in this study since the highest priming duration for this study was 5 hours, and it posted low final germination values. The effect of priming duration on mean germination time was negligible for all the crops, as there was no pattern in the distribution of values. All the values obtained “congregated’ around five (5) days. Hence, it does not make any agronomic sense to attach much importance to the significant results here. However, other researchers have reported better mean germination times in cucumber,50 tomato seeds,51 guava seeds,52 cowpea seeds.53 A high germination index shows that germinated seeds appeared faster. In the case of the cowpea seeds, there was a general decrease in values with increasing priming time for all isolates, and this decline was significant for some metabolites used. There was also a general but insignificant decline from lower to higher priming durations with the soybean seeds. However, priming time did not significantly affect the germination index of the sesame and okra seeds. Okra required a 48-hour priming duration for maximal germination index.48 The highest germination index was observed when soybean seeds were treated for 12 hours in PEG solution.54 Both studies utilized the traditional priming methods that involves seed re-drying. The vigor index reached its statistical highest at a lower priming duration in all the metabolites in the case of cowpea, then decreased afterward, and this same pattern was also obtained for the soybean and sesame seeds. Seeds can over-imbibe at longer priming durations, impeding germination. This can result in reduced seedling vigor index values since final germination percentage is a computational component of seedling vigor index. Over-imbibition is a potential priming pitfall for seeds with large endosperms, such as cowpeas and soybean. Six (6) hours was the best priming duration for biomass production in soybean,55 and 12 hours for the highest seedling vigor index in soybean by Sadeghi et al.54 There was generally no observable pattern in FPG for cowpea, soybean, sesame, and okra seeds with concentration. Erratic drops in values were observed for the okra seeds. Okra has irregular germination pattern.56 Similarly, germination was concentration-independent in the biopriming of canola seeds with varying concentrations of bacterial cell-free Page 17 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 supernatants of Devosia sp. (SL43).57 While increasing concentration did not impact germination negatively, it is usually not the case with chemical or hormonal priming where the impact of priming on germination and seedling parameters seems to always be concentration-dependent, with drastic effects at higher concentrations.58,59 Metabolite concentration did not produce an effect of considerable agronomic proportion on mean germination time in all the crops, as values clustered around five days. Concerning the germination index, there was no clear pattern for cowpea and, generally, for the soybean and okra seeds. For the metabolites from isolates L, M, and O used in the priming of the sesame seeds, there was a rare increase in GIX with increasing concentration, which was also significant. For the cowpea seeds, the seedling vigor index gradually peaked at lower concentration for all isolates, then decreased with increasing concentration, although the decreases were statistically insignificant at times. Similarly, the seedling vigor index peaked at a lower concentration, and then a significant decline was observed for the soybean seeds. Sesame recorded no clear pattern with increasing concentration; however, in the case of Isolate K, it peaked at a lower concentration, then decreased steadily afterward, and for Isolate L, it rose steadily and peaked significantly at the highest concentration. Mostly, there was no directional change in seedling vigor index with metabolite concentration for okra seeds treated in these metabolites. The hard seed coat of okra is responsible for the ambiguous response of okra to priming at different concentrations. The significantly highest value for okra was not limited to lower priming periods, which occurred either 3 or 4 h for virtually all the metabolite treatments. A high priming duration of 48 hours produced the best vigor index in okra.48 The seed coat of okra limits ample imbibition at short priming periods. The GC-MS analysis of the extracts detected the presence of several metabolites in the metabolome of each of them, some of which were common to all isolates. The compounds detected belong to different classes, such as, alkanes, alcohols, carboxylic acids, esters, and terpenes. The ability of alcohols such as 2,3-butanediol produced by Bacillus spp to promote the growth of Arabidopsis thaliana has been reported.60–62 Tetrahydrofuran-3-ol and 2-heptanone 2-ethyl-1-hexanol from Bacillus species can improve the growth of A. thaliana and tomato.63 Oleic acid was detected in the metabolome of some rhizobacteria.64 It was detected in all five strains in this study. n-hexadecanoic acid, a metabolite detected in the metabolome of two of the isolates in this study, and hexadecane can improve the growth of Vigna radiata.65 Conclusion This study revealed the dynamic of metabolite priming at different priming duration and metabolite concentration. The impact of priming duration in metabolite priming was revealed in this study. It showed that seeds with large endosperms can become over-bloated at longer priming durations, thereby impeding seed germination. Higher concentration was inhibitory to germination. Sesame and okra were the least affected seeds with regard to metabolite priming. However, the function of the various metabolites in the isolates sampled needs to be properly investigated to identify the bioactive metabolites responsible for growth promotion. The organisms themselves will have to be further studied to better understand the production of the bioactive metabolites. Data availability Figshare. Raw data on germinability parameters at different metabolite concentrations and priming duration in microbial metabolite. DOI: https://doi.org/10.6084/m9.figshare.23284865.v1.66 Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgements The authors are grateful to Afe Babalola University for providing the facilities for the study. Ethical approval The study did not involve humans or animals, hence ethical approval was not obtained from any ethics committee. The study proposal was however approved by the Board of the College of Postgraduate Studies, Afe Babalola University, Ado-Ekiti, Nigeria. Page 18 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 References 1. Alam AU, Ullah H, Himanshu SK, et al.: Seed priming enhances germination and morphological, physio-biochemical, and yield traits of cucumber under water-deficit stress. J. Soil Sci. Plant Nutr. 2023; 23: 3961–3968. Publisher Full Text 2. Mazhar MW, Ishtiaq M, Maqbool M, et al.: Seed priming with zinc oxide nanoparticles improves growth, osmolyte accumulation, antioxidant defence and yield quality of water-stressed mung bean plants. Arid. Land Res. Manag. 2023; 37(2): 222–246. Publisher Full Text 3. Sghayar S, Debez A, Lucchini G, et al.: Seed priming mitigates high salinity impact on germination of bread wheat (Triticum aestivum L.) by improving carbohydrate and protein mobilization. Plant Direct. 2023; 7(6): e497. Publisher Full Text 4. Shaffique S, Khan MA, Wani SH , et al.: Biopriming of maize seeds with a novel bacterial strain SH-6 to enhance drought tolerance in south Korea. Plants. 2022; 11(13): 1674. PubMed Abstract|Publisher Full Text|Free Full Text 5. Neha T, Mohapatra S, Sirhindi G, et al.: Seed priming with brassinolides improves growth and reinforces antioxidative defenses under normal and heat stress conditions in seedlings of Brassica juncea. Physiol. Plant. 2022; 174(6): e13814. PubMed Abstract|Publisher Full Text 6. Chunthaburee S, Sanitchon J, Pattanagul W, et al.: Alleviation of salt stress in seedlings of black glutinous rice by seed priming with spermidine and gibberellic acid. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2014; 42(2): 405–413. Publisher Full Text 7. Ibrahim EA: Seed priming to alleviate salinity stress in germinating seeds. J. Plant Physiol. 2016; 192: 38–46. Publisher Full Text 8. Forti C, Shankar A, Singh A, et al.: Hydropriming and biopriming improve Medicago truncatula seed germination and upregulate DNA repair and antioxidant genes. Genes. 2020; 11(3): 242. PubMed Abstract|Publisher Full Text|Free Full Text 20. Hu J, Xie XJ, Wang ZF, et al.: Sand priming improves alfalfa germination under high-salt concentration stress. Seed Sci. Technol. 2006; 34(1): 199–204. Publisher Full Text 21. Almeida OA, de Araujo NO, Mulato AT, et al.: Bacterial volatile organic compounds (VOCs) promote growth and induce metabolic changes in rice. Front. Plant Sci. 2023; 13: 1056082. PubMed Abstract|Publisher Full Text|Free Full Text 22. Bomfim CA, Coelho LG, Mendes IC, et al.: Secondary metabolites of Rhizobium tropici CIAT 899 added to Bradyrhizobium spp. inoculant promote soybean growth and increase yield. J. Soil Sci. Plant Nutri. 2021; 21(4): 3354–3366. Publisher Full Text 23. Choudhury A, Bordolui SK, Ray J: Optimizing priming concentration and duration of PEG 6000 for improving seed germination and vigour in chickpea (Cicer arietinum L.). Environ. Ecol. 2023; 41(3C): 1953–1959. Publisher Full Text 24. Ullah Z, Hassan I, Hafiz IA, et al.: Effect of different priming treatments on seed germination of sago palm (Cycas revoluta L.). World J. Microbiol. Biotechnol. 2020; 5(1): 1–3. 25. Aloui h, Souguir M, Hannachi C.: Determination of an optimal priming duration and concentration protocol for pepper seeds (Capsicum annuum L.). Acta Agriculturae Slovenica. 2014; 103(2): 213–221. 26. Sanders ER: Aseptic laboratory techniques: plating methods. JoVE (Journal of Visualized Experiments). 2012; 63: e3064. Publisher Full Text 27. William S, Feil H, Copeland A: Bacterial genomic DNA isolation using CTAB. Sigma. 2012; 50(6876). 28. Tamura K, Stecher G, Kumar S: MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021; 38(7): 3022–3027. Publisher Full Text 29. Akpor OB, Okonkwo MA, Ogunnusi TA, et al.: Production, characterization and growth inhibitory potential of metabolites produced by Pseudomonas and Bacillus species. Sci. Afr. 2022; 15: e01085. Publisher Full Text 9. Jisha KC, Vijayakumari K, Puthur JT: Seed priming for abiotic stress tolerance: an overview. Acta Physiologiae Plantarum. 2013; 35: 1381–1396. Publisher Full Text 30. 10. Robledo DA: Effects of halopriming on seed germination and seedling emergence of Capsicum frutescens. J. Bot. Res. 2020; 3(1): 114–118. Publisher Full Text Leggatt CW, Justice OL, Hay WD, et al.: Rules for testing seeds: association of official seed analysts. Proceedings of the Association of Official Seed Analysts. The Association of Official Seed Analysts 1949 Jan 1: 23–59. 31. 11. Nakkeeran S, Marimuthu T, Renukadevi P, et al.: Exploring the biogeographical diversity of Trichoderma for plant health. In Biocontrol agents and secondary metabolites. Woodhead Publishing; 2021 Jan 1; pp. 537–571. Publisher Full Text Ellis RH, Covell S, Roberts EH, et al.: The influence of temperature on seed germination rate in grain legumes: II. Intraspecific variation in chickpea (Cicer arietinum L.) at constant temperatures. J. Exp. Bot. 1986; 37(10): 1503–1515. Publisher Full Text 32. 12. Ali HM, Siddiqui MH, Al-Whaibi MH, et al.: Effect of proline and abscisic acid on the growth and physiological performance of Faba bean under water stress. Pak. J. Bot. 2013; 45(3): 933–940. Salehzade H, Shishvan MI, Ghiyasi M, et al.: Effect of seed priming on germination and seedling growth of wheat (Triticum aestivum L.) Research. J. Biol. Sci. 2009; 4(5): 629–631. 33. Abdul-Baki AA, Anderson JD: Vigor determination in soybean seed by multiple criteria 1. Crop Sci. 1973; 13(6): 630–633. Publisher Full Text 34. Shiade SR, Boelt B: Seed germination and seedling growth parameters in nine tall fescue varieties under salinity stress. Acta Agric. Scand. B Soil Plant Sci. 2020; 70(6): 485–494. Publisher Full Text 35. Afrin S, Tahjib-Ul-Arif M, Sakil MA, et al.: Hydrogen peroxide priming alleviates chilling stress in rice (Oryza sativa L.) by enhancing oxidant scavenging capacity. Fundam. Appl. Agric. 2019; 4(1): 713–722. Publisher Full Text 13. Wei LX, Lv BS, Li XW, et al.: Priming of rice (Oryza sativa L.) seedlings with abscisic acid enhances seedling survival, plant growth, and grain yield in saline-alkaline paddy fields. Field. Crop. Res. 2017; 203: 86–93. Publisher Full Text 14. Farahmandfar E, Shirvan MB, Sooran SA, et al.: Effect of seed priming on morphological and physiological parameters of fenugreek seedlings under salt stress. Int. J. Agric. Crop Sci. 2013; 5(8): 811. 15. Ulfat AN, Majid SA, Hameed A: Hormonal seed priming improves wheat (Triticum aestivum L.) field performance under drought and non-stress conditions. Pak. J. Bot. 2017; 49(4): 1239–1253. 36. 16. Dotto L, Silva VN: Beet seed priming with growth regulators. Semina: Ciências Agrárias. 2017; 38(4): 1785–1798. Publisher Full Text Pedrini S, Merritt DJ, Stevens J, et al.: Seed coating: science or marketing spin? Trends Plant Sci. 2017; 22(2): 106–116. PubMed Abstract|Publisher Full Text 37. 17. Heydariyan M, Basirani N, Sharifi-Rad M, et al.: Effect of seed priming on germination and seedling growth of the caper (Capparis Spinosa) under drought stress. Int. J. Adv. Biol. Biomed. Res. 2014; 2(8): 2381–2389. Hung R, Lee S, Rodriguez-Saona C, et al. : Common gas phase molecules from fungi affect seed germination and plant health in Arabidopsis thaliana. AMB Express. 2014; 4(1): 53–57. PubMed Abstract|Publisher Full Text|Free Full Text 38. Gfeller V, Huber M, Förster C, et al.: Root volatiles in plant–plant interactions I: High root sesquiterpene release is associated with increased germination and growth of plant neighbours. Plant Cell Environ. 2019; 42(6): 1950–1963. PubMed Abstract|Publisher Full Text|Free Full Text 39. Fincheira P, Parada M, Quiroz A: Volatile organic compounds stimulate plant growing and seed germination of Lactuca sativa. J. Soil Sci. Plant Nutr. 2017; 17(4): 8538–8567. 40. Pallaoro DS, Camili EC, Guimarães SC, et al. : Methods for priming maize seeds. J. Seed Sci. 2016; 38: 148–154. Publisher Full Text 18. Afzal I, Rehman HU, Naveed M, et al.: Recent advances in seed enhancements. New challenges in seed biology-basic and translational research driving seed technology. 2016: 47–74. Publisher Full Text 19. Basra SM, Farooq M, Tabassam R, et al.: Physiological and biochemical aspects of pre-sowing seed treatments in fine rice (Oryza sativa L.). Seed Sci. Technol. 2005; 33(3): 623–628. Publisher Full Text Page 19 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 41. Dekkers BJ, Costa MC, Maia J, et al.: Acquisition and loss of desiccation tolerance in seeds: from experimental model to biological relevance. Planta. 2015; 241: 563–577. PubMed Abstract|Publisher Full Text 42. Aminu MS, Ahmed AA, Bukar MA: Influence of seed hydro priming duration on growth and yield of soybean (Glycine max. L. Merr) in the Sudan Savannah. Fudma J. Sci. 2022; 6(4): 232–237. Publisher Full Text 43. 54. Sadeghi H, Khazaei F, Yari L, et al. : Effect of seed osmopriming on seed germination behavior and vigor of soybean (Glycine max L.). ARPN J. Agric. Biol. Sci. 2011; 6(1): 39–43. 55. Saeedipour S: Effect of phytohormone seed priming on germination and seedling growth of cowpea (Vigna sinensis L.) under different duration of treatment. Int. J. Biosci. 2013; 3(12): 187–192. Publisher Full Text Pradhan N, Moaharana RL, Ranasingh N, et al. : Effect of seed priming on different physiological parameters of Cowpea (Vigna unguiculata L. Walp) seeds collected from Western Odisha. Pharm. Innov. J. 2022; 11(6): 2338–2343. 56. Rahman I, Ali S, Adnan M, et al.: Effect of seed priming on growth parameters of Okra (Abelmoschus esculentus L.). Pure Appl. Biol. 2016; 5(1): 165–171. Publisher Full Text 44. Mehri S: Effect of seed priming on yield and yield components of soybean. Am.-Eurasian J. Agric. Environ. Sci. 2015; 15(3): 399–403. 57. 45. Tizazu Y, Ayalew D, Terefe G, et al.: Evaluation of seed priming and coating on germination and early seedling growth of sesame (Sesamum indicum L.) under laboratory condition at Gondar, Ethiopia. Cogent Food Agric. 2019; 5(1): 1609252. Publisher Full Text Shah A, Subramanian S, Smith DL: Seed priming with Devosia sp. cell-free supernatant (CFS) and citrus bioflavonoids enhance canola and soybean seed germination. Molecules. 2022; 27(11): 3410. PubMed Abstract|Publisher Full Text|Free Full Text 58. Dhakal P, Subedi R: Influence of mannitol priming on maize seeds under induced water stress. J. Agric. Crops. 2020; 6(3): 27–31. Publisher Full Text 59. Ebrahimi N, Kaboli SH: Effect of different times and KNO3 concentrations on Silybum marianum seedling enhancement. Journal of Medicinal Plants and By-product. 2020; 9(1): 51–58. 60. Ryu CM, Farag MA, Paré PW, et al.: Invisible signals from the underground: bacterial volatiles elicit plant growth promotion and induce systemic resistance. Plant Pathol. J. 2005; 21(1): 7–12. Publisher Full Text 61. Asari S, Matzén S, Petersen MA, et al.: Multiple effects of Bacillus amyloliquefaciens volatile compounds: plant growth promotion and growth inhibition of phytopathogens. FEMS Microbiol. Ecol. 2016; 92(6): fiw070. PubMed Abstract|Publisher Full Text 62. Rath M, Mitchell TR, Gold SE: Volatiles produced by Bacillus mojavensis RRC101 act as plant growth modulators and are strongly culture-dependent. Microbiol. Res. 2018; 208: 76–84. PubMed Abstract|Publisher Full Text 63. Jiang CH, Xie YS, Zhu K, et al.: Volatile organic compounds emitted by Bacillus sp. JC03 promote plant growth through the action of auxin and strigolactone. Plant Growth Regul. 2019; 87(2): 317–328. Publisher Full Text 64. Liu WW, Wei MU, Zhu BY, et al.: Antagonistic activities of volatiles from four strains of Bacillus spp. and Paenibacillus spp. against soil-borne plant pathogens. Agric. Sci. China. 2008; 7(9): 1104–1114. Publisher Full Text 65. Jishma P, Hussain N, Chellappan R, et al.: Strain-specific variation in plant growth promoting volatile organic compounds production by five different Pseudomonas spp. as confirmed by response of Vigna radiata seedlings. J. Appl. Microbiol. 2017; 123(1): 204–216. PubMed Abstract|Publisher Full Text 66. Akpor O: Raw data on germinability parameters at different metabolite concentrations and priming duration in microbial metabolite. Dataset. figshare. 2023. Publisher Full Text 46. Barupal S, Sharma R, Kumar M, et al.: Seed priming: A effective method for enhancing seed quality and plant stand establishment in okra (Abelmoschus esculentus L.). J. Pharm. Innov. 2022; 11(2): 1359–1364. 47. Kaur H, Chawla N, Pathak M: Effect of different seed priming treatments and priming duration on biochemical parameters and agronomic characters of okra (Abelmoschus esculentus L.). Int. J. Plant Physiol. Biochem. 2015; 7(1): 1–1. 48. Kujur AB, Lal GM: Effect of hydropriming and osmopriming on germination behaviour and vigor of soybean (Glycine max L.) seeds. Agric. Sci. Dig. 2015; 35(3): 207–210. Publisher Full Text 49. Gill PK, Sharma AD, Singh P, et al.: Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses. Plant Growth Regul. 2003; 40: 157–162. Publisher Full Text 50. Badu R, Malla S, Rawal S, et al.: Effect of seed priming on germination and seedling parameters of cucumber (Cucumis sativus L.) in Lamjung, Nepal. Turkish JAF Sci. 2022; 10(10): 1997–2000. Publisher Full Text 51. Santika P, Muhklisin I, Makama SD: Effect of aeration and KNO3 in seed priming on the germination of tomato (Solanum lycopersicum) Seeds. Agroteknika. 2022; 5(2): 151–160. Publisher Full Text 52. Sharma N, Sharma JR, Malik A, et al.: Effect of priming treatments on germination and seedling growth of artificially aged seed of guava (Psidium guajava). Indian J. Agric. Sci. 2022; 92(4): 516–520. Publisher Full Text 53. Arun MN, Bhanuprakash K, Hebbar SS, et al. : Effects of seed priming on biochemical parameters and seed germination in cowpea [Vigna unguiculata (L.) Walp]. Legum. Res. 2017; 40(3) Publisher Full Text Page 20 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Open Peer Review Current Peer Review Status: Version 3 Reviewer Report 10 July 2024 https://doi.org/10.5256/f1000research.167213.r285910 © 2024 Subramanian S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sowmyalakshmi Subramanian 1 2 Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada Comment 5: Statistical analysis Authors' response _ We are of the opinion that the alphabet separation method of statistical presentation is adequate. The addition of p-values for pairwise comparisons will make the results somewhat ambiguous, almost incomprehensible, and unsightly because it will require the inclusion of hundreds of p-values. One table alone will generate 90 p-values. Review comment - p-value is of importance in any statistical analysis and it needs to be included in the Tables. If not in the main text, it certainly has to be in the supplementary tables. It is not about how unsightly the tables look, but that they have a meaning to the data analyzed. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Plant-Microbe Interactions, Genomics, Transcriptomics, Proteomics, Metabolomics, Biostimulants, Biocontrol I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 19 Jul 2024 Oghenerobor B. Akpor Dear Reviewer, Thank you for the review, please find enclosed within this document our response to your comments. Page 21 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Comment: p-value is of importance in any statistical analysis and it needs to be included in the Tables. If not in the main text, it certainly has to be in the supplementary tables. It is not about how unsightly the tables look, but that they have a meaning to the data analyzed. Response: The p-vales have been included as tables in the supplementary files. Regards Competing Interests: No competing interests were disclosed. Version 2 Reviewer Report 23 May 2024 https://doi.org/10.5256/f1000research.160450.r274606 © 2024 Subramanian S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sowmyalakshmi Subramanian 1 Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada 3 Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada 2 The title should be changed – Seed priming is the term used for soaking seeds in water or any other soaking treatment to enhance germination. Change all the “steeping/steep” words to “priming”. Also, the work is about the germination potential of metabolites extracted from cell-free supernatant of bacteria and not just from the bacteria. This way, components of the medium in which the culture was grown will also be present in this metabolite extract. The authors have to justify clearly that the compounds/metabolites affecting the germination are from the bacteria and not from the medium. What growth medium was used to grow these bacteria? And was the growth medium also tested as a control? If water was used as control, in this case there should be two controls – one with just water for germination and the other with water as a seed prime. In fact, water as a seed prime works really well. If a treatment works better than seed primed water control, then seed priming makes sense. Kindly correct the SI units and the formatting. For example, 50℃ should be 50 ℃, 1000 mg L-1 etc. Be consistent with this type of formatting through out the text. The results section should have statistical analysis data associated with the germination data and with the p-values obtained by the analysis, to keep in accordance with a scientific publication. This part is lacking in this work, although Tables 1 (Cowpea), 2 (Soybean), 3 (Sesame) do represent alphabet separation but no p-values. Page 22 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests: No competing interests were disclosed. Reviewer Expertise: Plant-Microbe Interactions, Genomics, Transcriptomics, Proteomics, Metabolomics, Biostimulants, Biocontrol I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 28 May 2024 Oghenerobor B. Akpor Dear Reviewer, The responses to review comments are as follows: 1. Comment: The title should be changed – Seed priming is the term used for soaking seeds in water or any other soaking treatment to enhance germination. Response: The suggested correction has been made. 2. Comment: Change all the “steeping/steep” words to “priming”. Response: The necessary corrections have been made throughout the manuscript. 3. Comment: Also, the work is about the germination potential of metabolites extracted from cell-free supernatant of bacteria and not just from the bacteria. This way, components of the medium in which the culture was grown will also be present in this metabolite extract. The authors have to justify clearly that the compounds/metabolites affecting the germination are from the bacteria and not from the medium. What growth medium was used to grow these bacteria? And was the growth medium also tested as a control? If water Page 23 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 was used as control, in this case there should be two controls – one with just water for germination and the other with water as a seed prime. In fact, water as a seed prime works really well. If a treatment works better than seed primed water control, then seed priming makes sense. Response: Nutrient broth was used to grow the organisms. During preliminary investigation (data not reported) involving 75 isolates, two (2) controls were used. The controls were water and sterile nutrient broth. Elimination was based on the ability of an isolate to produce significantly higher vigor index than those obtained through both water and broth priming. It was this selection criteria that produced this group of 15 isolates used in this study. 4. Comment: Kindly correct the SI units and the formatting. For example, 50℃ should be 50 ℃, 1000 mg L-1 etc. Be consistent with this type of formatting through out the text. Response: Adequate attention has been paid to this and necessary corrections made. 5. Comment: The results section should have statistical analysis data associated with the germination data and with the p-values obtained by the analysis, to keep in accordance with a scientific publication. This part is lacking in this work, although Tables 1 (Cowpea), 2 (Soybean), 3 (Sesame) do represent alphabet separation but no p-values. Response: We are of the opinion that the alphabet separation method of statistical presentation is adequate. The addition of p-values for pairwise comparisons will make the results somewhat ambiguous, almost incomprehensible, and unsightly because it will require the inclusion of hundreds of p-values. One table alone will generate 90 p-values. Thanks. Competing Interests: No competing interests were disclosed. Version 1 Reviewer Report 06 October 2023 https://doi.org/10.5256/f1000research.150476.r201475 © 2023 Righini H. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hillary Righini 1 Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy 2 Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy Page 24 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 3 Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy 4 Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy Abstract First, why did the authors choose bacteria agents of human-infection for extraction of metabolite?? Could they represent risk also in agriculture for the environment and for the workers(in a view for future application)? This is an important issue that must be well argued and explained. The work consists of only a test on seed germination, so I think that materials and methods should be more and better explained. Then, the authors must pay attention to the language. The text is difficult to read and it does not flow well. In particular, the abstract is very difficult to read because of English language. A careful checking for spelling and grammatical is required throughout the text and often the sentences have no sense. Materials and methods lack of many important details. Here, I reported some suggestions, but MORE are needed. ABSTRACT ‘Vigorous germination and growth are linked to crop yield.’ This sentence should introduce better the scope of this work. In this way it doesn’t mean anything. ‘For concentration it was either a case of lower concentration being optimal or there was no detectable pattern with concentration.’ It is difficult to read and understand. Through out the manuscript, there are several sentence like this, very difficult to understand. I would like to underline that ‘a seed can be treated, not steeped’. So, seeds were treated by immersion/dipping/soaking for a period. MATERIALS AND METHODS ‘.. until when needed.’, it better ‘until use’. ‘The bacterial strains were isolated using the standard pour-plating procedure’, it needs a reference. ‘After isolation, distinct colonies were streaked on nutrient agar plates to obtain pure cultures,’. How was carried out the isolation? How did you identified the bacteria? Page 25 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 It is not clear the temporal succession of the methodological steps. Have you isolated the bacteria? And then identified? Or they were already isolated, identified and stored in your university?? ‘Prior to use, the respective seeds were subjected to viability tests.’ Change with ‘First, a test was carried out on seeds in order to assess their viability’. ‘Preliminary viability testing was carried out by soaking approximately 100 seeds from a lot in 200 mL of sterile distilled in a 400 mL beaker and allowed to stand for 2 min. ‘ I suggest to change with ‘Preliminary viability testing was carried out by soaking 100 seeds in 200 mL of sterile distilled in a beaker for 2 min’. ‘approximately’ is not a scientific term. ‘sterile distilled’ what? Water, compounds? It is only one ‘viability test’ that includes two steps: first step for excluding those seeds that floated, the second step for testing the germination percentage. This part should be explained better. ‘cotton wool’ was sterilized? Has the cotton been moistened? Which were the environmental conditions during the incubation for 7 days? Temperature (day/night), humidity. ‘known concentration’, which ones? ‘respective’ which ones? ‘under 1, 2, 3, 4, and 5 h’. Not correct. The right way is ‘FOR 1,2,3,4 or 5 hours.’ (For example, seeds were treated by immersion for 4 hours). Each formula needs a reference. It is not clear how the authors have weight the compounds to obtain 200 mg/L, 400 mg/L, 600 mg/L, 800 mg/L and 1000 mg/L. In materials and methods, there is no a statical paragraph. It is not clear how many repetitions. Where is the control? K, L, M, N, O: it is not clear their meaning. Moreover, in the results sometimes 'K, L, M, N, O' are reporte, sometimes the name of bacterial species. It is a confused way. ‘The bacterial strains were isolated using the standard pour-plating procedure’ explain the procedure and insert a reference for it.’ ‘from rhizospheres within Afe Babalola University environment’: rhizosphere of which plant? How was this environment managed? Was there any crop, are still any crops? Is it a resting soil? Page 26 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 ‘Afe Babalola University’ change with ‘Afe Babalola University (Ekiti, Nigeria)’ ‘All the seeds were sourced from local markets in Ado Ekiti, Ekiti State, Nigeria’. You must indicate the producer, the city, an the production and purchase date. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? No Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: Agriculture I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Author Response 20 Dec 2023 Oghenerobor B. Akpor Dear Reviewer, Please find enclosed our responses to your comments. First, why did the authors choose bacteria agents of human-infection for extraction of metabolite?? Could they represent risk also in agriculture for the environment and for the workers (in a view for future application)? This is an important issue that must be well argued and explained. Answer: Serratia is a genus of opportunistic pathogens of immunocompromised patients. The main pathogen in this genus is Serratia marcescens and Serratia liquefaciens are rarely implicated in infectious diseases. Providencia rettgeri is also a rare pathogen of humans and Page 27 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Bacillus cereus is a minor pathogen of foodborne illnesses. Moreover, strains of these organisms are common constituents of commercial preparations for plant growth promotion. This informed our decision to include them in this study. Nevertheless, the investigation of their pathogenicity will be carried out down the line in view of possible future application. Only then can their biosafety be ascertained. The work consists of only a test on seed germination, so I think that materials and methods should be more and better explained. Answer: The authors have gone to great lengths to expound the Material and Methods section. Then, the authors must pay attention to the language. The text is difficult to read and it does not flow well. In particular, the abstract is very difficult to read because of English language. Answer: The errors have been corrected. Significant improvements have been made to the composition and flow of the text. A careful checking for spelling and grammatical is required throughout the text and often the sentences have no sense. Answer: The authors have gone to great lengths to improve the grammar, style, and spelling in the text Materials and methods lack of many important details. Answer: Significant and important changes have been made. Here, I reported some suggestions, but MORE are needed. ABSTRACT ‘Vigorous germination and growth are linked to crop yield.’ This sentence should introduce better the scope of this work. In this way it doesn’t mean anything. Answer: Rectified. ‘For concentration it was either a case of lower concentration being optimal or there was no detectable pattern with concentration.’ It is difficult to read and understand. Through out the manuscript, there are several sentences like this, very difficult to understand. Answer: Corrected. I would like to underline that ‘a seed can be treated, not steeped’. So, seeds were treated by immersion/dipping/soaking for a period. Answer: The necessary has been made. Page 28 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 MATERIALS AND METHODS ‘.. until when needed.’, it better ‘until use’. Answer: Corrected. ‘The bacterial strains were isolated using the standard pour-plating procedure’, it needs a reference. Answer: Corrected. ‘After isolation, distinct colonies were streaked on nutrient agar plates to obtain pure cultures,’. How was carried out the isolation? How did you identified the bacteria? Answer: The requisite changes have been made. It is not clear the temporal succession of the methodological steps. Answer: Changes have been made. It now follows a logical succession. Have you isolated the bacteria? And then identified? Or they were already isolated, identified and stored in your university? Answer: They were isolated from a previous experiment separate from this study and stored on agar slants. They were identified through 16S rRNA. ‘Prior to use, the respective seeds were subjected to viability tests.’ Change with ‘First, a test was carried out on seeds in order to assess their viability’. Answer: The suggested correction has been made ‘Preliminary viability testing was carried out by soaking approximately 100 seeds from a lot in 200 mL of sterile distilled in a 400 mL beaker and allowed to stand for 2 min. ‘ I suggest to change with ‘Preliminary viability testing was carried out by soaking 100 seeds in 200 mL of sterile distilled in a beaker for 2 min’. Answer: The suggested correction has been made ‘approximately’ is not a scientific term. Answer: The suggested correction has been made ‘sterile distilled’ what? Water, compounds? Answer: Water. The necessary correction has been made It is only one ‘viability test’ that includes two steps: first step for excluding those seeds that floated, the second step for testing the germination percentage. This part should be explained better. Answer: The suggested correction has been made ‘cotton wool’ was sterilized? Page 29 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Answer: No. Has the cotton been moistened? Which were the environmental conditions during the incubation for 7 days? Temperature (day/night), humidity. Answer: Those were not measured. ‘known concentration’, which ones? Answer: This has been corrected ‘respective’ which ones? Answer: The 4 different seeds. It has been recast ‘under 1, 2, 3, 4, and 5 h’. Not correct. The right way is ‘FOR 1,2,3,4 or 5 hours.’ (For example, seeds were treated by immersion for 4 hours). Answer: The suggested correction has been made Each formula needs a reference. Answer: They were actually included in the original manuscript submitted. It is not clear how the authors have weight the compounds to obtain 200 mg/L, 400 mg/L, 600 mg/L, 800 mg/L and 1000 mg/L. Answer: Corrections made. In materials and methods, there is no a statical paragraph. It is not clear how many repetitions. Answer: The suggested correction has been made. Where is the control? Answer: No control because water is water, no concentration gradient. K, L, M, N, O: it is not clear their meaning. Moreover, in the results sometimes 'K, L, M, N, O' are reported, sometimes the name of bacterial species. It is a confused way. Answer: The full names were only used in the presentation of the results of metabolite characterization to ensure brevity, elsewhere in the Result section the letters were used. ‘The bacterial strains were isolated using the standard pour-plating procedure’ explain the procedure and insert a reference for it.’ Answer: Rectified ‘from rhizospheres within Afe Babalola University environment’: rhizosphere of which plant? How was this environment managed? Was there any crop, are still any crops? Is it a resting soil? Page 30 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Answer: 1. It’s a resting soil with various grasses. 2. The rhizospheres were not noted. 3. It was a managed grassland. ‘Afe Babalola University’ change with ‘Afe Babalola University (Ekiti, Nigeria)’ Answer: The suggested correction has been made ‘All the seeds were sourced from local markets in Ado Ekiti, Ekiti State, Nigeria’. You must indicate the producer, the city, an the production and purchase date. Answer: Unfortunately, the seeds were not labelled. But they were procured from a seed dealer who supplies farmers with fresh seeds. Nonetheless, in order to ascertain their viability, the germination test was carried out Competing Interests: The authors have no competing interest(s) Reviewer Report 04 September 2023 https://doi.org/10.5256/f1000research.150476.r193085 © 2023 Mitra D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Debasis Mitra 1 Department of Microbiology, Raiganj University, Raiganj, West Bengal, India Microbiology, Crop Production Division, International Rice Research Institute, Manila, Metro Manila, Philippines 3 Department of Microbiology, Raiganj University, Raiganj, West Bengal, India 4 Microbiology, Crop Production Division, International Rice Research Institute, Manila, Metro Manila, Philippines 5 Department of Microbiology, Raiganj University, Raiganj, West Bengal, India 6 Microbiology, Crop Production Division, International Rice Research Institute, Manila, Metro Manila, Philippines 7 Department of Microbiology, Raiganj University, Raiganj, West Bengal, India 8 Microbiology, Crop Production Division, International Rice Research Institute, Manila, Metro Manila, Philippines 2 The manuscript "Effects of steeping duration and concentration of metabolites from rhizosphere bacteria on germinability of cowpea (Vigna unguiculata), soybean (Glycine max), sesame (Sesamum indicum) and okra (Abelmoschus esculentus)" investigated a compelling topic to evaluate the impact of steeping duration and metabolite concentration on seed priming of five distinct crops, utilizing the metabolites of five bacterial isolates that were also characterized via Gas Chromatography- Page 31 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 Mass Spectrometry (GC-MS). The results indicated that longer steeping duration may have a negative effect on larger endosperm seeds, such as cowpea and soybean, and further research is needed to isolate and purify the active compounds for additional studies and practical applications. Despite the fact that this study has many positive aspects and is suitable for this journal's scope, however, the manuscript needs to be revised critically and some important details need to be addressed. Minor Comments: Correct all grammatical mistakes. Verify that every citation in the text is linked to an appropriate reference in the reference section. Ensure that every reference has a textual citation that corresponds to it. Major Comments: Revise the title, remove the crops scientific name from the title, and add all in abstract. ○ ○ ○ ○ The abstract is a too general statement that does not convey the objective of your study and highlights the major flaws in your discussion of findings. Add Gas Chromatography-Mass Spectrometry (GC-MS) in Keywords “The bacterial strains were isolated from rhizospheres within Afe Babalola University environment.” rewrite it and give proper isolation and identification details. ○ Metabolite extraction protocol must be explained in detail alone with GC-MS procedure. ○ “Ethical approval” put it in end. ○ NCBI accession numbers show in one place only, no need to write in every place. It's recommended to use it with the strain name or code. Example. OP830504 - Serratia liquefaciens AYO-O or AYO-O; OP830503 - Serratia liquefaciens AYO-N or AYO-N; OP830491 - Providencia rettgeri AYO-B /AYO-B.... ○ The conclusion is poorly written and lacks properly investigated findings and discussion regarding the role of metabolites in crop growth and development. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Page 32 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: Plant Microbe Interactions, Soil Microbiology,Arbuscular Mycorrhizal Fungi, Biocontrol, Environmental Microbiology, Strigolactone I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Author Response 20 Dec 2023 Oghenerobor B. Akpor Dear Reviewer, Please find enclosed within this document our response to your comments. Minor comments: Correct all grammatical mistakes. Verify that every citation in the text is linked to an appropriate reference in the reference section. Ensure that every reference has a textual citation that corresponds to it. Response: Adequate attention has been paid to the grammar, citation, and referencing. Major Comments: Revise the title, remove the crops scientific name from the title, and add all in abstract. Response: This has been attended to. The abstract is a too general statement that does not convey the objective of your study and highlights the major flaws in your discussion of findings. Response: Rectified Add Gas Chromatography-Mass Spectrometry (GC-MS) in Keywords ○ ○ ○ Response: Rectified “The bacterial strains were isolated from rhizospheres within Afe Babalola University environment.” rewrite it and give proper isolation and identification details. Response: Corrected Metabolite extraction protocol must be explained in detail alone with GC-MS procedure. Response: Revised “Ethical approval” put it in end. ○ ○ ○ Response: The stipulated correction has been made NCBI accession numbers show in one place only, no need to write in every place. It's ○ Page 33 of 34 F1000Research 2024, 12:781 Last updated: 23 JUL 2024 recommended to use it with the strain name or code. Example. OP830504 - Serratia liquefaciens AYO-O or AYO-O; OP830503 - Serratia liquefaciens AYO-N or AYO-N; OP830491 - Providencia rettgeri AYO-B /AYO-B.... Response: Corrections have been made The conclusion is poorly written and lacks properly investigated findings and discussion regarding the role of metabolites in crop growth and development. Response: It has been reworked ○ Competing Interests: The authors have no competing interest(s) The benefits of publishing with F1000Research: • Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com Page 34 of 34