F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.137322.1 Research Article Articles 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)

[version 1; peer review: 1 approved with reservations, 1 not approved]

 Akpor Oghenerobor B. Conceptualization Formal Analysis Investigation Methodology Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4256-1549 a 1 Ajinde Ayotunde Formal Analysis Investigation Methodology Resources Validation Writing – Original Draft Preparation 1 Ogunnusi Tolulope A. Supervision Writing – Original Draft Preparation Writing – Review & Editing 1 Department of Biological Sciences, Afe Babalola University, Ado Ekiti, Ekiti, 360102, Nigeria akporob@abuad.edu.ng

No competing interests were disclosed.

 5 7 2023 2023 12 781 28 6 2023 Copyright: © 2023 Akpor OB et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Vigorous germination and growth are linked to crop yield. This study was carried out to assess the effects of steeping duration and metabolite concentration on priming of 5 different crops, using the metabolites of five (5) bacterial isolates that were also characterized through Gas Chromatography-Mass Spectrometry (GC-MS). The crop seeds were steeped in cold-extracted metabolites of the 5 isolates for a known period (1, 2, 3, 4, and 5 h) and then also in different metabolites concentrations for a known duration determined as optimal in the first experiment. Characterization of cold-extracted metabolites was also carried out using GCMS. The results of this study revealed that steeping cowpea and soybean for longer durations (< 3 h) could be inhibitory to growth and development. For concentration, it was either a case of lower concentration being optimal or there was no detectable pattern with concentration. The metabolites of the different isolates revealed the present of some common molecules, and some of the GCMS-identified metabolites (e.g., Hexadecanoic acid) have been shown to possess growth promotion properties in other studies. This study highlights that large endosperm seeds such as cowpea and soybean are more prone to the negative effects of steeping for longer durations, and further experiments should be carried out to isolate and purify the bioactive moieties for further studies and onward application.

 Germinability rhizobacteria germinability seed vigor The author(s) declared that no grants were involved in supporting this work. Introduction

Seeds are critical inputs in agricultural production. However, poor crop stand, a major production constraint in the developing world 1 can result from low-quality seeds and unfavorable edaphic and abiotic factors. This indicates that effort must be made to ensure that seeds are properly prepared, so that their germination and growth and crop yield are not negatively impacted. To this end, seed priming has been proposed and used. The beneficial effects of seed priming on a variety of crops have been confirmed. 2 – 5 Although, priming, as a plant growth enhancement technique, can be applied at numerous stages in the developmental cycle of a plant, it is typically utilized on seeds for reasons of practicality and simplicity.

Priming usually involves soaking seeds in a solution to kickstart various pre-germinative activities, 6 , 7 and it typically involves re-drying the seed before plant. 8 Several methods of priming that have been shown to enhance the agro-morphic parameters of various crops exist. Hydropriming involves steeping the seeds in water 9 ; osmopriming involves the use of an osmoticum 10 ; halopriming involves soaking in salt solutions 11 ; solid-matrix priming involves priming on a solid material 12 ; and hormonal priming, which involves the use of plant growth regulators such as abscisic acid (ABA), 13 , 14 gibberellic acids (GAs), 6 or salicylic acid (SA). 15 – 17 Biopriming involving the use of microbial products is a relatively new priming strategy that comes with the advantage of environmental friendliness and may be also be less expensive than most of the priming methods available.

Seed priming has been shown to enhance germination, reduce germination time, and improve seedling vigor. 18 Priming also increase the resistance of seeds to environmental stress. 19 Positive priming effects have been reported for many crops. 20 – 25

Although, the exact mechanism of priming is not well-understood, it is generally understood to involve specific physiological and biochemical reactions. 26 , 27 The synchronous germination and high germination rate seen as a result of seed priming is attributable to metabolic repair that occur during the priming process, 28 increase in the concentration of germination-promoting metabolites, 26 and osmotic fine-tuning. 29 Also, seed priming leads to an increase in the concentration of α-amylase, an enzyme that play huge role in starch metabolism, in order to meet the energy demand of the growing embryo. 28

Optimal priming duration is plant-specific due to seed structures that are unique morphologically and physiologically. Since bacteria secret substances that can promote the growth of plants, 30 the determination of effective concentration is also key towards the development of the wholesome application of these substances. Optimal concentrations of chemicals used in priming have been determined. 31 , 32

However, while there is ample amount of research works existing on other priming methods, microbial priming is less-well research and the optimization of priming parameters is even less so. Hence, we set out to understand the impact of steeping duration and metabolite concentration on the growth promotion activity of secondary metabolites on selected crops (cowpea, soybean, sesame, and okra) vis-a-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 huge economic importance in the tropical region where they are a vital source of dietary requirements. Therefore, there is the need to boost their production and seed priming is a veritable tool to achieving this end.

 Methods Test metabolites

The metabolites used for the study were extracted from cultures of Serratia liquefaciens, (OP830504), S. liquefaciens (OP830503), Providencia rettgeri (OP830491), P. rettgeri (OP830498), and Bacillus cereus (OP830501). The bacterial strains were isolated from rhizospheres within Afe Babalola University environment.

The bacterial strains were isolated using the standard pour-plating procedure. After isolation, distinct colonies were streaked on nutrient agar plates to obtain pure cultures, after which they were stored on agar slants at 4 °C±2 °C until when needed.

For metabolite extraction, the cold extraction method as reported by Ref. 33 was adopted. The respective extracts were put in clean sterile universal bottles and stored 4 °C±2 °C until when needed.

Characterization of the metabolites was carried using gas chromatography mass-spectroscopy procedure. The equipment (Varian 3800/4000) was equipped with an Agilent splitter split/splitless BP5 (30 m × 0.25 mm × 0.25 microns) capillary column with nitrogen used as a gas carrier.

 Test seeds

The seeds used for the study were cowpea ( Vigna unguiculata), soy-bean ( Glycine max), sorghum ( Sorghum bicolor), sesame ( Sesamum indicum), and okra ( Abelmoschus esculentus). All the seeds were sourced from local markets in Ado-Ekiti, Ekiti State, Nigeria.

Prior to use, the respective seeds were subjected to viability tests. 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. Seeds that floated were discarded and considered non-viable while those that settled at the bottom of the beaker were subjected to further viability testing. Further viability testing of the seeds was carried out by planting approximately seven seeds in transparent plastic containers that contained absorbent cotton wool as blotters in triplicates and incubating under fluorescent light for 7 d. Seed lots that showed average percent germination of at least 65% were considered as viable and used for germinability studies.

 Germinability studies

Germinability experiments were carried out by investigating the effects of steeping duration and metabolite concentration on the seeds.

The effect of steeping duration of seeds in the respective metabolites on germinability was carried out under 1, 2, 3, 4, and 5 h. The respective seeds from the viable lots were steeped in a known concentration of metabolite and allowed to stand for 5 h duration. Every one hour, for a 5 h duration, approximately seven seeds were withdrawn and planted in transparent plastic cups and incubated for 7 d. At the expiration of incubation, final percent germination, mean germination time, germination index and vigor index were estimated as follows:

Final germination % FGP = total number of germinated seeds total number of seeds sown × 100 % 34

Mean germination time MGT = ∑ fx f 35

Where f is the number of seeds germinated on day x

Germination index GIX = 8 × N 1 + 7 × N 2 + 6 × N 3 + … + 1 × N 8 36

Where N 1 , N 2 , N 3 … N 8 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 height 37

With respect to effect of metabolite concentration, 200 mg/L, 400 mg/L, 600 mg/L, 800 mg/L and 1000 mg/L were used for the study. The seeds were steeped in the respective metabolite concentrations and allowed to stand for the optimal steeping time obtained in the first experiment before planting and incubation. At the expiration of the 7 day incubation period, final percent germination, mean germination time, germination index and vigor index were estimated were estimated.

 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.

 Results Effect of steeping duration in metabolites

Generally, final percent germination of the cowpea seeds showed significantly highest and lowest values in setups steeped for 2 and 5 h, 1 and 5 h, 1, 2, and 3 and 4 h in metabolites from Isolates K, L and M, respectively. There was no significantly difference between final percent germination at the different steeping durations for seeds treated in metabolite from Isolate N. In addition, seeds treated with metabolite from Isolate O showed significantly lowest final percent germination at 3 and 5 h. With respect to mean germination time, significantly lowest values were recorded for seeds steeped 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).

Germinability of the cowpea seeds at the different steeping durations in the respective metabolites.
Parameter Time Isolate K Isolate L Isolate M Isolate N Isolate O
FPG 1 h 71.43 a (±0.00) 78.57 a (±7.82) 85.71 a (±0.00) 64.29 a (±7.82) 78.57 a (±7.82)
2 h 85.71 b (±15.65) 64.29 b (±7.82) 71.43 ac (±15.65) 78.57 a (±7.82) 78.57 a (±7.82)
3 h 71.43 a (±0.00) 42.86 c (±15.65) 71.43 ac (±31.30) 71.43 a (±0.00) 50.00 b (±23.47)
4 h 71.43 a (±0.00) 57.14 b (±0.00) 50.00 b (±7.82) 78.57 a (±23.47) 78.57 a (±7.82)
5 h 54.29 c (±15.65) 21.43 d (±7.82) 64.29 bc (±7.82) 78.57 a (±7.82) 64.29 ab (±23.47)
MGT 1 h 5.14 a (±0.05) 5.45 a (±0.25) 5.04 a (±0.04) 5.14 a (±0.06) 5.24 a (±0.06)
2 h 5.51 b (±0.15) 5.37 ab (±0.01) 5.34 b (±0.02) 5.36 b (±0.08) 5.25 a (±0.17)
3 h 5.32 c (±0.04) 5.23 b (±0.00) 5.39 b (±0.06) 5.40 b (±0.17) 5.40 b (±0.16)
4 h 5.43 b (±0.08) 5.11 c (±0.00) 5.41 b (±0.10) 5.21 a (±0.11) 5.25 a (±0.07)
5 h 5.38 bc (±0.08) 5.25 bc (±0.27) 5.57 c (±0.07) 5.24 a (±0.06) 5.45 b (±0.15)
GIX 1 h 129.50 a (±3.83) 120.00 a (±29.58) 164.50 a (±3.83) 116.00 ab (±18.62) 133.50 a (±8.22)
2 h 125.00 a (±33.96) 101.50 a (±11.50) 115.50 b (±26.84) 124.00 ab (±18.62) 133.00 a (±0.00)
3 h 116.00 a (±3.29) 73.50 b (±26.84) 112.50 b (±53.13) 110.00 b (±10.95) 79.00 b (±44.91)
4 h 109.00 a (±4.38) 105.00 a (±0.00) 77.00 c (±7.67) 133.50 a (±31.22) 131.00 a (±9.86)
5 h 84.40 b (±19.72) 35.00 c (±7.67) 89.50 bc (±16.98) 133.50 a (±8.22) 93.00 b (±25.20)
VIX 1 h 818.88 a (±69.86) 862.35 a (±163.31) 1578.37 a (±104.63) 783.78 a (±215.62) 943.06 ab (±2.46)
2 h 1212.14 b (±240.22) 575.61 b (±130.67) 697.76 bc (±202.77) 1390.10 b (±258.55) 1064.49 a (±77.35)
3 h 788.78 a (±32.42) 323.67 c (±239.21) 888.57 c (±625.97) 700.51 a (±100.04) 608.47 b (±535.09)
4 h 877.55 a (±125.19) 507.35 b (±66.62) 343.98 b (±156.16) 1305.20 b (±800.23) 556.43 c (±266.37)
5 h 434.12 c (±247.37) 37.55 d (±29.51) 353.88 b (±68.41) 999.18 ab (±229.37) 466.53 c (±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.

In the case of the soybean seeds, final percent germination of seeds showed the highest values at 2 h steeping duration when treated in the respective metabolites, apart from those treated in Isolate O, where 1 h steeping duration was observed to show highest values. Furthermore, significantly lowest mean germination times were recorded in seeds steeped for 1, 2, and 3 h, 1 and 2 h, 1, 4, and 5 h in metabolites from isolates L, M and N, respectively, and 2 h in metabolites from isolate K and 1, 3, and 4 h in metabolites from isolate O. However, significantly highest germination index was observed in 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).

Germinability of the soybean seeds at the different steeping durations in the respective metabolites.
Parameter Time Isolate K Isolate L Isolate M Isolate N Isolate O
FPG 1 h 64.29 a (±7.82) 50.00 ad (±7.82) 57.14 ac (±15.65) 50.00 a (±7.82) 85.71 a (±0.00)
2 h 71.43 a (±15.65) 64.29 b (±7.82) 71.43 b (±0.00) 78.57 b (±7.82) 57.14 bc (±0.00)
3 h 42.86 b (±15.65) 42.86 a (±0.00) 71.43 b (±0.00) 71.43 b (±15.65) 50.00 b (±7.82)
4 h 57.14 ab (±15.65) 57.14 bd (±0.00) 50.00 a (±7.82) 57.14 a (±0.00) 50.00 b (±7.82)
5 h 57.14 ab (±0.00) 50.00 ad (±7.82) 64.29 bc (±7.82) 50.00 a (±7.82) 64.29 c (±7.82)
MGT 1 h 5.37 a (±0.01) 5.34 a (±0.02) 5.31 a (±0.14) 5.50 a (±0.25) 5.51 ab (±0.21)
2 h 5.27 b (±0.05) 5.44 ac (±0.06) 5.37 a (±0.10) 5.69 b (±0.10) 5.61 a (±0.12)
3 h 5.67 c (±0.06) 5.41 ac (±0.10) 5.53 b (±0.15) 5.72 b (±0.15) 5.34 b (±0.12)
4 h 5.48 d (±0.02) 5.65 b (±0.04) 5.55 b (±0.06) 5.61 ab (±0.12) 5.52 ab (±0.23)
5 h 5.50 e (±0.00) 5.50 c (±0.20) 5.60 b (±0.01) 5.50 a (±0.00) 5.54 a (±0.08)
GIX 1 h 101.50 ab (±11.50) 80.50 a (±11.50) 95.00 ac (±33.96) 71.50 a (±0.55) 124.50 a (±16.98)
2 h 119.00 b (±23.00) 98.00 b (±15.34) 112.50 a (±7.12) 99.00 b (±4.38) 78.00 bc (±6.57)
3 h 57.00 c (±23.00) 66.50 c (±3.83) 101.00 ac (±12.05) 91.00 bc (±27.39) 81.00 bd (±18.62)
4 h 84.50 a (±23.55) 75.50 ac (±2.74) 70.50 b (±8.22) 78.00 ac (±6.57) 71.00 b (±1.10)
5 h 84.00 a (±0.00) 71.50 ac (±1.64) 88.50 c (±11.50) 73.50 a (±11.50) 92.00 cd (±15.34)
VIX 1 h 490.00 a (±60.14) 326.63 a (±28.50) 607.45 a (±339.48) 241.73 ad (±85.74) 1027.96 a (±329.30)
2 h 926.73 b (±416.05) 593.67 b (±120.95) 845.41 b (±39.68) 897.14 b (±367.98) 494.69 b (±66.17)
3 h 243.67 a (±223.11) 252.24 a (±91.88) 805.10 ab (±60.36) 711.63 bc (±437.06) 418.88 b (±196.62)
4 h 474.59 a (±374.35) 305.71 a (±75.56) 422.45 a (±135.48) 556.73 c (±15.20) 243.67 c (±44.26)
5 h 346.94 a (±76.01) 279.08 a (±85.06) 440.00 a (±110.89) 232.96 d (±115.02) 494.59 b (±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.

For the sesame seeds, remarkably high final percent germination values (>78%) were observed in the respective treatments, irrespective of the steeping duration. However, significantly lowest mean germination time was observed for seeds steeped 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 steeped 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 seedling vigor index, seeds steeped 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 significantly highest values ( Table 3).

Germinability of the sesame seeds at the different steeping durations in the respective metabolites.
Parameter Time Isolate K Isolate L Isolate M Isolate N Isolate O
FPG 1 h 92.86 a (±7.82) 100.00 a (±0.00) 100.00 a (±0.00) 92.86 a (±7.82) 100.00 a (±0.00)
2 h 100.00 a (±0.00) 100.00 a (±0.00) 100.00 a (±0.00) 100.00 b (±0.00) 100.00 a (±0.00)
3 h 92.86 a (±7.82) 85.71 b (±0.00) 92.86 b (±7.82) 100.00 b (±0.00) 92.86 b (±7.82)
4 h 100.00 a (±0.00) 78.57 c (±7.82) 100.00 a (±0.00) 100.00 b (±0.00) 92.86 b (±7.82)
5 h 92.86 a (±7.82) 100.00 a (±0.00) 92.86 b (±7.82) 100.00 b (±0.00) 100.00 a (±0.00)
MGT 1 h 5.33 ab (±0.14) 5.42 a (±0.00) 5.30 a (±0.04) 5.00 a (±0.00) 5.16 a (±0.04)
2 h 5.27 ab (±0.08) 5.20 b (±0.00) 5.13 b (±0.15) 5.42 a (±0.00) 5.38 b (±0.04)
3 h 5.25 a (±0.02) 5.27 c (±0.05) 5.37 a (±0.06) 5.34 a (±0.00) 5.37 b (±0.04)
4 h 5.34 b (±0.00) 5.35 d (±0.04) 5.34 a (±0.08) 5.34 a (±0.00) 5.53 c (±0.03)
5 h 5.33 ab (±0.01) 5.34 d (±0.08) 5.50 c (±0.00) 5.42 a (±0.00) 5.38 b (±0.04)
GIX 1 h 148.50 a (±0.55) 154.00 a (±0.00) 164.50 a (±3.83) 182.00 a (±15.34) 178.50 a (±3.83)
2 h 168.00 b (±7.67) 175.00 b (±0.00) 182.00 b (±15.34) 154.00 b (±0.00) 157.50 b (±3.83)
3 h 157.50 a (±11.50) 143.50 c (±3.83) 147.00 c (±7.67) 161.00 b (±0.00) 147.00 c (±15.34)
4 h 161.00 b (±0.00) 126.00 d (±15.34) 161.00 a (±7.67) 161.00 b (±0.00) 133.50 d (±8.22)
5 h 150.50 a (±11.50) 161.00 a (±7.67) 136.50 c (±11.50) 154.00 b (±0.00) 157.50 b (±3.83)
VIX 1 h 528.67 a (±111.00) 800.00 a (±178.40) 952.86 a (±7.82) 666.53 a (±93.00) 756.43 a (±71.20)
2 h 711.43 b (±12.52) 660.00 b (±115.80) 887.86 a (±2.35) 506.43 bd (±44.60) 877.86 b (±28.95)
3 h 678.16 b (±33.31) 615.31 b (±2.01) 721.63 b (±42.48) 829.29 c (±97.81) 791.43 ab (±78.25)
4 h 592.86 a (±31.30) 339.18 c (±22.80) 582.86 c (±50.08) 553.57 b (±44.60) 447.55 c (±138.83)
5 h 511.73 a (±95.12) 455.71 d (±0.00) 461.73 d (±106.08) 450.71 d (±22.69) 496.43 c (±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.

When okra seeds were steeped in the metabolites for varying durations, final percent germination 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 between final germination of the seeds treated with metabolite from isolate M at the different steeping durations. Also, mean germination time showed no significant difference at the different steeping durations for seeds treated with metabolites from isolates K, M and O. However, significantly lowest mean germination times were observed for seeds steeped for 2 and 5 h and 3-5 h, when treated with metabolites from isolates L and N, respectively. Furthermore, seeds steeped for 3 h showed significantly highest germination and vigor indices when treated with metabolites from isolates K respectively. Significantly highest germination index values were observed for seeds steeped 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).

Germinability of the okra seeds at the different steeping durations in the respective metabolites.
Parameter Time Isolate K Isolate L Isolate M Isolate N Isolate O
FPG 1 h 28.57 a (±15.65) 57.14 ad (±15.65) 85.71 a (±0.00) 85.71 a (±0.00) 71.43 ab (±15.65)
2 h 71.43 b (±0.00) 71.43 ab (±0.00) 78.57 a (±7.82) 64.29 b (±7.82) 71.43 ab (±15.65)
3 h 85.71 c (±0.00) 85.71 bc (±0.00) 78.57 a (±7.82) 71.43 bc (±0.00) 85.71 a (±15.65)
4 h 64.29 b (±7.82) 92.86 c (±7.82) 78.57 a (±7.82) 64.29 b (±23.47) 78.57 ab (±7.82)
5 h 71.43 b (±15.65) 50.00 d (±23.47) 78.57 a (±23.47) 78.57 ac (±7.82) 64.29 b (±7.82)
MGT 1 h 5.41 a (±0.10) 5.28 ac (±0.01) 5.30 a (±0.08) 5.47 a (±0.20) 5.22 a (±0.24)
2 h 5.33 a (±0.16) 5.14 bd (±0.05) 5.39 a (±0.33) 5.91 b (±0.06) 5.29 a (±0.23)
3 h 5.29 a (±0.07) 5.36 a (±0.02) 5.30 a (±0.13) 5.35 c (±0.04) 5.28 a (±0.30)
4 h 5.33 a (±0.11) 5.39 a (±0.06) 5.19 a (±0.01) 5.27 c (±0.05) 5.20 a (±0.05)
5 h 5.28 a (±0.31) 5.19 cd (±0.21) 5.38 a (±0.17) 5.34 c (±0.02) 5.35 a (±0.26)
GIX 1 h 45.50 a (±26.84) 93.00 a (±24.10) 140.50 a (±7.12) 124.00 a (±18.62) 120.00 ab (±8.76)
2 h 114.00 b (±13.15) 129.50 b (±3.83) 121.00 bc (±13.15) 71.00 b (±5.48) 122.50 ab (±42.17)
3 h 141.00 c (±6.57) 133.00 b (±1.10) 127.50 abd (±1.64) 113.00 a (±1.10) 139.00 ab (±1.10)
4 h 102.50 b (±4.93) 144.50 b (±18.07) 137.00 ab (±12.05) 108.50 a (±42.17) 137.00 a (±18.62)
5 h 114.50 b (±2.74) 84.00 a (±30.67) 120.00 cd (±24.10) 126.50 a (±14.79) 102.00 b (±3.29)
VIX 1 h 108.27 a (±92.67) 335.31 a (±154.70) 580.41 ac (±45.61) 743.27 a (±73.77) 488.98 ac (±140.40)
2 h 495.92 bd (±36.89) 557.65 b (±73.22) 539.29 ad (±126.87) 303.88 b (±9.17) 522.65 ac (±230.94)
3 h 869.39 c (±8.05) 836.33 c (±12.07) 643.88 ab (±133.02) 511.22 c (±4.47) 818.88 b (±292.31)
4 h 537.45 b (±112.12) 754.08 c (±109.77) 713.67 b (±61.93) 553.47 ac (±336.68) 710.20 ab (±140.84)
5 h 410.82 d (±171.02) 250.41 a (±222.00) 488.47 cd (±104.96) 559.90 ac (±100.27) 392.55 c (±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.

 Effect of concentration of metabolite

At the different concentrations of the respective metabolites, significantly lowest final precent germination values were observed for cowpea seeds treated with 800 mg/L of metabolites from isolates K, L, and N. Final percent germination of cowpea seeds treated with metabolites from isolates M and O did not differ significantly between the respective concentrations. Also, significantly lowest mean germination time was 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 s M), 200 – 800 mg/L (metabolite N), and 200-600 mg/L (metabolite O). Significantly highest germination index values were observed in 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).

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 71.43 ab (±0.00) 64.29 a (±23.47) 50.00 a (±7.82) 64.29 a (±23.47) 64.29 a (±7.82)
400 mg/L 57.14 ac (±31.30) 42.86 b (±0.00) 42.86 a (±0.00) 85.71 b (±0.00) 57.14 a (±46.95)
600 mg/L 85.71 b (±0.00) 85.71 c (±0.00) 57.14 a (±31.30) 85.71 b (±15.65) 78.57 a (±7.82)
800 mg/L 50.00 c (±7.82) 14.29 d (±0.00) 50.00 a (±7.82) 42.86 a (±0.00) 64.29 a (±7.82)
1000 mg/L 71.43 abc (±15.65) 64.29 a (±23.47) 57.14 a (±0.00) 78.57 ab (±23.47) 71.43 a (±15.65)
MGT 200 mg/L 5.32 a (±0.25) 5.67 a (±0.09) 5.61 a (±0.12) 5.45 a (±0.05) 5.37 ab (±0.01)
400 mg/L 5.62 b (±0.12) 5.38 bc (±0.07) 5.25 b (±0.27) 5.47 ab (±0.11) 5.28 a (±0.31)
600 mg/L 5.39 ab (±0.09) 5.47 b (±0.08) 5.54 a (±0.04) 5.47 ab (±0.05) 5.40 ac (±0.24)
800 mg/L 5.52 ab (±0.23) 5.25 c (±0.27) 5.69 a (±0.04) 5.41 a (±0.10) 5.52 bc (±0.07)
1000 mg/L 5.64 b (±0.35) 5.51 ab (±0.01) 5.55 a (±0.06) 5.56 b (±0.07) 5.75 d (±0.16)
GIX 200 mg/L 116.50 a (±18.07) 84.00 a (±35.05) 67.50 a (±4.93) 98.00 a (±38.34) 101.50 ab (±11.50)
400 mg/L 79.00 b (±47.10) 67.00 a (±3.29) 73.50 a (±11.50) 127.00 a (±7.67) 84.50 a (±61.89)
600 mg/L 133.50 a (±7.12) 127.00 b (±6.57) 81.00 a (±42.72) 125.50 a (±26.84) 120.50 b (±6.02)
800 mg/L 71.00 b (±1.10) 24.50 c (±3.83) 64.50 a (±8.22) 66.50 b (±3.83) 92.00 ab (±7.67)
1000 mg/L 88.50 b (±3.83) 92.50 a (±32.32) 81.00 a (±3.29) 109.50 a (±27.93) 85.50 a (±8.22)
VIX 200 mg/L 675.00 a (±123.52) 584.69 ab (±234.07) 318.98 a (±122.73) 741.12 a (±481.21) 765.92 a (±137.94)
400 mg/L 588.98 ac (±540.57) 308.57 ac (±42.92) 331.53 a (±147.89) 1279.59 b (±34.88) 1042.55 a (±1097.35)
600 mg/L 1100.20 b (±10.06) 918.37 b (±490.94) 719.18 b (±627.31) 1406.33 b (±612.78) 1065.82 a (±254.30)
800 mg/L 320.92 c (±27.61) 16.94 c (±0.22) 330.41 a (±73.55) 302.76 a (±66.73) 796.73 b (±137.71)
1000 mg/L 525.31 ac (±178.40) 744.49 ab (±618.37) 411.84 ab (±97.03) 1240.41 b (±443.99) 863.06 b (±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.

For the soybean seeds, significantly lowest final percent germination values were observed in treatments 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 from N), and 1000 mg/L (metabolite from isolate O). In the case of mean germination time, significantly lowest values were recorded in 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 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 seeds treated with metabolites from isolates K, M, N, and O, significantly highest seedling vigor index values were observed in seeds treated with 400 mg/L, 600 mg/L, 400 mg/L, and 600 mg/L, respectively ( Table 6).

Germinability of the soybean 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 42.86 ab (±15.65) 42.86 a (±31.30) 71.43 a (±15.65) 50.00 ac (±7.82) 50.00 a (±7.82)
400 mg/L 50.00 a (±7.82) 42.86 a (±31.30) 64.29 a (±7.82) 50.00 ad (±7.82) 57.14 a (±15.65)
600 mg/L 42.86 ab (±0.00) 42.86 a (±15.65) 85.71 b (±0.00) 14.29 b (±0.00) 85.71 b (±0.00)
800 mg/L 35.71 b (±7.82) 35.71 a (±23.47) 42.86 c (±15.65) 57.14 a (±0.00) 71.43 c (±0.00)
1000 mg/L 42.86 ab (±15.65) 42.86 a (±15.65) 85.71 d (±0.00) 42.86 cd (±15.65) 35.71 d (±7.82)
MGT 200 mg/L 5.43 a (±0.08) 5.50 a (±0.00) 5.52 ab (±0.00) 5.59 ab (±0.10) 5.39 a (±0.08)
400 mg/L 5.41 a (±0.10) 5.59 ab (±0.10) 5.55 a (±0.10) 5.55 a (±0.06) 5.35 a (±0.04)
600 mg/L 5.66 bc (±0.17) 5.59 ab (±0.10) 5.47 b (±0.00) 5.75 bcd (±0.27) 5.36 a (±0.05)
800 mg/L 5.52 ab (±0.31) 5.93 b (±0.62) 5.55 a (±0.06) 5.61 ad (±0.00) 5.54 b (±0.05)
1000 mg/L 5.73 c (±0.00) 5.68 ab (±0.24) 5.51 ab (±0.01) 5.81 c (±0.09) 5.59 b (±0.15)
GIX 200 mg/L 66.50 ab (±26.84) 63.00 a (±46.01) 101.00 a (±23.00) 68.00 a (±5.48) 77.50 a (±8.22)
400 mg/L 77.00 b (±7.67) 57.00 a (±39.44) 89.50 a (±4.93) 70.50 ac (±8.22) 91.00 ac (±23.00)
600 mg/L 57.00 a (±6.57) 57.50 a (±16.98) 127.00 b (±0.00) 18.00 b (±3.29) 136.50 b (±3.83)
800 mg/L 48.50 a (±0.55) 50.50 a (±44.37) 60.00 c (±19.72) 78.00 c (±0.00) 102.00 c (±3.29)
1000 mg/L 54.00 a (±19.72) 58.00 a (±29.58) 124.00 b (±2.19) 49.00 d (±14.24) 50.00 d (±15.34)
VIX 200 mg/L 237.35 a (±216.63) 153.98 a (±133.13) 847.55 ac (±398.16) 305.10 a (±53.88) 439.29 a (±98.25)
400 mg/L 390.10 b (±115.47) 355.31 a (±351.88) 581.94 ad (±139.39) 183.27 b (±62.15) 450.61 a (±234.07)
600 mg/L 146.94 a (±12.74) 238.17 a (±160.29) 2450.20 b (±1569.39) 25.31 c (±17.44) 1251.43 b (±29.51)
800 mg/L 154.29 a (±32.19) 304.90 a (±317.90) 266.12 a (±202.99) 319.59 a (±68.41) 703.57 c (±124.63)
1000 mg/L 149.18 a (±68.19) 280.82 a (±229.82) 1249.59 cd (±98.59) 62.45 c (±11.18) 180.82 d (±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.

In the case of the sesame seeds, remarkably high final germination (> 90 %) was observed at all steeping duration for all steeping 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).

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 96.43 ab (±3.91) 85.71 a (±0.00) 100.00 a (±0.00) 100.00 a (±0.00) 92.86 a (±7.82)
400 mg/L 100.00 a (±0.00) 85.71 a (±0.00) 100.00 a (±0.00) 100.00 a (±0.00) 92.86 a (±7.82)
600 mg/L 100.00 a (±0.00) 92.86 b (±7.82) 92.86 b (±7.82) 100.00 a (±0.00) 92.86 a (±7.82)
800 mg/L 92.86 b (±7.82) 100.00 c (±0.00) 92.86 b (±7.82) 92.86 b (±7.82) 92.86 a (±7.82)
1000 mg/L 92.86 b (±7.82) 100.00 c (±0.00) 100.00 a (±0.00) 100.00 a (±0.00) 100.00 a (±0.00)
MGT 200 mg/L 5.20 a (±0.00) 5.32 a (±0.13) 5.30 a (±0.04) 5.23 ac (±0.04) 5.21 ab (±0.02)
400 mg/L 5.20 a (±0.08) 5.18 b (±0.03) 5.38 b (±0.04) 5.22 a (±0.05) 5.17 a (±0.11)
600 mg/L 5.06 b (±0.07) 5.03 c (±0.03) 5.33 ab (±0.01) 5.22 a (±0.05) 5.21 ab (±0.06)
800 mg/L 5.14 c (±0.01) 5.21 b (±0.09) 5.37 b (±0.06) 5.32 b (±0.10) 5.25 b (±0.02)
1000 mg/L 5.17 ac (±0.03) 5.13 b (±0.00) 5.16 c (±0.04) 5.30 bc (±0.04) 5.13 c (±0.00)
GIX 200 mg/L 168.25 a (±7.39) 138.00 a (±10.95) 164.50 a (±3.83) 171.50 a (±3.83) 161.00 a (±15.34)
400 mg/L 175.00 a (±7.67) 151.00 b (±3.29) 157.50 a (±3.83) 172.00 a (±4.38) 164.50 a (±3.83)
600 mg/L 189.00 b (±7.67) 178.50 c (±11.50) 150.50 b (±11.50) 172.00 a (±4.38) 161.00 a (±7.67)
800 mg/L 168.00 a (±15.34) 172.50 c (±10.41) 147.00 b (±7.67) 150.50 b (±3.83) 157.50 a (±11.50)
1000 mg/L 164.50 a (±11.50) 182.00 c (±0.00) 178.50 d (±3.83) 164.50 c (±3.83) 182.00 b (±0.00)
VIX 200 mg/L 470.20 a (±54.55) 377.14 a (±59.02) 542.86 ad (±10.95) 529.29 a (±10.17) 437.96 a (±39.79)
400 mg/L 603.57 b (±151.01) 380.20 a (±3.35) 458.57 bc (±104.85) 478.57 b (±28.17) 433.16 a (±37.22)
600 mg/L 682.14 b (±66.51) 491.63 bc (±137.49) 450.82 c (±53.88) 445.71 b (±20.34) 455.51 a (±45.61)
800 mg/L 470.00 a (±17.21) 409.29 ab (±44.60) 407.96 c (±25.71) 457.45 b (±35.66) 432.76 a (±22.02)
1000 mg/L 437.86 a (±85.29) 567.86 c (±88.42) 525.00 d (±30.52) 450.71 b (±69.64) 524.29 b (±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.

Furthermore, final percent germination 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 metabolites from isolates M and N showed no significance difference at the different concentrations, while 800 mg/L and 200, 400, and 1000 mg/L of metabolites from isolates K and O showed significantly highest values, respectively. With respect to germination index, significantly highest values were recorded in 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, seeds treated in 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).

Germinability of the okra 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 28.57 a (±0.00) 28.57 a (±0.00) 42.86 a (±31.30) 42.86 a (±15.65) 28.57 a (±0.00)
400 mg/L 42.86 b (±15.65) 50.00 b (±7.82) 42.86 a (±0.00) 42.86 a (±15.65) 42.86 b (±0.00)
600 mg/L 42.86 b (±15.65) 64.29 c (±7.82) 42.86 a (±15.65) 28.57 b (±0.00) 35.71 c (±7.82)
800 mg/L 7.14 c (±7.82) 28.57 a (±0.00) 28.57 ab (±0.00) 7.14 c (±7.82) 64.29 d (±7.82)
1000 mg/L 50.00 b (±7.82) 35.71 a (±7.82) 14.29 b (±0.00) 64.29 d (±7.82) 71.43 e (±0.00)
MGT 200 mg/L 5.37 a (±0.15) 5.37 a (±0.15) 5.44 a (±0.06) 5.26 a (±0.17) 5.50 a (±0.00)
400 mg/L 5.37 a (±0.15) 5.43 ab (±0.08) 5.41 a (±0.10) 5.67 a (±0.06) 5.50 a (±0.00)
600 mg/L 5.23 a (±0.00) 5.54 b (±0.05) 5.50 a (±0.00) 5.86 a (±0.15) 5.41 b (±0.10)
800 mg/L 5.75 b (±3.01) 5.37 a (±0.15) 5.50 a (±0.00) 5.75a (±3.01) 5.32 c (±0.04)
1000 mg/L 5.27 a (±0.05) 5.44 ab (±0.23) 5.50 a (±0.00) 5.50 a (±0.00) 5.47 ab (±0.09)
GIX 200 mg/L 45.50 a (±3.83) 45.50 a (±3.83) 66.50 a (±49.84) 74.00 a (±33.96) 42.00 a (±0.00)
400 mg/L 66.50 b (±19.17) 77.00 b (±15.34) 66.50 a (±3.83) 57.00 a (±23.00) 63.00 b (±0.00)
600 mg/L 73.50 b (±26.84) 91.50 c (±8.22) 63.00 a (±23.00) 33.00 b (±3.29) 56.00 b (±15.34)
800 mg/L 10.50 c (±11.50) 45.50 a (±3.83) 42.00 ab (±0.00) 10.50 bc (±11.50) 105.00 c (±15.34)
1000 mg/L 84.00 b (±15.34) 53.00 a (±4.38) 21.00 b (±0.00) 94.50 c (±11.50) 106.00 c (±6.57)
VIX 200 mg/L 83.06 a (±9.61) 106.94 a (±20.12) 285.10 a (±275.65) 217.96 a (±124.30) 84.08 a (±16.54)
400 mg/L 181.02 b (±105.74) 201.94 b (±2.12) 253.16 ab (±34.54) 174.08 ab (±76.68) 196.53 a (±55.00)
600 mg/L 212.24 b (±126.09) 410.41 c (±160.74) 205.51 ab (±109.32) 93.06 ab (±15.20) 205.71 a (±81.82)
800 mg/L 10.82 a (±11.85) 66.73 a (±14.53) 99.18 bc (±21.01) 15.20 b (±16.66) 479.39 b (±223.78)
1000 mg/L 232.04 b (±70.42) 126.43 ab (±45.94) 29.69 c (±7.49) 621.53 c (±274.87) 589.80 b (±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.

 Detected compounds in the metabolites

In the metabolite from S. liquefaciens (OP830504), 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).

Detected compounds in the metabolites.
Peak # RT Compound detected Comp. wt% RT Compound detected Comp. wt%
Metabolite from S. liquefaciens (OP830504) 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 43.50 Stigmasterol 15.00
17 44.23 β-Sitosterol 12.19
18 44.99 Squalene 2.78
Metabolite from S. liquefaciens (OP830503) Metabolite from P. rettgeri (OP830498),),
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
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)
1 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

In addition, the metabolites from S. liquefaciens (OP830503) 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. In the case of the metabolite from P. rettgeri (OP830498), 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 ( Table 9).

The metabolites from B. cereus (OP830501) were Tetradecanoic acid (15.00%), Oleic acid (26.69%) and 11,14,17-Eicosatrienoic acid, methyl ester (19.22%) as the most dominant compounds ( Table 9).

 Discussion

Seeds that germinate vigorously offers better crop yield. 38 For cowpea, generally, final percent germination reached a significantly highest value at shorter steeping duration, then decreased afterwards. However, for Isolate N and O, all values were statistically the same or nearly so. With soybean, it increased with increasing steeping duration until 1, 2, or 3 h, then decreasing afterward, even though this decrease was sometimes insignificant. Although, microbial metabolites are known to promote germination, 39 – 41 this pattern of result for cowpea and soybean could be ascribed to nutrient and electrolyte leakage at prolonged steeping duration. 42 The risk of bacterial and fungal proliferation which can lead to seed decay and spoilage is increased when large endosperm seeds such as soybean and cowpea are primed at longer steeping periods and sown immediately as in this study.

Priming for a shorter period may not allow enough water and bioactive metabolites to enter the seeds and too long a priming period may cause seeds not to germinate. 43 The results of this study shows that steeping soybean and cowpea for shorter periods is sufficient for maximal germination values. Steeping duration did not seem to have an impact on final germination pattern on sesame, as high values were distributed throughout without order. There was also no order to the distribution of FPG values for okra, indicating that duration of steeping in the metabolites did not significantly influence germination. This uneven germination pattern could be linked to the hard seed coat of okra, 44 which perhaps limited imbibition. This hard seed coat resulted in very high maximal steeping durations of 12 h 45 and 48 h 46 for okra. Insufficient imbibition can lead to germination delay, 47 a situation that could have happened with a hardy seed such as okra in this study, since the highest steeping duration in this study was 5 h and low final germination values were recorded for it.

The effect of steeping duration on mean germination time was negligible for all the crops, as no patterns were observed. All the values obtained “congregated’ around 5. It therefore makes no agronomic sense to attach any importance to significant results here. However, other researchers have reported better mean germination times. 48 , 49

A higher germination index shows that germinated seeds appeared faster. In the case of cowpea, there was a general decrease in values with increasing steeping time for all isolates and this decline was significant for some isolates. There was also a general but insignificant decline from lower to higher steeping durations with soybean. However, for sesame and okra, steeping time was not significantly affecting the values. Okra required a 48-hour of steeping duration for maximal germination index. 46

Vigor index reached its statistical highest at a lower steeping duration for all the isolates in the case of cowpea, then decreased afterwards, and this same pattern was also observed for soybean and sesame. The highest value for okra was not limited to lower steeping periods. In fact, the significantly highest value was obtained at either 3 or 4 h for virtually all isolates. A high steeping duration of 48 hours produced the best vigor index in okra. 46 The seed coat of okra limit ample imbibition at short steeping periods.

There was generally no observable pattern for cowpea, soybean, and sesame, and abrupt drop in values were observed for okra with regards to concentration. This may have a lot to do with the erratic emergence of okra seeds 50 than any other thing. Similarly, germination was also found to be concentration-independent in the biopriming of canola seeds with varying concentrations of bacterial cell-free supernatants of Devosia sp. (SL43). 51 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 negative and drastic effects at higher concentrations. 52 , 53

Concentration was not effective in producing an impact of significant agronomic proportion on mean germination time in all the crops, as values clustered around 5. With respect to germination index, there was no clear pattern for cowpea, and generally for soybean and okra, too. For metabolites from isolates L, M, and O used in the priming of sesame seeds, there was a rare increase in GIX with increasing concentration, that was also significant.

For cowpea, seedling vigor index gradually peaked at lower concentration for all isolates, then decreased with increasing concentration, although the decreased was statistically insignificant at times. Similarly, for soybean it peaked at a lower concentration, then a significant decline was observed. Sesame recorded no clear pattern with increasing concentration, however, in the case of isolate K, it peaked at a lower concentration, then decreased steadily afterwards, and for isolate L, it rose steadily and peaked significantly at the highest concentration. Mostly, there was no directional change with concentration for seedling vigor index of okra seeds steeped in these metabolites. The hard seed coat of okra is responsible for the ambiguous response of okra to priming at different concentrations.

The GC-MS analysis of the extracts detected the presence of a number metabolites in the metabolome of each of them, some of which were common to all isolates. The compounds detected belong to categories such as alkanes, alcohols, carboxylic acids, esters, and terpenes. The ability of alcohols such as 2,3-butanediol produced by various species of Bacillus to promote the growth of Arabidopsis thaliana has been reported. 54 – 56 Tetrahydrofuran-3-ol and 2-heptanone 2-ethyl-1-hexanol from Bacillus species as well have also been reported to improve the growth of A. thaliana and tomato. 57

Oleic acid has been commonly detected in the metabolomes of some rhizobacteria. 58 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, have been shown to possess the ability to improve the growth of Vigna radiata. 59

Conclusion

This study enables us to understand the dynamics surrounding the effectiveness of microbial metabolites, something that is not possible with typical screening assays that employs a constant concentration. 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 steeping duration in microbial metabolite. DOI: https://doi.org/10.6084/m9.figshare.23284865.v1. 60

Acknowledgements

The authors are grateful to Afe Babalola University for providing the facilities for the study

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Rahman I Ali S Adnan M : Effect of seed priming on growth parameters of Okra ( Abelmoschus esculentus L.). Pure Appl. Biol. 2016;5(1):165–171. 10.19045/bspab.2016.50021 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. 35684348 10.3390/molecules27113410 PMC9182190 Dhakal P Subedi R : Influence of mannitol priming on maize seeds under induced water stress. J. Agric. Crops. 2020;6(3):27–31. 10.32861/jac.63.27.31 Ebrahimi N Kaboli SH : Effect of different times and KNO 3 concentrations on Silybum marianum seedling enhancement. Journal of Medicinal plants and By-product. 2020;9(1):51–58. Ryu CM Farag MA Paré PW : Invisible signals from the underground: bacterial volatiles elicit plant growth promotion and induce systemic resistance. Plant Pathol. J. 2005;21(1):7–12. 10.5423/PPJ.2005.21.1.007 Asari S Matzén S Petersen MA : Multiple effects of Bacillus amyloliquefaciens volatile compounds: plant growth promotion and growth inhibition of phytopathogens. FEMS Microbiol. Ecol. 2016;92(6):fiw070. 27053756 10.1093/femsec/fiw070 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. 29551214 10.1016/j.micres.2017.12.014 Jiang CH Xie YS Zhu K : 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. 10.1007/s10725-018-00473-z Liu WW Wei MU Zhu BY : 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. 10.1016/S1671-2927(08)60153-4 Jishma P Hussain N Chellappan R : 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. 28423218 10.1111/jam.13474 Akpor O : Raw data on germinability parameters at different metabolite concentrations and steeping duration in microbial metabolite.Dataset. figshare. 2023. 10.6084/m9.figshare.23284865.v1 10.5256/f1000research.150476.r201475 Reviewer response for version 1 Righini Hillary 1 Referee Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy

Competing interests: No competing interests were disclosed.

 6 10 2023 Copyright: © 2023 Righini H 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

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?

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?

‘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

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

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

No

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.

Akpor Oghenerobor B. Biological Sciences, Afe Babalola University, Ado Ekiti, Ekiti, Nigeria

Competing interests: The authors have no competing interest(s)

 20 12 2023

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 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.

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?

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?

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

10.5256/f1000research.150476.r193085 Reviewer response for version 1 Mitra Debasis 1 2 Referee https://orcid.org/0000-0003-0525-8812 Department of Microbiology, Raiganj University, Raiganj, West Bengal, India Microbiology, Crop Production Division, International Rice Research Institute, Manila, Metro Manila, Philippines

Competing interests: No competing interests were disclosed.

 4 9 2023 Copyright: © 2023 Mitra D 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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-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

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

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.

Akpor Oghenerobor B. Biological Sciences, Afe Babalola University, Ado Ekiti, Ekiti, Nigeria

Competing interests: The authors have no competing interest(s)

 20 12 2023

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 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