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

doi:10.12688/f1000research.137322.1

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Research Article

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]

Oghenerobor Akpor ¹, Ayotunde Ajinde¹, Tolulope Ogunnusi¹

PUBLISHED 05 Jul 2023

Author details Author details

¹ Department of Biological Sciences, Afe Babalola University, Ado Ekiti, Ekiti, 360102, Nigeria

Oghenerobor Akpor
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Ayotunde Ajinde
Roles: Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Original Draft Preparation

Tolulope Ogunnusi
Roles: Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

Abstract

Vigorous germination and growth are linked to crop yield. This study was carried out to assess the effect 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.

Keywords

Germinability, rhizobacteria, germinability, seed vigor

Corresponding author: Oghenerobor Akpor

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2023 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: Akpor O, Ajinde A and Ogunnusi T. 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]. F1000Research 2023, 12:781 (https://doi.org/10.12688/f1000research.137322.1) First published: 05 Jul 2023, 12:781 (https://doi.org/10.12688/f1000research.137322.1) Latest published: 07 Oct 2024, 12:781 (https://doi.org/10.12688/f1000research.137322.5)

Introduction

Seeds are critical inputs in agricultural production. However, poor crop stand, a major production constraint in the developing world¹ 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 water⁹; osmopriming involves the use of an osmoticum¹⁰; halopriming involves soaking in salt solutions¹¹; solid-matrix priming involves priming on a solid material¹²; 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:

• $Final germination % (FGP) = \frac{total number of germinated seeds}{total number of seeds sown} \times 100 %$ ³⁴
• $Mean germination time (MGT) = \sum \frac{fx}{f}$ ³⁵
Where $f$ is the number of seeds germinated on day $x$
• $Germination index (GIX) = (8 \times N 1) + (7 \times N 2) + (6 \times N 3) + \dots + (1 \times N 8)$ 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.
• $Vigor index (VIX) = FGP \times average plant height$ 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).

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)

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).

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)

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).

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)

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).

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)

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 isolates 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).

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)

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).

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)

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).

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)

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).

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)

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).

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 h⁴⁵ and 48 h⁴⁶ 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 seeds⁵⁰ 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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Version 5

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Author details Author details

¹ Department of Biological Sciences, Afe Babalola University, Ado Ekiti, Ekiti, 360102, Nigeria

Oghenerobor Akpor
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Ayotunde Ajinde
Roles: Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Original Draft Preparation

Tolulope Ogunnusi
Roles: 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.

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Akpor O, Ajinde A and Ogunnusi T. 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]. F1000Research 2023, 12:781 (https://doi.org/10.12688/f1000research.137322.1)

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Hillary Righini, Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy

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https://doi.org/10.5256/f1000research.150476.r201475

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
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.

CITE

Report a concern

Author Response 17 Jan 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

17 Jan 2024

Author Response

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 ... Continue reading 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
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
Competing Interests: The authors have no competing interest(s) Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 17 Jan 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

17 Jan 2024

Author Response

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 ... Continue reading 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
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
Competing Interests: The authors have no competing interest(s) Close
Report a concern

Views

Reviewer Report 04 Sep 2023

Debasis Mitra, Department of Microbiology, Raiganj University, Raiganj, West Bengal, India; Microbiology, Crop Production Division, International Rice Research Institute, Manila, Metro Manila, Philippines

Approved with Reservations

https://doi.org/10.5256/f1000research.150476.r193085

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
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?

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.

CITE

Report a concern

Author Response 17 Jan 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

17 Jan 2024

Author Response
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 ... Continue reading
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
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
Competing Interests: The authors have no competing interest(s) Close
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COMMENTS ON THIS REPORT

Author Response 17 Jan 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

17 Jan 2024

Author Response
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 ... Continue reading
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
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
Competing Interests: The authors have no competing interest(s) Close
Report a concern

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Version 5

VERSION 5 PUBLISHED 05 Jul 2023

Open Peer Review

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Version 5 (revision) 07 Oct 24				read
Version 4 (revision) 23 Jul 24			read	read
Version 3 (revision) 03 Jun 24			read
Version 2 (revision) 09 Jan 24			read
Version 1 05 Jul 23	read	read

Debasis Mitra, Raiganj University, Raiganj, India; International Rice Research Institute, Manila, Philippines
Hillary Righini, University of Bologna, Bologna, Italy
Sowmyalakshmi Subramanian, McGill University, Montreal, Canada
Olatunde Samuel Dahunsi, Bowen University, Iwo, Nigeria

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Reviewer Report

2 Views

17 Oct 2024 | for Version 5

Olatunde Samuel Dahunsi, Microbiology Programme, College of Agriculture, Engineering and Science, Bowen University, Iwo, Osun, Nigeria

2 Views Cite this report Responses(0)

Approved

Authors have satisfactorily addressed all my previous concerns. I now approve this submission for acceptance and indexing.

Competing Interests

No competing interests were disclosed.

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.

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Reviewer Report

6 Views

03 Oct 2024 | for Version 4

Sowmyalakshmi Subramanian, Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada

6 Views Cite this report Responses(0)

Approved

Thank you for making the changes.

Competing Interests

No competing interests were disclosed.

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.

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Reviewer Report

9 Views

20 Sep 2024 | for Version 4

Olatunde Samuel Dahunsi, Microbiology Programme, College of Agriculture, Engineering and Science, Bowen University, Iwo, Osun, Nigeria

9 Views Cite this report Responses(1)

Approved With Reservations

I have reviewed the manuscript appropriately. The study was well conceived, designed and executed while the presented results are commendable. I have no challenge reading through the manuscript and can recommend acceptance after some minor revision. My comments includes:

There is need to improve the English language usage throughout the manuscript
The keyword section is not acceptable in the current state all keywords are directly taken from the manuscript title besides that the number are few. I encourage authors to overhaul the section by selecting words or phrases that best describe the content of the manuscript and that can be used for indexing and abstracting purposes.
In the method section, details of bacterial isolation and characterization should be given as well as those of metabolite characterization.
The provided ascension numbers should be well clarified.
Generally, authors need to overhaul the grammar in this manuscript.

Is the work clearly and accurately presented and does it cite the current literature?

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

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

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

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

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

English usage, methods and general formatting

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Author Response

07 Oct 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

Dear Reviewer,
Please find within this document our responses to your comments.

Comment: There is need to improve the English language usage throughout the manuscript
Response: The manuscript has been further reviewed to improve presentation style.

Comment: The keyword section is not acceptable in the current state all keywords are directly taken from the manuscript title besides that the number are few. I encourage authors to overhaul the section by selecting words or phrases that best describe the content of the manuscript and that can be used for indexing and abstracting purposes.
Response: The necessary corrections have been made throughout the manuscript.

Comment: In the method section, details of bacterial isolation and characterization should be given as well as those of metabolite characterization.
Response: This section has been overhauled.

Comment: The provided ascension numbers should be well clarified.
Response: The necessary corrections have been made.

Comment: Generally, authors need to overhaul the grammar in this manuscript.
Response: The manuscript has been extensively reviewed to improve grammar and style.

Thanks.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

12 Views

10 Jul 2024 | for Version 3

Sowmyalakshmi Subramanian, Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada

12 Views Cite this report Responses(1)

Approved With Reservations

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

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Author Response

23 Jul 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

Dear Reviewer,

Thank you for the review, please find enclosed within this document our response to your comments.

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

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

13 Views

23 May 2024 | for Version 2

Sowmyalakshmi Subramanian, Department of Plant Sciences, MacDonald Campus, McGill University, Montreal, Québec, Canada

13 Views Cite this report Responses(1)

Approved With Reservations

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.

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

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Author Response

21 Jun 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

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 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.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

18 Views

06 Oct 2023 | for Version 1

Hillary Righini, Department of Agricultural and Food Sciences, Alma Mater Studiorum, University of Bologna, Bologna, Italy

18 Views Cite this report Responses(1)

Not Approved

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

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Author Response

17 Jan 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

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

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Competing Interests

The authors have no competing interest(s)

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Reviewer Report

25 Views

04 Sep 2023 | for Version 1

25 Views Cite this report Responses(1)

Approved With Reservations

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?

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

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Author Response

17 Jan 2024

Oghenerobor B. Akpor, Department of Biological Sciences, Afe Babalola University, Ado Ekiti, 360102, Nigeria

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

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Competing Interests

The authors have no competing interest(s)

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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[9] 9. Afzal I, Basra SM, Ahmad NA, et al.: Effect of priming and growth regulator treatments on emergence and seedling growth of hybrid maize (Zea mays L.). Int. J. Agric. Biol. 2002; 4(2): 303–306.

[10] 10. Rouhi HR, Aboutalebian MA, Sharif-Zadeh F: Effects of hydro and osmopriming on drought stress tolerance during germination in four grass species. Int. J. AgriScience. 2011; 1(2): 107–114.

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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)

Abstract

Keywords

Introduction

Methods

Test metabolites

Test seeds

Germinability studies

Ethical approval

Results

Effect of steeping duration in metabolites

Table 1. Germinability of the cowpea seeds at the different steeping durations in the respective metabolites.

Table 2. Germinability of the soybean seeds at the different steeping durations in the respective metabolites.

Table 3. Germinability of the sesame seeds at the different steeping durations in the respective metabolites.

Table 4. Germinability of the okra seeds at the different steeping durations in the respective metabolites.

Effect of concentration of metabolite

Table 5. Germinability of the cowpea seeds at the different concentrations of the respective metabolites.

Table 6. Germinability of the soybean seeds at the different concentrations of the respective metabolites.

Table 7. Germinability of the sesame seeds at the different concentrations of the respective metabolites.

Table 8. Germinability of the okra seeds at the different concentrations of the respective metabolites.

Detected compounds in the metabolites

Table 9. Detected compounds in the metabolites.

Discussion

Conclusion

Data availability

Acknowledgements

References

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