Eco-friendly soil amendments improve growth, antioxidant activities, and root colonization in lingrain (Linum Usitatissimum L.) under drought conditions.

This study's primary purpose was to investigate the possible amelioration of limited irrigation conditions by mycorrhiza (AMF), vermicompost, and green manure for lingrain plants. This experiment was accomplished as a factorial based on the completely randomized design with three replications. The first factor was green manure (without green manure and with Trifolium pratense as green manure); the second factor consisted of Rhizophagus irregularis mycorrhiza, vermicompost, a combination of mycorrhiza and vermicompost and none of them, and also the third factor was irrigation regime (full irrigation and late-season water limitation). Green manure, vermicompost, and mycorrhiza single-use enhanced the plant's underwater limitation conditions compared to the control. However, vermicompost and green manure or mycorrhiza developed a positive synergistic effect on most traits. Combining green manure with the dual fertilizer (mycorrhiza + vermicompost) resulted in the vermicompost and mycorrhiza synergistic effects, especially under limited irrigation. Consequently, the combination of green manure, mycorrhiza, and vermicompost experienced the highest amount of leaf relative water content, root colonization, leaf nitrogen, chlorophyll a, chlorophyll b, carotenoids, antioxidant enzymes activity, grain yield, and oil yield, which would lead to more resistance of plants to limited irrigation conditions.

1. Introduction

Climate change and water scarcity have sparked drought as the most critical environmental stress affecting crop growth, development, and productivity worldwide [1]. Along with the increase in greenhouse gases emissions under the constant global climate change, an increase in the drought intensity and incidence is expected soon. Approximately half of the agricultural lands worldwide are constantly endangered by water shortages, resulting in a 50% reduction in grain yield [2]. Drought stress can seriously disturb secondary metabolites concentration, also along with the morphological and physiological traits [3, 4].

Plants could create different defined mechanisms against water stress, such as root development increasing for more water absorption, free radical scavenging enzymes activity, and osmolytes accumulation to moderate the osmotic pressure [5, 6].

Plant resistance to drought can also be increased using other external methods, for instance, by different soil amendments of which the eco-friendly treatments (for example, mycorrhizal inoculation and organic fertilizer like vermicompost and green manure) have great importance.

Organic fertilizers increase product yield and quality by improving soil structure and nutrient availability [7]. The use of organic sources for enhancing nutrient supply may help maintain the organic matter balance and improve the soil’s chemical, physical, and biological properties [8].

Green manure plays a significant role in nutrients supply for crops, decreasing dependency on chemical fertilizers, enhancing crop yield, enriching the cultivated field’s ecological environment, and lowering soil destruction and contamination [9].

Vermicompost, derived from the organic wastes of the earthworm, has nutrients that are readily absorbable by a plant. Vermicompost is a nutrient-rich and nature-friendly substance that has several potential applications as soil conditioners [10]. It can enhance soil’s physical, chemical, and biological fertility. Physically, vermicompost can improve porosity, bulk density, aeration, and water retention of the soil. Chemical properties such as electrical conductivity, organic matter content, and pH may also improve crop performance [11].

Arbuscular mycorrhizal fungi (AMF) are omnipresent in land-dwelling ecosystems and indicate symbiotic interactions with many plant species roots [12]. The AMF can protect plants against biotic and abiotic stresses by different mechanisms such as increasing nutrient absorption, photosynthetic activity, osmolytes accumulation, antioxidant enzymes production and improving the rhizosphere environment [13]. The methods used by the AM fungi for increasing the host plants-water relationships are not clear enough. However, the cumulative water absorption may cause this through exterior hyphae, the adjustment of the stomatal system, the increased antioxidant enzymes activity, and nutrient uptake, mainly phosphorus [14].

Lingrain (Linum usitatissimum) has been extensively cultivated for its fine, cellulose-rich, bast fibers and its nutritious oil [15]. Lingrain oil is a superb dietary supplement being rich in omega-3 fatty acids and α-linolenic acid. The oil is also benefited in industrial raw materials production [16]. The world’s lingrain harvested area and production went down from 1994 to 2007. However, from 2008 to 2019, both the area under cultivation and total production has increased. Hence it was cultivated on 3.2 million hectares of land worldwide with 3000000 tons of grain production [17].

In most semi-arid countries, including Iran, there is often a severe shortage of water supply for irrigation of crops in the late growing season, which coincides with mid and late-summer. The significant impact of various soil amendments, including organic and biologic fertilizers, has been reported to improve plants’ performance under water deficit stress. Due to the severe shortage of organic matter in soils of semi-arid regions such as Iran and despite much research on organic fertilizers, little information is available about the simultaneous use of different types of organic fertilizers (vermicompost and green manure) and especially their combination with biological fertilizer (mycorrhiza). Therefore, this study was aimed to investigate the effect of single and combined use of these eco-friendly fertilizers for possible mitigation of lingrain performance under late-season irrigation limitation.

2. Materials and methods

2.1. Experimental design

Field experiments were accomplished in 2017 and 2018 at the Saatlou Station (44″° N, 45°10’95" E), Urmia Agricultural Research Centre in Iran. The average rainfall was 17.5 mm and 32.07 mm during the lingrain growing season in 2017 and 2018. This experiment was factorial based on a randomized complete block design with three replications. Factors included (1) irrigation regimes (full irrigation (Iw), limited irrigation (Is) (irrigation cut-off from the flowering stage), (2) green manure (no green manure (Gc), Trifolium pratense as green manure (Gr), and (3) mycorrhiza inoculation ((Rhizophagus irregularis) (Fm) 300 g m-2), vermicompost (Fv) (5 t/ha), their combination (mycorrhiza + vermicompost (Fm+v)) and no fertilizer (Fc). The total plots number was 36.

2.2. Soil amendments

The soil texture was silty loam with a pH of 7.8. Other soil physical and chemical properties are indicated in Table 1.

Table 1

Soil physicochemical properties.

Characteristic	Saturation extract	pH	EC	P	K	N	Organic carbon	Sand	Silt	Clay	Texture
			(dS/m)	(mg/kg)	(mg/k)	(%)	(%)	(%)	(%)	(%)
0–30 cm	42	7.8	0.58	7.1	184	0.09	0.91	23	42	25	Silty loam

Red clover (Trifolium pratense) was grained in August 2016 and 2017. Also, fresh Gr was manually incorporated into 20 cm depth of surface soil in March 2017 and 2018, at the flowering stage.

Mycorrhizal inoculum included a blend of sterile sand, mycorrhizal hyphae, and spores (30 spores g-1 inoculum). It colonized fragments of canola root that were isolated from an AMF community of a canola farm by the plant protection department of Agricultural and Natural Resources Research Centre, West Azerbaijan province, Urmia, Iran. Vermicompost was prepared according to the method of Ayyobi et al. [18]. The chemical properties of the vermicompost that was used in these experiments are mentioned in Table 2.

Table 2

Vermicompost properties.

Properties	pH	EC (dS/m)	Organic matter (%)	Nitrogen (%)	P (%)	K (%)	Mg (%)	Ca (mg/kg)	Cu (mg/kg)	Zn (mg/kg)
Vermicompost	7.64	5.22	12.3	1.3	0.88	0.67	1.1	8.75	3.14	46.76

2.3. Plant culture

After one month of green manure integration, the land was manually prepared. In the first and second years, green manure measured dry biomass production at 4.72 t ha-1 and 4.95 t ha-1, respectively. Also, inoculum (300 g m−2) was incorporated into the soil of the grain bed before the grain sowing. The exact amounts of vermicompost were manually spread onto the soil surface and incorporated into the top 20 cm of the soil.

Lingrain Grains were obtained from the Agricultural and Natural Resources Research Centre of West Azerbaijan province, Urmia, Iran. The grains were sown in 8 rows on 21 April 2017 and 24 April 2018, respectively (with two cm intra-row and 25 cm inter-row spaces).

The full irrigation treatment included five irrigations (after sowing, at the 4-leaf stage, at stem elongation, at flowering and capsule formation stages), and limited irrigation treatment (irrigation cut-off from the flowering stage) had three irrigations (after sowing, at 4-leaf and stem elongation stages). The use of a water meter measured the irrigation water amount in each plot. Moreover, the average humidity at the surface and depths of 20 and 40 cm of soil was determined using a soil moisture meter (EXTECH MO750) in each plot before each irrigation. After that, the amount of water that was required for each irrigation was calculated by the following equation:

Where,

V, the volume of irrigation water (m3),

Fc moisture content at the field capacity point (volume),

θ soil moisture (volume),

A area of the plot (m2) and

h, Effective depth of lingrain root (m).

The water used for irrigation was 2346 and 1420 m3/ha (for the first year) and 1750 and 600 m3/ha (for the second year) for full and limited irrigation, respectively.

All the practices were manually handled in this experiment in order to prevent interference with all materials.

2.4. Parameters measured

Leaf relative water content (LRWC)

Leaf relative water content was assayed in leaf samples with respect to García-Mata and Lamattina [18]. LRWC measurements were determined according to the formula:

Fresh weight (FW) was measured one week after the late season drought stress, and dry weight (DW) was obtained after drying the samples at 75°C for at least 24 h. Turgor weight (TW) was determined by subjecting leaves to rehydration for 2 h, after drought treatments.

2.5. AMF colonization

The mycorrhizal root colonization percentage was determined for each plot in five roots. For this, roots were cleared by 10% KOH at 90°C for 10 min, and after that, were stained in 0.05% lactic acid-glycerol-Trypan Blue [19]. The mycorrhizal colonization was measured according to the method of Giovannetti and Mosse [20] gridline intersect method.

2.6. Nutrient content

The leaf samples were dried in an oven for 72 h at 70°C, and after that, were grounded using an electric mill. The Kjeldahl method was used for Nitrogen measurement [21]. Spectrophotometry and flame photometry methods were applied in order to determine P and K contents, respectively.

2.7. Photosynthetic pigment content assay

Approximately 10 ml of acetone 80% was gradually added to extracts of fresh leaf tissue samples (0.2 g) followed by centrifugation at 4000 rpm for 10 min, and the absorbance was recorded using a spectrophotometer at 645, 663, and 470 nm. Chlorophyll and carotenoids were obtained based on the following equations:

2.8. Proline

Leaf fresh tissue (1 g) was used for leaf proline extracting in sulfosalicylic acid 5% (w/v). Leaf proline was determined using spectrophotometric analysis at 520 nm of the ninhydrin reaction using toluene as blank. A standard curve for concentration from 0–512 μL (20–100 μg/ml) of L-Proline was used [22].

2.9. Total soluble sugars (TSS)

Leaf total soluble sugar was measured by the Anthrone method [23]. One mL of aqueous sample was added to a test tube. Also one mL water was added to triplicate tubes to serve as reagent blank. 4 mL ice-cold anthrone reagent was added to all tubes, vortexed, and placed at 100°C for exactly 10 min. Tubes were placed into an ice bath and rapidly cooled. They were mixed well and were read the absorbance of green to dark green blanks, standards, and samples at 630 nm. The TSS concentration was determined using the glucose standard curve.

2.10. Hydrogen peroxide (H2O2)

The fresh leaf tissue (0.5 g) was grounded in a pre-chilled mortar with 5 mL of 0.1% (w/v) TCA. After that, this homogenate was centrifuged at 12000 × g for 15 min at 4°C. Also, 0.5 ml of the supernatant was added to 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 1 ml of 1 M KI, and the OD of the suspension was read at 390 nm [24].

2.11. Malondialdehyde (MDA)

Malondialdehyde was measured as per Heath and Packer method [25]. where 532 nm represented the maximum absorbance of the TBA- MDA complex, 600 nm the correction for nonspecific turbidity, and 155 000 the molar extinction coefficient for MDA.

2.12. Glycine betaine (GB)

Glycine betaine analysis was performed according to the method of Grieve and Grattan [26]. Leaf glycinebetaine was extracted from the dry leaf material with warm distilled water (70°C). The extract (0.25 ml) was mixed with 0.25 ml of 2 N HCl and 0.2 ml of potassium triiodide solution. The contents were shaken and cooled in an ice bath for 90 min. Then 2.0 ml of ice cooled distilled water and 20 ml of 1,2- dichloromethane (cooled at −10°C) were added to the mixture. The two layers were formed in the mixture. The upper aqueous layer was discarded and optical density of the organic layer was measured at 365 nm. The concentrations of glycinebetaine were calculated on fresh weight basis.

2.13. Estimation of catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD)

The fresh leaf tissues (5.0 g) were macerated in 10 mL potassium phosphate buffer [pH 7.8; 50 mM] using an ice-cooled sterilized pestle and mortar. After that, the homogenates were centrifuged for 20 min at 4°C at12000 g. The supernatant was used to assay the antioxidant enzymes activity assay.

Catalase activity was assayed according to the Method of Maehly and Chance [27]. Fifty ml enzyme extract was added to 2.5 ml of 50 mM phosphate buffer (pH 7.4) and 0.1 ml of 1% hydrogen peroxide in the ice bath to determine CAT activity. The H2O2 content reduction was obtained at 240 nm for 1 min.

Ascorbate peroxidase activity was determined by absorption reducing at 290 nm due to ascorbate oxidation in 3 min, according to Chen and Asada [28]. The reaction mixture involved 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbate, 1.54 mM hydrogen peroxide, and 50ml enzyme extract.

Superoxide dismutase activity was measured following the method of Dhindsa et al. [29] method.

2.14. Grain oil extraction

Grain oil was extracted using the Soxhlet technique [30], and the oil yield was calculated by using the following formula:

2.15. Grain yield

At plant maturity, above-ground biomass was harvested from 1m2 of each experimental plot by ignoring marginal effects. Each plot’s grains were separated and weighed in order to measure grain yield.

2.16. Statistical analysis

Combined factorial analysis of variance (three factors) including those gathered data for about two years (with one year as a random factor) was performed using SAS software and by applying the general linear model (GLM) procedure to establish these three factors main and interactive influences. After that, this model was approved by checking the residues’ normality and homogeneity (using the Bartlett test). Also, no data transformation was performed. The mean values were compared using the protected least significant difference (PLSD) at P ≤ 0.05 because the treatment factors were qualitative.

3. Results

3.1. Leaf relative water content (LRWC)

LRWC considerably decreased in water-limited conditions. However, Green manure addition noticeably increased the LRWC in mycorrhizal plants (Fm and Fm+v). In both irrigation conditions, single and combined use of vermicompost and mycorrhiza (Fm, Fv, and Fm+v) increased the LRWC significantly compared to the control, with the highest increase (92.56%) belonging to the Fm+v as shown in Table 3.

Table 3

Comparison between 2-year mean values of physiological traits of lingrain affected by green manure× mycorrhiza × vermicompost under full and limited irrigation.

Treatment	Leaf Relative	Colonization (%)	Leaf N	Leaf K	Chlorophyll a	Chlorophyll b	Carotenoids
	water content (%)		% on dry matter	% on dry matter	(mg/g FW)	(mg/g FW)	(mg/g FW)
IW× Gc× Fc	66.64f	7.50ij	2.42fg	0.18h	2.19h	1.68c	1.87h
IW× Gc× Fm	79.66c	56.25c	3.04c	0.25cd	3.35d	2.08b	2.9d
IW× Gc× Fv	75.04d	13.03g	2.77de	0.25cd	2.71f	1.96b	2.67e
IW× Gc× Fm+v	83.71b	58.25c	3.22b	0.28ab	4.42b	2.11b	3.83b
IS× Gc× Fc	51.35h	4.94k	1.89i	0.9i	1.19m	1.06g	1.02l
IS× Gc× Fm	63.16g	42.55e	2.3g	0.21ef	1.66j	1.54cd	1.63i
IS× Gc× Fv	62.73g	9.40hi	2.41fg	0.22ef	1.34kl	1.31ef	1.24k
IS× Gc× Fm+v	65.15fg	52.91c	2.41fg	0.22ef	1.83i	1.6cd	1.85h
IW× Gr× Fc	73.69d	8.29ij	2.49f	0.20fg	2.41g	1.94b	2.16g
IW× Gr× Fm	81.98bc	67.40b	3.27b	0.27abc	3.68c	2.1b	3.17c
IW× Gr× Fv	75.56d	18.39f	2.88b	0.26bc	2.98d	1.96b	2.7e
IW× Gr× Fm+v	92.56a	71.08a	3.38a	0.29a	4.86a	2.44a	4.37a
IS× Gr× Fc	53.74h	5.92jk	2.17h	0.18h	1.23ml	1.22fg	1.13kl
IS× Gr× Fm	71.08e	44.04e	2.79f	0.23de	2.46g	1.96b	2.29g
IS× Gr× Fv	62.96g	11.57gh	2.41e	0.23de	1.43k	1.46de	1.44j
IS× Gr× Fm+v	72.95de	56.58c	2.90de	0.23de	2.71f	1.98b	2.46f

Where Iw (full irrigation), Is (limited irrigation), Gc (no green manure), Gr (green manure), Fc (no fertilizer), Fm (mycorrhiza inoculation), Fv (vermicompost application), and Fm+v (combination of mycorrhiza and vermicompost). Means followed by the same letter in each column are not significantly different using

PLSD method at P ≤ 0.05.

3.2. Colonization

Single and combined usage of mycorrhiza and vermicompost (Fv, Fm, and Fm+v) led to more AMF colonization with lingrain roots in comparison with non-fertilized treatment (Fc) (Table 3). In full irrigation conditions, green manure usage increased the colonization in all fertilizer treatments except Fc. The most significant colonization was achieved in the combination of mycorrhiza and vermicompost (Fm+v) with green manure, as indicated in Table 3. Moreover, limited- irrigation significantly declined the colonization in all fertilizer treatments (Table 3).

3.3. Leaf N content

Water limitation decreases the levels of leaf nitrogen. The single and combined usage of vermicompost and mycorrhiza resulted in increased leaf nitrogen in both irrigation conditions compared to Fc. Application of green manure and mycorrhiza significantly elevated leaf nitrogen in both irrigation conditions except Fv. The combination of mycorrhiza and vermicompost could generate the highest leaf nitrogen percentage, as indicated in Table 3.

3.4. Leaf K content

Water stress strongly decreased the leaf K content in all fertilizer treatments. Under normal irrigation conditions, Fm+v was more effective in leaf K content increasing in comparison with single vermicompost and mycorrhiza. However, they were not significantly different from each other under limited irrigation conditions. On the other hand, they all (Fm+v, Fm, and Fv) produced more leaf K than the control treatment (Fc).

3.5. Photosynthetic pigments

Water limitation significantly reduced chlorophyll a, chlorophyll b, and carotenoid contents. Green manure application soared chlorophyll content in both irrigation conditions. Also, Fm+v had the highest chlorophyll-a content in all conditions; after that, Fm and Fv were in second and third place, respectively. In addition, the lowest chlorophyll-a quantity belonged to Gc×Fc (Table 3).

Using green manure significantly increased the level of carotenoid in Fm+v in both full irrigation and late-season water stress. Green manure increased the amount of chlorophyll b, and single-use of mycorrhiza and vermicompost could not affect the chlorophyll b under full irrigation conditions, but mycorrhizal plants resulted in a significant increase of chlorophyll b content under limited irrigation (Table 3). The combined treatment of vermicompost and mycorrhiza produced the highest carotenoid content in both conditions, as shown in Table 3.

3.6. Leaf P content

Water inadequacy could reduce the leaf P concentrations. Under normal irrigation conditions, single and double fertilizers (Fm, Fv, and Fm+v) brought approximately a higher percentage of leaf P in comparison with control (Fc), but there was no significant difference amongst the fertilizer treatments (Table 4). Although dual usage of mycorrhiza and vermicompost (Fm+v) accumulated more P in the leaf, it indicated no significant difference with the single applications of mycorrhiza and vermicompost (Fm and Fv) in water-limited conditions (Table 4). In the second year, leaf P increased significantly compared to the first year (Table 5).

Table 4

Comparison amongst 2-year mean values of physio-biochemical traits, leaf P, total soluble sugars, catalase, and superoxide dismutase activity affected by mycorrhiza × vermicompost under full and limited irrigation.

Treatment	Leaf P	Proline (μmol/g FW)	Total soluble sugars (μmol/g FW)	Glycine betaine (μmol/g FW)	Hydrogen peroxide (μmol/g FW)	CAT activity (mmol min-1/g FW)	SOD activity (Units/g FW)
	% on dry matter
Iw× Fc	0.47d	23.04d	22.78e	19.80cd	10.91c	4.76f	19.14e
Iw× Fm	0.65ab	18.45e	15.93f	16.43e	5.59f	7.89b	23.21c
Iw× Fv	0.64b	18.63e	15.26f	19.62d	7.86d	7.23d	21.71d
Iw× Fm+v	0.67a	12.68f	10.89g	15.82e	6.53e	7.71bc	23.21c
Is× Fc	0.25f	25.09c	27.85d	21.94c	18.06a	5.33e	23.72c
Is× Fm	0.44de	27.80b	39.10b	27.54b	10.52c	8.61a	32.91a
Is× Fv	0.42e	27.42b	33.74c	26.47b	12.82b	7.42cd	29.08b
Is× Fm+v	0.53c	36.47a	44.12a	34.39a	11.01c	8.71a	33.22a

Where Iw (full irrigation), Is (limited irrigation), Fc (no fertilizer), Fm (mycorrhiza inoculation), Fv (vermicompost application), and Fm+v (combination of mycorrhiza and vermicompost). Mean values that were followed by the same letter in each column are not significantly different using PLSD method at P ≤ 0.05.

Table 5

Comparison between 2-year mean values of physiological traits of lingrain affected by green manure× mycorrhiza × vermicompost under full and limited irrigation.

Treatment	Glycine betaine (μmol/g FW)	Hydrogen peroxide (μmol/g FW)	Malondialdehyde (μmol/g FW)	Ascorbate Peroxidases activity (mmol min-1/g FW)	Guaiacol peroxidase activity (mmol min-1/g FW)	Grain yield (kg h-1)	Oil yield (kg h-1)
IW× Gc× Fc	19.20gh	11.02e	31.80fg	1.35j	0.65g	3298gh	870.12gh
IW× Gc× Fm	13.05j	7.32g	24.13hi	3.43fg	2.38e	4228bc	1209.92d
IW× Gc× Fv	18.27h	7.67g	26.22h	2.78i	2.36c	3995cd	1102.8ef
IW× Gc× Fm+v	11.99j	5.65h	16.93j	3.24gh	3.45c	4456b	1287.72c
IS× Gc× Fc	21.95e	19.02a	70.05a	3.97e	1.75f	2050j	452.09j
IS× Gc× Fm	26.67cd	12.18cd	36.56d	5.51b	2.59de	3233gh	842.91h
IS× Gc× Fv	25.47d	13.05c	43.45c	4.8d	2.47e	2350i	604.20i
IS× Gc× Fm+v	33.93a	10.80e	34.26e	5.06c	3.69bc	3237gh	868.69g
IW× Gr× Fc	20.40fg	9.67f	29.69g	1.71j	3.24c	3463fg	1095.92ef
IW× Gr× Fm	19.81fg	5.74h	23.94i	3.81ef	4.08b	4359b	1422.83b
IW× Gr× Fv	20.98ef	7.93g	24.10hi	2.93h	3.1cd	3999cd	1290.68c
IW× Gr× Fm+v	16.03i	5.52h	16.40j	3.37fgh	4.24b	4862a	1602.66a
IS× Gr× Fc	21.93e	17.10b	59.19a	4.48d	3.37c	2312i	637.43i
IS× Gr× Fm	28.41b	11.37de	33.21ef	5.92b	4.13b	3587ef	1052.97f
IS× Gr× Fv	27.48bc	12.45c	42.04c	5.61c	4.06b	3187h	928.2g
IS× Gr× Fm+v	34.84a	9.85e	31.14fg	7.15a	5.27a	3806d	1129.08e

Where Iw (full irrigation), Is (limited irrigation), Gc (no green manure), Gr (green manure), Fc (no fertilizer), Fm (mycorrhiza inoculation), Fv (vermicompost application), and Fm+v (combination of mycorrhiza and vermicompost). Means followed by the same letter in each column are not significantly different using

PLSD method at P ≤ 0.05.

3.7. Proline

Water limitation significantly increased but green manure did not change the proline concentration. The single and dual application of vermicompost and mycorrhiza increased the proline content in water-limited conditions; also, Fm+v indicated the highest proline accumulation under water limitation. Similarly, the single usage of fertilizers had the same effect on proline concentration in both irrigation regimes (Table 4).

3.8. Total soluble sugar

Total soluble sugar concentration was enhanced in the second year compared to the first year in both irrigation regimes (Table 6). Solo application (F) and (F) did not significantly affect TSS concentration in the full irrigation regime. Also, F decreased TSS concentration in this condition. All fertilizer sources increased TSS concentration in irrigation cut-off conditions. So that the dual fertilizer treatment (F) produced more TSS than the others under irrigation cut-off conditions (Table 4).

Table 6

Comparison between 2-year mean values of physiological traits of lingrain affected by green manure× mycorrhiza × vermicompost under full and limited irrigation.

Treatment	Glycine betaine (μmol/g FW)	Hydrogen peroxide (μmol/g FW)	Malondialdehyde (μmol/g FW)	Ascorbate Peroxidases activity (mmol min-1/g FW)	Guaiacol peroxidase activity (mmol min-1/g FW)	Grain yield (kg h-1)	Oil yield (kg h-1)
IW× Gc× Fc	19.20gh	11.02e	31.80fg	1.35j	0.65g	3298gh	870.12gh
IW× Gc× Fm	13.05j	7.32g	24.13hi	3.43fg	2.38e	4228bc	1209.92d
IW× Gc× Fv	18.27h	7.67g	26.22h	2.78i	2.36c	3995cd	1102.8ef
IW× Gc× Fm+v	11.99j	5.65h	16.93j	3.24gh	3.45c	4456b	1287.72c
IS× Gc× Fc	21.95e	19.02a	70.05a	3.97e	1.75f	2050j	452.09j
IS× Gc× Fm	26.67cd	12.18cd	36.56d	5.51b	2.59de	3233gh	842.91h
IS× Gc× Fv	25.47d	13.05c	43.45c	4.8d	2.47e	2350i	604.20i
IS× Gc× Fm+v	33.93a	10.80e	34.26e	5.06c	3.69bc	3237gh	868.69g
IW× Gr× Fc	20.40fg	9.67f	29.69g	1.71j	3.24c	3463fg	1095.92ef
IW× Gr× Fm	19.81fg	5.74h	23.94i	3.81ef	4.08b	4359b	1422.83b
IW× Gr× Fv	20.98ef	7.93g	24.10hi	2.93h	3.1cd	3999cd	1290.68c
IW× Gr× Fm+v	16.03i	5.52h	16.40j	3.37fgh	4.24b	4862a	1602.66a
IS× Gr× Fc	21.93e	17.10b	59.19a	4.48d	3.37c	2312i	637.43i
IS× Gr× Fm	28.41b	11.37de	33.21ef	5.92b	4.13b	3587ef	1052.97f
IS× Gr× Fv	27.48bc	12.45c	42.04c	5.61c	4.06b	3187h	928.2g
IS× Gr× Fm+v	34.84a	9.85e	31.14fg	7.15a	5.27a	3806d	1129.08e

Where Iw (full irrigation), Is (limited irrigation), Gc (no green manure), Gr (green manure), Fc (no fertilizer), Fm (mycorrhiza inoculation), Fv (vermicompost application), and Fm+v (combination of mycorrhiza and vermicompost). Means followed by the same letter in each column are not significantly different using PLSD method at P ≤ 0.05.

3.9. Catalase (CAT)

Water limitation increased CAT activity. The incorporation of green manure under water limitation increased the activity of the CAT enzyme in all treatments (Fig 1).

Fig 1

Interaction of green manure and irrigation regimes on catalase activity.

Using mycorrhiza, with or without vermicompost, indicated a higher influence on the enzyme activity under water limitation. Furthermore, the highest activities of CAT (8.71, 8.61. mmol min-1/g FW) were observed in mycorrhizal plants (Table 4).

3.10. Superoxide dismutase (SOD)

In both years, SOD activity was significantly increased by water limitation, as shown in Table 6. The application of green manure enhanced the activity of SOD in the limited irrigation situation (Fig 2).

Fig 2

Interaction of green manure and irrigation regimes on superoxide dismutase activity.

The synergistic effects of the co-application and the separate usages of mycorrhiza were more than that of vermicompost under limited irrigation (Table 4).

3.11. Glycine betaine (GB)

Water limitation considerably enhanced the levels of GB in lingrain leaves. Although vermicompost and mycorrhiza increased GB content under the limited-irrigation circumstance, the highest GB was obtained from dual fertilizer treatment (Fm+v). Additionally, only mycorrhizal fertilizers (Fm, Fm+v) under full irrigation led to an increase in GB content (Table 5).

3.12. Hydrogen peroxide

Drought stress considerably increased the level of leaf hydrogen peroxide. However, using fertilizer treatments (Fm, Fv and Fm+v) caused a significant decline in its amount compared to the control (Fc). Dual fertilizer (Fm+v) resulted in the highest reduction percentage for hydrogen peroxide. The mycorrhiza-containing treatments more effectively reduced the amount of hydrogen peroxide compared to vermicompost (Table 5). Green manure usage significantly affected H2O2 concentration in all treatments except (Fv) in both irrigation regimes (Fig 3).

Fig 3

Interaction effects of green manure * fertilizers on hydrogen peroxide concentration.

3.13. Malondialdehyde (MDA)

Water limitation enormously increased the levels of MDA, which was a result of lipid peroxidation increasing. However, single and dual usage of the fertilizers resulted in a remarkable drop in MDA content compared to the control (Fc). Dual fertilizer (Fm+v) brought approximately the maximum reduction for MDA. The mycorrhizal treatments can decrease the level of MDA more effectively in comparison with vermicompost. Also, the single application of green manure was helpful in the MDA level reduction. Green manure usage decreased MDA level only in mycorrhizal treatments in irrigation cut-off condition (Table 5).

3.14. Ascorbate peroxidase (APX)

Green manure increased the APX activity only underwater limitation conditions in all treatments except Fm. Water deficit elevated the APX activity. Furthermore, single or combined vermicompost and mycorrhiza had a more stimulatory effect on the enzyme activity under water limitation. The mycorrhiza and vermicompost combination caused the highest activity of APX (Table 5).

3.15. Guaiacol peroxidase (GPOX)

Water deficit elevated the Guaiacol peroxidase activity. Green manure increased the enzyme activity in all treatments except vermicompost in a full irrigation regime. Furthermore, enzyme activity was equal when using mycorrhiza under both irrigation conditions. The mycorrhiza and vermicompost combination caused the highest activity of GPOX (Table 6).

3.16. Grain yield

Limited irrigation significantly reduced the grain yield compared to full irrigation; however, green manure increased grain yield relative to the non-application of green manure in this condition. Although, co-application of green manure with vermicompost and mycorrhiza did not influence grain yield in full irrigation. Green manure application under complete irrigation situation produced the maximum grain yield (4862 kg ha-1) in Fm+v treatment. In addition, using green manure reduced the adverse effects of water restriction on grain yield, and the presence of mycorrhiza and vermicompost will additionally mitigate these adverse effects (Table 5).

3.17. Oil yield

The sole and conjoint usage of mycorrhiza and vermicompost, along with co-application of green manure under full and limited irrigation, increased oil yield by comparing them with their corresponding controls (Table 5).

Using mycorrhiza was more effective in increasing oil yield compared to vermicompost, but the dual fertilizer produced the highest oil yield in both irrigation regimes with or without green manure. However, adding green manure to them produced more oil yield than control (Gc×Fc). Furthermore, the green manure inclusion to Fm+v produced the maximum level of oil yield (1602.66 kg h-1) under full irrigation conditions (Table 5). This treatment also significantly improved the content of Leaf P content, and total soluble sugar and improved the activity of SOD (Table 7)

Table 7

Comparison amongst 2-year mean values of physio-biochemical traits, leaf P, total soluble sugars, superoxide dismutase activity under full and limited irrigation.

Treatment	Leaf P	Total soluble sugars (μmol/g FW)	SOD activity (Units/g FW)
	% on dry matter
Y1× Iw	0.57b	15.72d	21.6c
Y1× Ic	0.37d	35.10b	22.03c
Y2× Iw	0.66a	16.71c	28.86b
Y2× Ic	0.45c	37.31a	30.6a

Where Y1 (first year), Y2 (second year), Iw (full irrigation), Is (limited irrigation). Means values that were followed by the same letter in each column are not significantly different using PLSD method at P ≤ 0.05.

4. Discussion

Water deficit significantly reduced the leaf relative water contents (LRWC) [31]. By considering the high water absorption efficiency of the soil by peripheral hyphae [32], using different types of mycorrhizal fungi could cause a substantial rise in the water status of inoculated plants, which is in association with non-mycorrhizal plants, under water-limited conditions [33].

Using organic fertilizers improves the water potential and maintains leaf turgor pressure, which is in agreement with this study result [34]. Regarding the green manure and vermicompost roles in soil organic matter and water content enhancement, dual-fertilized plants (Fm+v) indicated a high percentage of LRWC in green manured plots.

This study results demonstrated that water stress caused to inhibit mycorrhizal colonization in lingrain roots by 21.4%. These results are in agreement with earlier findings, proposing that mycorrhization will be decreased at low levels of soil moisture [35]. The lower colonization may be due to two reasons; the inadequate carbon availability from the host plants under drought stress and the inhibition of the fungal spore germination and hyphal growth in the rhizosphere soil due to drought stress [36]. To the best of our knowledge, organic matter can affect the activity and frequency of soil microorganisms like AMF, increasing the plant nutrients [37]. Vermicompost promotes mycorrhiza colonization in the roots [38, 39]. The highest percentage of AMF colonization (66%) in Ocimum basilicum roots was observed using vermicompost+ G. intraradices combination compared with control [40]. Therefore, the combination of vermicompost, mycorrhiza, and green manure exhibit high root colonization (63.83%) under full irrigation conditions in this research.

Nutrient absorption directly relates to the soil water status. Thus, the nutrient flow in the soil and uptake by roots decreases by water shortage [41]. Concerning better solubility of phosphorus in irrigated soil, the phosphorus concentration of the leaves indicates a reduction along with water restriction, which is probably caused by reduced solubility, mobility (mass flow or propagation), transfer between the roots and branches, and also the absorption of P under drought conditions [42]. Mycorrhizal inoculation can increase the infection percentage and absorption range in the extra metrical mycelium, and after that, increase the nitrogen absorption rate and phosphorus and improve the nutrition in plants [43]. However, inoculated plants under limited irrigation contained less nitrogen and phosphorus content, which may be due to drought constraints in mycelium development and reduced absorption range compared to infected plants in well-watered conditions [44].

Green manure will increase the soil holding capacity for water and nutrients content and mobility in the upper layer of soil by providing organic matter. Plants can use these elements, and therefore increase their growth and productivity [45]. Moreover, there is an extensive record of the green manure advantages for nitrogen nutrition of succeeding crops. Mineralized nitrogen from leguminous plant residues can provide considerable quantities of N to a subsequent crop [46-Field. Crops. Res. 2018 ">50]. Green manure may protect soil from surface runoff and reduce the rate of rainwater infiltration into the soil and thus reduce nitrogen movement. In addition, Azotobacter, which is present in the rhizosphere of leguminous green manure but not in non-leguminous green manure, can fix large amounts of N from the atmosphere and create relatively N-rich areas in the soil, thus significantly increase the amount of N in the soil [51].

The use of green manure significantly raised the content of soil nutrients compared to the control. As necessary soil elements for crop growth, P and K bioavailabilities were significantly enhanced using green manure, especially in soil layers deeper than 20 cm. The deficiency of available phosphorus, one of the easily fixed elements in the soil, is a significant issue that limits the growth of crops [52-54]. Green manure’s effect on phosphorous availability could be caused by its P uptake and unavailable inorganic P changing to more available organic forms to the subsequent crop [52].

Vermicompost provided high nutrition by balancing the nutrients due to the fact that it may contain all macro and some micro-nutrients. In line with our results, vermicompost treatments increased the uptake of N by the corn plant during vegetative growth and the uptake of P and K during the reproductive growth period of corn, which plays an important part in enhancing corn yield [55].

Water limitation reduced the leaf chlorophyll (by 60%) and also carotenoid (by 55%) concentrations. One of the common symptoms of oxidative stress under drought conditions is chlorophyll content reduction, which may be due to photo-oxidation and pigment degradation [56]. This study results indicated that carotenoids, chlorophyll b, and a significantly decreased under water limitation. These results are not consistent with several studies reported that carotenoid increased under water deficit conditions [57].

Earlier research [58-J. Plant. Nutr. 2018 ">60] approved our results that vermicompost application in soil brought about a remarkable growth in carotenoid content under water deficit conditions. Mycorrhizal inoculation resulted in maintaining a very high chlorophyll content relative to non-mycorrhizal plants in those plants subjected to late-season water limitation. These study results are in agreement with those reported by Metwally et al. [31]. Using green manure can enhance nitrogen availability, which can be used by lingrain in chlorophyll production. Chlorophyll plays a crucial role in photosynthesis, such that increased chlorophyll formation will improve photosynthetic reactions and finally raise plant growth and production. Also, Subaedah and Aladin [45] clarified the enhancement of chlorophyll content by using green manure in maize, which is in parallel with our findings.

Amassing proline under stress conditions is identified as one of the plant remedies for osmotic regulation improving in the leaves. Proline plays a leading role during stress in the plant. In addition to great osmolyte, it acts as a metal chelator, signaling, and antioxidant [61]. Having a higher capacity for osmotic regulation is one of those plants’ characteristics with higher drought tolerance. In this study, AMF-treated plants indicated an enhanced accumulation of proline in water-limited conditions. These results are in agreement with Gheisari Zardak et al. [62] and Tyagi et al. [63] findings. Consequently, AM plants can be physiologically more tolerant against drought and indicate more osmotic regulation during exposure to stress conditions. Bidabadi et al. [64] demonstrated a substantial elevation in proline concentration in pomegranate using vermicompost.

Increasing the TSS concentrations occurred in those plants treated with one of Fm and Fv, along with their combination in both irrigation regimes. However, under limited irrigation conditions, TSS increasing was remarkably higher in mycorrhizal treatments and the presence of green manure. AMF enhances TSS and electrolyte concentrations in host plants. Furthermore, carbohydrates increasing in mycorrhizal plants may be attributed to increased photosynthetic carbon and enzymes activation reduction. Other researchers have reported increased carbohydrates in AM plants [65]. Furthermore, Tejada et al. [66] reported that soluble carbohydrate contents of maize plants were the highest in Trifolium pratense L. amended soils. The increment of leaf TSS of plants treated with vermicompost was also reported by Salehi et al. [67].

The primary role of the GB could be considered as protecting the plasma membrane integrity from drought stress injury and contributing to osmotic regulation. Dual application of mycorrhiza and vermicompost increased the GB content under the water-limited conditions and thus indicating that the fertilizers could alleviate drought stress impacts. Moreover, Hashem et al. [14] reported that enhanced GB accumulation leads to better growth and salt tolerance and subsequently improves the performance of photosynthetic traits.

Lower accumulation of H2O2 proves that increasing the activity of the antioxidant enzymes brings about the removal of ROS and the protection of host plants against oxidative stress [68]. A higher concentration of MDA in leaves, which can indicate the amount of peroxidation of membrane lipids, may be accompanied by higher H2O2 accumulation in drought stress plants (Ma, Ma, et al. [55]). Additionally, fertilized plants (Fm and Fv) indicated less MDA than control, indicating the fertilizers’ involvement in ROS metabolism [64, 69, 70]. In addition, mycorrhiza inoculation reduced the H2O2, and MDA concentration in foxtail millet leaves in drought [71]. Inconsistent with our results, Bidabadi et al. [64] demonstrated a reduction in H2O2 content in vermicompost application.

Moreover, using green manure would result in the reduction of MDA and H2O2 concentrations and, along with that, enhance the catalase and peroxidase activities in water limitation. It appeared that green manure could impede H2O2 accumulation and lipid peroxidation in plants by increasing the water content of the soil. Limited irrigation causes oxidative stress by increasing the production of ROS in plant cells, and oxidative damage is also eased by antioxidant enzymes like APX, CAT, and SOD [72].

In this study, the triple application of mycorrhiza, vermicompost, and green manure increased the CAT activity in both irrigation conditions. Consequently, they can facilitate quick sweeping of ROS, especially H2O2, so that metabolic reactions are less affected. Earlier results confirmed that the mycorrhizal and vermicompost-applied plants indicated considerably higher enzyme activities in stress conditions [59, 69, 73, 74].

Applying the green manure in soils at high doses increased maize grain yield compared to the control soil [75]. Similar results were obtained with the green manure application in wheat grain yield by Gruter et al. [76].

Vermicompost, along with biofertilizers-treated plots, indicated a substantial increase in essential oil content, essential oil yield, and biomass of Ocimum basilicum in comparison with control [40]. Similarly, Doan et al. [77] and Goswami et al. [78] reported an increase in grain yield by using vermicompost.

Mycorrhizal symbiosis enhanced the grain and oil yields in comparison with non-mycorrhizal plants under both irrigation conditions. The results demonstrated that mycorrhizal colonization was a significant factor for lingrain development, especially under limited water conditions with respect to playing a significant role in nutrient and water absorption in water-limited plants [79].

5. Conclusions

These results demonstrated that water limitation could reduce lingrain pigments, nutrient contents, LRWC, colonization percentage, and (grain and oil) yields. The inclusion of vermicompost increased the lingrain performance for most of the investigated traits compared to the control (Fc). Although Fv had no advantage compared to mycorrhizal fertilizers (Fm and Fm+v), its presence and green manure or mycorrhiza could develop a positive synergistic effect on these traits. Moreover, green manure increased the vermicompost efficacy under stress conditions. Also, combining green manure with the dual fertilizer (Fm+v) led to the synergistic effects of vermicompost and mycorrhiza.

In drought stress conditions, adding the green manure improved mycorrhiza efficiency on elevating RWC, leaf nitrogen, chlorophyll a, chlorophyll b, grain yield, oil yield, and antioxidant enzymes activity. All fertilizer treatments (FV, Fm, and Fm+v) imposed less hydrogen peroxide and malondialdehyde on lingrain in both irrigation conditions compared to control.

The co-application of eco-friendly soil amendments would result in profitable lingrain production underwater limitations. Therefore, as a result, the triple fertilizer (Gr×Fm+v) experienced the highest amount of LRWC, leaf nitrogen, chlorophyll a, chlorophyll b, carotenoids, grain yield, and oil yield. In addition, GrFm+v enjoyed the maximum quantity of and also antioxidant enzymes activity under drought stress conditions. Consequently, it can be concluded that the combined use of organic and biological fertilizers (green manure, vermicompost, and mycorrhiza) can also be applied as a solution to maintain the lingrain plant performance under the water-limited condition.

19 Oct 2021

PONE-D-21-27855Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities and Root Colonization in Lingrain (Linum Usitatissimum L.) Under Drought ConditionsPLOS ONE

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Reviewer comments:

Reviewer #1: In the manuscript PONE-D-21-27855, the authors investigated Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities and Root Colonization in Lingrain (Linum Usitatissimum L.) Under Drought Conditions. This work seems interesting, but there are some shortcomings in the manuscript. The authors should revise the complete manuscript in the light of suggested comments.

Abstract: For abbreviation like AMF, when describing the term first time, write complete word and abbreviation in brackets, and then onwards the abbreviation.

Introduction: A lot of research already conducted on green manure, vermicompost and mycorrhiza. Therefore, properly justify your work.

Materials and methods: According to authors, factorial study (three factors) was conducted. How many total plots were used in this experiment?

What standard was used for proline measurement? Please explain to the readers.

Discussion: In this section, the authors should explain the mode of action of green manures and vermicompost on enhancing the drought tolerance.

Conclusion: In the conclusion, no needs to repeat the results. It should be concise and comprehensive.

Typographic and syntax corrections in the text need careful attention of the authors.

Reviewer #2: Dear Authors

Manuscript "Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities and Root

Colonization in Lingrain (Linum Usitatissimum L.) Under Drought Conditions" was received for evaluation. It was a well planned experiment but there are some comments for improvement.

English need to be thoroughly improved.

Materials and methods section needs improvement. Design mentioned in this section is different from that mentioned in abstract.

Cross check statistical analysis, particularly PLSD test results for several traits mentioned in the attached file.

Discussion needs drastic change and there are some suggestions in the attached file.

Conclusion must be brief

It will be more beneficial if authors provide % change due to treatments compared to control instead of repeating same results in the discussion section.

Reviewer #3: General Comments:

Overall, the manuscript presented lot of data and parameters studies during the experiment, but analytical methods section and results section are key areas of concern in this manuscript. The author needs a through revision/redrafting of methodology and results section to make it reader friendly and self-explanatory. Lot of grammatical errors are frequently spotted in the manuscript and whole article needs proof reading before submission.

Specific Comments:

Abstract:

Abstract of manuscript is not clear for readers, it reflects the confusion in elaboration of factors and treatments as treatments are not clear. Line no 29 reflects green manure with dual fertilizer while dual fertilizer treatment is not elaborated in abstract (do you mean by vermicompost and green manure?). The abstract must be very clear and self-explanatory for the reader.

Line No. 21, green manure for ingrain plants, do you mean lingrain? Its typing error or you are using ingrain as verb. You can use either ingrain or plant, cant use ingrain plants as it is repetition error. If you are intended to use ‘lingrain’ plant then you can mention as lingrain plant.

Line No. 23, first factor was green manure (without green manure and Trifolium pratense) its confusing for the reader, its with green manure and indicating without green manure. Please make it very specific and self-explanatory for the readers

Introduction:

Line no 43. Use word greenhouse gases rather gas. As multiple gases contribute in greenhouse effect.

Line no 54, what do you mean by increasing soil structure? How organic fertilizers increase soil structure. Please reconsider such statements.

Line No. 55 and 56, using nutrients from organic sources help maintain organic matter…… Please rephrase the statement may be like use of organic sources for enhance nutrients supply may help maintain organic matter. Your statement reflects that nutrient help maintain organic matter which is ambiguous.

Line No. 66, suggest to write as Arbuscular mycorrhizal fungi (AMF) in omnipresent in land dwelling……..

Line No 77, again using ingrain, its intentional or typing mistake?

Material and Methods:

The material is methods sections is not uniformly drafted. Some analytical methods only reference is quoted while some methods are explained briefly. You need to have harmonized pattern for whole section and its recommended to have briefly drafted analytical procedures that were adopted rather reference only.

Line No. 122, lingrain grain were achieved from…. Does it make sense? Please rephrase may be like lingrain seeds were collected from….

Results and Discussion:

Results are elaborated in a very confusing way, you need to thoroughly revisit and redraft the results sections in specific and clear way that may attract the reader attention. Your results presentation is not understandable for someone who don’t know about your experiment.

Line no 226, colonization of what? its AMF colonization with roots?

Line no 227, sole and dual usage of mycorrhizae…. What is this? Its not explained anywhere in methodology or experimental design.

Line No 227, Sole and dual usage of mycorrhiza and vermicompost (Fv, Fm, and Fm+v) would lead to more colonization in comparison with non-fertilized treatment… Its an assumption or presenting result, it presenting result then it must be is past tense rather future tense.

Line no 229, green manure usage increased colonization in all fertilizer treatments….. you are presenting results here please make it very specific, colonization of what and where? May be AMF colonization with lingrain roots??

Line No. 235, single and dual use of vermicompost… do you mean by combined use of vermicompost and mycorrhizae?? Dual use is not making sense here. You need to rephrase..

Line No. 236, again using future tense in presenting the results, these are not assumption.. please use past tense what was done and what was the result.

Line 242, you are presenting the comparison of year one and year two while in above parameters you have presented the comparison of treatments with control and within treatments. I think you need to have comparison of treatment and with control and if comparing the results of two year then explain for all parameters rather any single parameter.

Line No. 245, On the other hand, they all produced more leaf K than the control treatment? What do you mean by they all?? Explain it properly.

Line No. 275, At this stage, Water limitation significantly increased.

What does it mean? What do you mean by at this stage? Which stage? Water limitation significantly increased, what was increased? You must present the results in very systematic way rather making vague statements.. this does not make sense.

Line No. 282, Application solo? What is this application.

Line No. 307, ingrain or lingrain??

Line No. 320, what is fertilizer treatment?? Not explained anywhere.

Line No. 365, again future tense in results presentation, these are result not assumptions. Present results like you concrete finding rather ambiguous statements

Conclusion:

This section is quite long than required. Same repetition of results in conclusion. Last concluding statement is good. You need to cut it short.

**********

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Submitted filename: PONE-D-21-27855-reviewed file.pdf

Click here for additional data file.

7 Nov 2021

Response to Review Comments

We appreciated PONE’s feedback on our paper. Clearly, some unresolved issues still remained after our first revision. As requested, we further revised the paper by improving the abstract, providing more experimental details as requested, and improving our presentation of the results and related discussions. Many other minor editorial changes and corrections were included to improve the writing style and readability. Below are our responses (in blue) to the review comments.

Reviewer comments:

Reviewer #1:

In the manuscript PONE-D-21-27855, the authors investigated Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities and Root Colonization in Lingrain (Linum Usitatissimum L.) Under Drought Conditions. This work seems interesting, but there are some shortcomings in the manuscript. The authors should revise the complete manuscript in the light of suggested comments.

Abstract: For abbreviation like AMF, when describing the term first time, write complete word and abbreviation in brackets, and then onwards the abbreviation.

Response: Thank you. Done.

Introduction: A lot of research already conducted on green manure, vermicompost and mycorrhiza. Therefore, properly justify your work.

Response: Thank you. Done.

Materials and methods: According to authors, factorial study (three factors) was conducted. How many total plots were used in this experiment?

Response: Total plots number was added to the text.

What standard was used for proline measurement? Please explain to the readers.

Response: The standard was added.

Discussion: In this section, the authors should explain the mode of action of green manures and vermicompost on enhancing the drought tolerance.

Response: Done.

Conclusion: In the conclusion, no needs to repeat the results. It should be concise and comprehensive.

Response: According to your comment we revised the conclusion.

Typographic and syntax corrections in the text need careful attention of the authors.

Response: Typographic and syntax errors in the text were corrected.

Reviewer #2:

Manuscript "Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities and Root

Colonization in Lingrain (Linum Usitatissimum L.) Under Drought Conditions" was received for evaluation. It was a well planned experiment but there are some comments for improvement.

English need to be thoroughly improved.

Response: We re-read the text and fixed any errors.

Materials and methods section needs improvement. Design mentioned in this section is different from that mentioned in abstract.

Response: Done.

Cross check statistical analysis, particularly PLSD test results for several traits mentioned in the attached file.

Response: Done.

Discussion needs drastic change and there are some suggestions in the attached file.

Response: Done

Conclusion must be brief

Response: Done.

It will be more beneficial if authors provide % change due to treatments compared to control instead of repeating same results in the discussion section.

Response: We provided percentage change due to the treatments when possible.

Reviewer #3: General Comments:

Overall, the manuscript presented lot of data and parameters studies during the experiment, but analytical methods section and results section are key areas of concern in this manuscript. The author needs a through revision/redrafting of methodology and results section to make it reader friendly and self-explanatory.

Response: Done.

Lot of grammatical errors are frequently spotted in the manuscript and whole article needs proof reading before submission.

Response: Done.

Specific Comments:

Abstract:

Abstract of manuscript is not clear for readers, it reflects the confusion in elaboration of factors and treatments as treatments are not clear. Line no 29 reflects green manure with dual fertilizer while dual fertilizer treatment is not elaborated in abstract (do you mean by vermicompost and green manure?). The abstract must be very clear and self-explanatory for the reader.

Response: We are grateful for the comment. Done.

Line No. 21, green manure for ingrain plants, do you mean lingrain? Its typing error or you are using ingrain as verb. You can use either ingrain or plant, cant use ingrain plants as it is repetition error. If you are intended to use ‘lingrain’ plant then you can mention as lingrain plant.

Response: We thank you; it was a typing error. Lingrain is correct.

Line No. 23, first factor was green manure (without green manure and Trifolium pratense) its confusing for the reader, its with green manure and indicating without green manure. Please make it very specific and self-explanatory for the readers

Response: Done.

Introduction:

Line no 43. Use word greenhouse gases rather gas. As multiple gases contribute in greenhouse effect.

Response: Done.

Line no 54, what do you mean by increasing soil structure? How organic fertilizers increase soil structure.

Response: We are grateful for the comment. ‘Improving’ was replaced.

Please reconsider such statements.

Line No. 55 and 56, using nutrients from organic sources help maintain organic matter…… Please rephrase the statement may be like use of organic sources for enhance nutrients supply may help maintain organic matter. Your statement reflects that nutrient help maintain organic matter which is ambiguous.

Response: Thank you for the suggestion. Done.

Line No. 66, suggest to write as Arbuscular mycorrhizal fungi (AMF) in omnipresent in land dwelling……..

Response: Done.

Line No 77, again using ingrain, its intentional or typing mistake?

Response: We are sorry for typing mistakes. Done.

Material and Methods:

The material is methods sections is not uniformly drafted. Some analytical methods only reference is quoted while some methods are explained briefly. You need to have harmonized pattern for whole section and its recommended to have briefly drafted analytical procedures that were adopted rather reference only.

Response: Brief explanations of analytical procedures were added.

Line No. 122, lingrain grain were achieved from…. Does it make sense? Please rephrase may be like lingrain seeds were collected from….

Response: Done.

Results and Discussion:

Results are elaborated in a very confusing way, you need to thoroughly revisit and redraft the results sections in specific and clear way that may attract the reader attention. Your results presentation is not understandable for someone who don’t know about your experiment.

Response: The results presentation was improved.

Line no 226, colonization of what? its AMF colonization with roots?

Response: Done.

Line no 227, sole and dual usage of mycorrhizae…. What is this? Its not explained anywhere in methodology or experimental design.

Response: “Single and dual usage of mycorrhiza and vermicompost” was replaced.

Line No 227, Sole and dual usage of mycorrhiza and vermicompost (Fv, Fm, and Fm+v) would lead to more colonization in comparison with non-fertilized treatment… Its an assumption or presenting result, it presenting result then it must be is past tense rather future tense.

Response: We are grateful for the comment. Done.

Line no 229, green manure usage increased colonization in all fertilizer treatments….. you are presenting results here please make it very specific, colonization of what and where? May be AMF colonization with lingrain roots??

Response: Done.

Line No. 235, single and dual use of vermicompost… do you mean by combined use of vermicompost and mycorrhizae?? Dual use is not making sense here. You need to rephrase.

Response: Done.

Line No. 236, again using future tense in presenting the results, these are not assumption. please use past tense what was done and what was the result.

Response: Done.

Line 242, you are presenting the comparison of year one and year two while in above parameters you have presented the comparison of treatments with control and within treatments. I think you need to have comparison of treatment and with control and if comparing the results of two year then explain for all parameters rather any single parameter.

Response: Done.

Line No. 245, On the other hand, they all produced more leaf K than the control treatment? What do you mean by they all?? Explain it properly.

Response: Thank you for the comment. They were clarified.

Line No. 275, At this stage, Water limitation significantly increased.

What does it mean? What do you mean by at this stage? Which stage? Water limitation significantly increased, what was increased? You must present the results in very systematic way rather making vague statements.. this does not make sense.

Response: We are sorry. The statements were corrected.

Line No. 282, Application solo? What is this application.

Response: We are sorry. The statement was corrected.

Line No. 307, ingrain or lingrain??

Response: lingrain is correct.

Line No. 320, what is fertilizer treatment?? Not explained anywhere.

Response: We clarified the fertilizer treatments.

Line No. 365, again future tense in results presentation, these are result not assumptions. Present results like you concrete finding rather ambiguous statements

Response: Done.

Conclusion:

This section is quite long than required. Same repetition of results in conclusion. Last concluding statement is good. You need to cut it short.

Response: We revised the conclusion section and made it shorter.

Submitted filename: Response to Reviewer comments.docx

Click here for additional data file.

29 Nov 2021

Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities and Root Colonization in Lingrain (Linum Usitatissimum L.) Under Drought Conditions

PONE-D-21-27855R1

Dear Dr. Arminia,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Sajid Ali

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PLOS ONE

Reviewers' comments:

Reviewer #1: In the revised manuscript, Eco-friendly soil amendments improve growth, antioxidant activities and root colonization in lingrain (Linum Usitatissimum L.) under drought conditions (PONE-D-21-27855R1), the authors mostly incorporated the suggestions. Now, the manuscript could be considered for publication.

Reviewer #3: I appreciate the significant effort made by the author to revise the manuscript. Authors have address all the comments and correction very nicely thus the paper can be considered for publication

3 Dec 2021

PONE-D-21-27855R1

Eco-Friendly Soil Amendments Improve Growth, Antioxidant Activities, and Root Colonization in Lingrain (Linum Usitatissimum L.) Under Drought Conditions

Dear Dr. Arminia:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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Academic Editor

PLOS ONE