Impact of varying levels of soil salinity on emergence, growth and biochemical attributes of four Moringa oleifera landraces.

Salinity in soil and water is one of the environmental factors that severely hinder the crop growth and production particularly in arid and semi-arid regions. A pot experiment was conducted to investigate the impact of salinity levels (1.5 dS m-1, 3.5 dS m-1, 7.5 dS m-1 and 11.5 dS m-1) on emergence, growth and biochemical traits of moringa landraces under completely randomized design having three replications. Four landraces of Moringa oleifera (Faisalabad black seeded moringa [MFB], Patoki black seeded moringa [MPB], Faisalabad white seeded moringa [MFW] and Rahim Yar Khan black seeded moringa [MRB]) were selected for experimentation. All the salinity levels significantly affected the emergence parameters (time to emergence start, time to 50% emergence, mean emergence time, emergence index and final emergence percentage) of moringa landraces. However, 1.5 dS m-1 and 3.5 dS m-1 were found more favorable. Higher salinity levels (7.5 dS m-1 and 11.5 dS m-1) significantly minimized the root surface area, root projected area, root volume and root density as compared to 1.5 dS m-1, 3.5 dS m-1. Number of branches, leaves, leaflets and leaf length were also adversely affected by 7.5 dS m-1 and 11.5 dS m-1. Maximum seedling fresh and dry weights, and seedling length were recorded at 1.5 dS m-1 followed by 3.5 dS m-1. Chlorophyll a and b contents, carotenoids and membrane stability index were also observed highest at salinity level of 1.5 dS m-1. In case of moringa landraces, MRB performed better regarding emergence attributes, growth parameters, and biochemical analysis followed by MFW as compared to MFB and MPB. Moringa landraces i.e. MRB and MFW were found more tolerant to salinity stress as compared to MFB and MPB.

Introduction

Urbanization is a major threat for agriculture sustainability because agricultural land is being ruthlessly used for commercial purposes [1]. At the same time, various abiotic stresses like soil salinity, drought, extreme temperature and high wind are adversely influencing the production and cultivation of field crops. Among these, salinity is one of the most destructive environmental stress, which causes the reduction of crop yield and its quality [1]. Salinization is considered as a major abiotic stress, especially in semi-arid and arid regions, that affects plant growth throughout the world [2, 3]. Salt stress affects the water uptake, decreases photosynthetic rate and synthesis of photosynthetic pigments resulting in reduction of plant growth [4]. It also reduces the stomatal conductance, thereby limiting CO2 supply to leaves as well inhibits PSII activities [5]. Salinity leads to cause osmotic stress and ionic toxicity which adversely affects growth and economic yield of plants [6]. In Pakistan, salinity stress is at an alarming rate because total cultivated area is 22.1 Mha out of which 6.28 Mha area is salt affected [7]. Human activities are also responsible for secondary salinization by mishandling the land and primary source of salinity [8]. As salt concentration increases in soil, uptake of Zn is decreased and Cd toxicity increases in plants which have deleterious effects on plant growth and development [9].

Moringa oleifera is a perennial tree, commonly known as horseradish found in tropical and sub-tropical regions. It is source of herbal medicine because it is rich in antioxidant like ascorbic acid, tocopherols (Vitamin E) and minerals, and also used as vegetable [10]. There are several clinical studies on moringa trees focused on its pharmacological role in osteoporosis, anemia and diabetes mellitus. Moringa leaves have different therapeutic applications such as antiparasitic, anti-oxidant, anti-inflammatory, antibacterial, antifungal and cardioprotective activities [11]. Moringa leaf extract and root extract are also being used as crop growth enhancer either through foliar application and /or seed priming agents to improve the growth and productivity of field crops [12-15]. Moringa plant may resistant to moderate saline condition [16] but when it is exposed to high salt concentration it behaves as a salt sensitive crop [17]. Under high salinity, uptake of K+ and Ca2+ is restricted in moringa plants with low dry weight, less root and shoot length [18]. In some moringa landraces, photosynthetic pigments also affected under high salinity [19].

To overcome the impact of salinity, various agronomic and genetic approaches are being practiced worldwide [20-22]. Among those, one of them is cultivation of salt resistant flora. According to our best knowledge there is no study about salinity tolerance of different moringa landraces at various salinity levels. This study was planned to explore the impact of various levels of salt stress on growth and development of different moringa landraces present in Pakistan. The present study also investigated the growth and physiological response of different moringa landraces in saline conditions and to determine the optimum, lethal and sub-lethal levels of salt stress.

Materials and methods

Experimental particulars

Experiment was conducted in the wire house, Department of Agronomy, University of Agriculture, Faisalabad to evaluate the adverse effect of sodium chloride on different landraces of moringa. Four landraces of Moringa oleifera (Faisalabad black seeded moringa [MFB], Patoki black seeded moringa [MPB], Faisalabad white seeded moringa [MFW] and Rahim Yar Khan black seeded moringa [MRB]) were selected for experimentation and their seeds were collected from their respective places. Completely randomized design (CRD) with three replications was followed to conduct the experimentation. Four salinity levels i.e. 1.5 dS m-1, 3.5 dS m-1, 7.5 dS m-1 and 11.5 dS m-1 were under study. Salinity level of 1.5 dS m-1 was considered as control. Fifteen seeds of each landrace were sown in the pots having 5 kg of soil on March 8, 2019. The plants were grown for a period of one month under natural conditions in wire house. During this period of growth each pot was irrigated by 150 ml of water daily. Four salinity levels (1.5 ds m-1, 3.5 dS m-1, 7.5 dS m-1 and 11.5 dS m-1) were created by adding calculated amount of NaCl salt and mixing manually according to procedure mentioned in USDA laboratory manual [23]. Three plants from each replication were randomly selected to record the data regarding seedling emergence, growth, physiological attributes and biochemical analyses.

Emergence parameter

Daily emerged seeds were counted until the final seed emerged and following parameters were recorded. Emergence was counted on daily basis until a constant count achieved. Time to start emergence and time taken for 50% germination (T50) were measured according to Farooq et al. [24] formula; [T50 = ti + {(N/2 − ni)/ (nj − ni)} × (tj − ti)]. Mean emergence time (MET) was calculated [25] (MGT = Dn/n). Emergence index (EI) was calculated by AOSA [26] [EI = (number of germinated seed(s)/day of first count) + …. + (number of germinated seeds/days of final count)]. Final emergence percentage was calculated according to formula (FEP = Final no. of seeds germinated / total number of seeds) [27].

Growth, physiological and biochemical analysis

Randomly selected three plants from each pot were observed for their physiological and biochemical analysis. The growth attributes were recorded after one month of emergence. Seedlings fresh weight was recorded at the time of harvesting and dry weight was measured by oven drying the samples until the constant weight obtained. Growth parameters like root length, plant height, leaflet length, number of leaves per plant, number of leaflets per leaf, number of branches per plant were recorded at harvesting stage. Root length, surface area, projected area, average density and volume were measured by using root scanner (STD4800). To determine the chlorophyll a and b and carotenoids, the leaves were taken from each replication and kept in freezer overnight for chilling rupture. The 0.1 g sample form these leaves was taken and dipped in 5 ml of 80% acetone solution and centrifuged for 15 minutes at 10,000 x g. After that, the data were recorded using the UV-visible spectrophotometer against a blank containing 80% acetone at wavelength 480, 645 and 663 nm [28].

Statistical analysis

Collected data regarding emergence, growth, physiological and biochemical attributes were analyzed and evaluated statistically by using statistical package “Statistic 8.1” by employing the Fisher’s analysis of variance (ANOVA) technique at 5% probability level under completely randomized design [29]. Treatments’ means were compared using Tukey Honest Significant Difference (HSD). Microsoft Excel was used for calculation and graphical presentation.

Results

In present study, effect of four salinity levels (1.5, 3.5, 7.5 and 11.5 dS m-1) were observed on emergence and seedling vigor of four moringa landraces (MFB, MPB, MFW and MRB). The significance of salinity levels and moringa landraces as well as their interaction is presented in Table 1. It has been observed that salinity levels significantly affected the emergence parameters (time to emergence start, time to 50% emergence, mean emergence time, emergence index and final emergence percentage) of all moringa landraces at various extent. Overall, at highest salinity level (11.5 dS m-1), moringa seeds took maximum time for emergence than at control (lowest salinity level; 1.5 dSm-1). Similarly, emergence index (EI) and final emergence percentage (FEP) were also recorded best at lowest salinity level as compared to highest salinity level. Moreover, all moringa landraces showed different emergence rate at different salinity level, thus maximum emergence attributes were recorded in MFB followed by MPB, MFW and MRB (Fig 1).

Table 1

Mean sum of squares of emergence, growth and physiological parameters of moringa landraces grown under varying salinity levels.

SOV	DF	TSE	T50	MET	EI	FEP	RSA	RPA	RV	RD	NB
Salinity levels (SL)	3	100.13**	21.25**	36.31**	24.07**	1211.39**	703.12**	69.89**	0.077**	0.16**	46.46**
Landraces (LR)	3	8.47**	35.06**	88.36**	5.24**	49.28**	137.87**	12.81**	0.022**	0.013**	3.62**
SL × LR	9	2.28NS	0.47NS	1.50NS	0.21NS	6.26NS	232.25**	3.17**	0.012**	0.0042**	0.403NS
		NL	NLL	LL	SFW	SDW	SL	Chl a	Chl b	CAR	MSI
Salinity levels (SL)	3	54.83**	72.35**	29.83**	2782.06NS	315.970**	4242.06**	6.97NS	0.0829**	1.55021	1855.10**
Landraces (LR)	3	3.94**	5.41**	0.716**	7.75**	5.388**	84.71**	0.0057**	0.034**	0.0096 NS	18.35**
SL × LR	9	0.33NS	1.15NS	0.329*	11.88NS	0.633NS	16.42NS	0.060**	0.0187**	0.0094*	3.02**

SOV = Source of variance, DF = Degree of freedom, Chl = Chlorophyll, CAR = Carotenoids, TSE = Time to start emergence, T50 = 50% emergence time, MET = Mean emergence time, EI = Emergence index, FEP = Final emergence percentage, RSA = Root surface area, RPA = Root projected area, RV = Root volume, RD = Root density, NL = No of leaves per plant, NB = No of branches, NLL = no of leaflets per plant, LL = leaf length, SFW = Seedling fresh weight, SDW = Seedling dry weight, SL = Seedling length, MSI = Membrane stability index, NS = Non-significant

* = Significant at P ≤ 0.05

** = Significant at P ≤ 0.01.

Fig 1

Impact of salinity on time to start emergence (TSE), time to 50% emergence time (T50), mean emergence time (MET), emergence index and final emergence percentage (FEP) of moringa landraces.

The effect of various salinity levels (1.5, 3.5, 7.5 and 11.5 dSm-1) was observed on morphological attributes of moringa landraces (MFB, MPB, MFW, MRB). Data recorded for seedling fresh weight demonstrated that all moringa landraces showed maximum fresh biomass at minimum salinity level (1.5 dS m-1), while least seedling fresh weight was observed at highest salinity level (11.5 dS m-1). Similarly, the results obtained for seedling dry weight was showed that among different moringa landraces maximum seedling dry weight was noted at 1.5 dS m-1 salinity level, while least was observed at 11.5 dS m-1 salinity level. However, the order of improvement of seedling dry weight among landraces was: MBR > MPB > MFW > MFB. Data, recorded for seedling length, showed significant difference between moringa landraces and salinity levels but their interaction was non-significant. Among the salinity levels the plant showed the maximum seedling length at minimum salinity level (1.5 dS m-1), while the maximum seedling length of MBR landrace was noted as compared to other landraces. However, at highest salinity level (11.5 dS m-1) maximum reduction in seedling length was recorded (Fig 2).

Fig 2

Impact of salinity on time to seedling fresh weight, seedling dry weight and seedling length of moringa landraces.

Data, recorded for chlorophyll contents, showed that salinity levels decreased the chlorophyll a content of all landraces. All the landraces showed different behavior under salinity levels, the order reduction among all moringa landraces at 1.5 dS m-1 salinity level was: MPB > MFB > MRB > MFW, while order of reduction at 3.5 dS m-1 was: MFB > MBP > MFW > MRB, at 7.5 dS m-1 salinity level reduction order was: MRB > MFB > MFW > MPB, similar at 11.5 dS m-1 salinity level reduction order was: MFW > MRB > MPB > MFB. Thus overall, maximum chlorophyll a content was noted in MFW landrace and MPB synthesized minimum chlorophyll a pigment. Moreover, data recorded for chlorophyll b showed that among all moringa landraces, maximum chlorophyll b pigments were observed in MFW while MBP had minimum chlorophyll b contents. Results obtained for carotenoids concentration showed that among moringa landraces, MRB showed maximum carotenoids while MFW showed minimum. However highest carotenoids concentration was noted at 7.5 dS m-1 salinity level and lowest concentration was observed at 11.5 dS m-1 salinity level. Membrane stability index (MSI) was significantly different among the salinity level, lowest reduction of MSI noted at 3.5 dS m-1 salinity level and highest reduction was observed at 11.5 dS m-1. Among all moringa landraces, MRB showed more MSI, while MPB had least MSI value (Fig 3).

Fig 3

Impact of salinity on chlorophyll a and b contents, carotenoids and membrane stability index of moringa landraces.

Data showed that higher soil salinity significantly decreased the root surface area (RSA) of all moringa landraces. Among all salinity level maximum RSA was observed at 1.5 dS m-1 salinity level, while minimum RSA was noted at highest salinity level i.e. 11.5 dS m-1. However, among all moringa landraces the order of RSA was: MRB > MFB > MFW > MPB. Data noted for root projected area (RPA) showed significant difference among salinity levels and moringa landraces. Maximum reduction in RPA was observed at 11.5 dS m-1. Maximum RPA was observed in MRB landrace followed by MFB, MFW and MPB. Results obtained for root volume (RV) demonstrated that there was significant difference between salinity levels as well as moringa landraces. Salinity levels significantly decreased the RV of all moringa landraces, but highest reduction was noted at 11.5 dS m-1 salinity level. Moreover, maximum RV was observed in MRB landrace followed by MPB, MFW and MFB landraces. Similarly, salinity levels significantly affect the root density (RD) of moringa landraces, maximum reduction was observed in MFW landrace at 11.5 dS m-1 salinity level, while highest RD was noted in MFB landrace at minimum salinity level i.e. 1.5 dS m-1 (Table 2). Salinity significantly decreased the total number of branches, leaves, leaflets and leaf length of all moringa landraces, but maximum reduction was recorded at 11.5 dS m-1 salinity level and least reduction was observed at 1.5 dS m-1 salinity level. There was also significant difference among the landraces. Highest number of branches, leaves and leaf length was noted in MRB moringa, while maximum number of leaflets was observed in MFW moringa landrace (Table 3).

Table 2

Impact of various salinity levels on root attributes of moringa landraces.

Treatments	Root surface area (cm 2 )					Root projected area (cm 2 )
	MFB	MPB	MFW	MRB	Mean (SL)	MFB	MPB	MFW	MRB	Mean (SL)
1.5 d Sm-1	26.36a	13.99de	23.23ab	21.69bc	21.19A	7.05a	3.67d-f	4.06c-e	7.9633a	5.69A
3.5 d Sm-1	16.69de	6.92f	13.00e	17.73cd	13.58C	6.59ab	4.58b-d	6.41ab	5.88a-c	5.86A
7.5 d Sm-1	7.32f	5.48fg	5.65fg	13.99de	8.11C	2.28e-g	1.58fg	1.39g	4.43b-e	2.42 B
11.5 d Sm-1	3.99fg	1.91g	2.82g	4.78fg	3.37D	1.18g	0.812g	0.67g	1.405g	1.02C
Mean (LR)	13.591 A	6.949 C	11.174 B	14.548 A		4.27A	2.6632 B	3.1337 B	4.92A
HSD	SL = 1.6128, LR = 1.4506, SL×LR = 3.9738					SL = 0.5387, LR = 0.8057, SL×LR = 2.2071
Treatments	Root volume (cm 3 )					Root density
	MFB	MPB	MFW	MRB	Mean (SL)	MFB	MPB	MFW	MRB	Mean (SL)
1.5 d Sm-1	0.15de	0.29a	0.28a-c	0.33a	0.2567 A	0.5127 a	0.4660 b	0.4550 b	0.4427 b	0.4691 A
3.5 d Sm-1	0.102d-g	0.26ab	0.135d-f	0.27a	0.1931 B	0.5377 a	0.3967 c	0.3697 c	0.4453 b	0.4373 B
7.5 d Sm-1	0.17b-d	0.09d-g	0.039g	0.17cd	0.1193 C	0.4590 b	0.3743 c	0.3987 c	0.4613 b	0.4233 B
11.5 d Sm-1	0.09d-g	0.06fg	0.66e-g	0.09d-g	0.0753 D	0.2050 d	0.2057 d	0.2187 d	0.2187 d	0.2123 C
Mean (LR)	0.1280 C	0.1769 B	0.1246 C	0.2149 A		0.4286 A	0.3607 C	0.3605 C	0.3922 B
HSD	SL = 0.0417, LR = 0.0322, SL×LR = 0.0883					SL = 0.0218, LR = 0.0149, SL×LR = 0.0408

Table 3

Impact of various salinity levels on growth attributes of moringa landraces.

Treatments	Number of branches					Number of leaves
	MFB	MPB	MFW	MRB	Mean (SL)	MFB	MPB	MFW	MRB	Mean (SL)
1.5 d Sm-1	7.67	7.00	6.67	8.67	7.5A	8.33	7.67	8.33	9.67	8.50 A
3.5 d Sm-1	7.00	6.33	7.00	7.67	7.0A	7.0	7.33	7.00	8.33	7.42 B
7.5 d Sm-1	4.67	3.67	4.17	5.33	4.46A	5.0	4.67	4.00	5.33	4.75 C
11.5 d Sm-1	3.33	3.33	3.33	3.67	3.42C	4.0	3.67	3.67	4.67	4.00 C
Mean (LR)	5.67 AB	5.08 B	5.29 B	6.33 A		6.08 B	5.83 B	5.75 B	7.00 A
HSD	SL = 1.0174, LR = 1.0239, SL×LR = 2.8048					SL = 0.7603, LR = 0.8904, SL×LR = 2.4391
Treatments	Number of leaflets					Leaf length
	MFB	MPB	MFW	MRB	Mean (SL)	MFB	MPB	MFW	MRB	Mean (SL)
1.5 d Sm-1	10.0	9.66	11.33	10.0	10.25A	8.33ab	7.67a-c	8.3333 ab	9.67a	8.5A
3.5 d Sm-1	7.0	7.667	10.0	9.33	8.5B	7.0b-e	7.33a-d	7.0b-e	8.33ab	7.42B
7.5 d Sm-1	5.33	5.667	5.67	6.0	5.67C	5.0d-f	4.66ef	4.0f	5.33c-f	4.75C
11.5 d Sm-1	4.33	4.667	5.33	5.67	5.0C	4.0f	3.66f	3.67f	4.66ef	4.0C
Mean (LR)	6.66B	6.92B	8.08A	7.75AB		6.08B	5.83B	5.75B	7.0A
HSD	SL = 1.0617, LR = 1.0854, SL×LR = 2.9733					SL = 0.3841, LR = 0.4178, SL×LR = 1.1446

Discussion

Salinity is also known to impair seed germination, emergence and early seedling growth [30]. The presence of more salt in the soil negatively affected physiological and biochemical attributes in plants which decreased plant biomass and economic yield [31]. Various plants have different salt tolerance levels, as do most cereals, including moringa. The results of present study depict that, the speed and spread of seedling of different moringa landraces were reduced with increasing salinity (Fig 1). These results corroborate the findings of previous studies that higher salt stress retards seed germination and seedling emergence rate, emergence index due to osmotic effect, which is deleterious and prevents the plant in maintaining their proper nutritional requirements necessary for seedling vigor [32, 33]. Salt stress delayed the seedling germination, lower seedling growth rate resulting in hampered crop growth and yield, because salinity lowered the soil water potential [34, 35] as well as increase Na+ level in plant cells which limit plant productivity, in severe plant death occur [36].

A stronger root system could probably lead to more plant biomass production on normal growth conditions or less yield reduction under abiotic stress and nutrient-deficient conditions. Root development requires uptake, translocation, and metabolism of essential nutrients. The development of root system architecture is a biological process that interacts with ion homeostasis [37]. In this study, root surface area, root projected area, root volume and density significantly affected by increasing the salinity concentration in soil in all moringa landraces (Fig 2). Minimum root attributes were affected in MRB moringa landrace, this may be due to genetically strong adaptation as compared to other landraces, while root length was severely affected by salt stress. Similarly, salt stress affected the root architecture in wheat cultivars due to ionic imbalance [37]. Moreover, it has been found that salinity reduced the root density and root surface area [38] and root density [39, 40] in plants. It is also reported that organic amendments play a vital role to reduce the impact of abiotic stress [41, 42].

Generally, salinity is known to cause a marked reduction in photosynthetic (chlorophyll a, b) and accessory pigments (carotenoids) as noted in present study (Fig 3). High salt concentration caused the formation of chlorophyllase and hampered nitrogen absorption which disturb the biosynthesis of photosynthetic pigments which adversely affect photosynthetic rate and ultimately limited crop yield [43, 44]. Salt stress changed the ultrastructure of cellular components by enhancing the reactive oxygen species (ROS) which cause lipid peroxidation of plasma membrane, ultimately photosynthetic machinery disturbed, resulting growth and economic yield of plants reduced [45]. At high salinity levels, crude protein, carotenoids and chlorophyll were reduced while antioxidants, enzymes, non-enzymes, proline and total carbohydrates were also increased [19]. Sustainable production is possible if integrated application of agronomic and cultural practices is insured [46].

The general effect of salinity is to reduce the growth rate resulting in smaller leaves, shorter stature, and sometimes fewer leaves. Moringa plants may resistant to moderate saline condition [16] but when it is expose to high salt concentration its growth decreased [17] which was in line with results of current study in which salinity stress effects the growth and development of moringa landraces. Because high salinity level inhibits the uptake of K+ and Ca2+ as well as enhance Na+ toxicity, which hamper plant crucial mechanism [47], as a consequence plant length and fresh and dry biomass affected [18]. Plant growth decreased by increasing the level of salinity, all landraces showed lower seedling length and fresh dry biomass. It is also reported that unfavorable conditions significantly reduced the emergence and growth attributes [48].

Nevertheless, MRB landrace had improved seedling growth compared to other landraces. The reduction in plant length, fresh and dry weight may be due to increase of Na+ ion toxicity, loss of cell membrane permeability and senescence [49-51]. Environmental stress is responsible for reduction in the growth and development of crops [52]. It has been reported that Na+ toxicity significantly hampered the photosynthesis process, resulting in energy supply to plant body reduced, hence shoot and root growth decreased [36]. The results of the present study show that the growth of moringa landraces (number of branches, leaves, leaflets and leaf length) was decreased significantly with increasing salinity levels in all landraces, resulting limited plant growth. Overall, MRB showed better adaptation as compared to other landraces. Similar findings were also reported on various plants which were in line with our results [53, 54].

Conclusion

In present study, impact of four salinity levels (1.5, 3.5, 7.5 and 11.5 dS m-1) were observed on seedling emergence, growth and biochemical attributes of four landraces of Moringa oleifera (Faisalabad black seeded moringa [MFB], Patoki black seeded moringa [MPB], Faisalabad white seeded moringa [MFW] and Rahim Yar Khan black seeded moringa [MRB]). It can be concluded from the results of current experimentation that moringa can tolerate the salinity up to 3.5 dS m-1 and retain optimum growth. Its growth is adversely affected as the salinity level increases up to 7.5 dS m-1. Regarding moringa landraces, Rahim Yar Khan black seeded moringa and Faisalabad white seeded moringa are more resistant to salinity stress as compare to Faisalabad black seeded moringa and Patoki black seeded moringa.

13 Jan 2022

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Article entitled “Impact of Varying Levels of Soil Salinity on Emergence, Growth and Biochemical Attributes of Four Moringa Oleifera Landraces” is of worth reading for farmers and scientific community. It can be considered for publication after changes because some suggestion and comments are given below.

Abstract

Line 1 replace “or” with “and”

Line 1 replace “major adversely” with “adverse”

Line 2 replace semiarid with semi-arid

Line 3 replace explore with investigate/inspect

Line 11 replace reduced with minimized

Line 11 misspelled room change with root

Introduction

Line 1 use one word urbanization or over population

Line 2 replace industrialization with commercial purposes.

Line 8 remove of roots

2nd paragraph line 1 remove reached

3rd paragraph line 1 remove herb

Line 2 it’s a source of herbal medicine because it is rich in antioxidants like….. and also used as vegetable.

Line 6 remove frequently

Line 8 mistake remove line root extract of moringa is also

Line 9 misspelled trees not tress

Line 11 it behaves as a

Line 13 replace Ca2+ with Ca2+

Last paragraph last line saline conditions

Italic “et al’’ in whole intro

Material and methods

Line 6 Experiment not experiments

Sowing and treatment imposition

Line 2 completely randomized block design

Line 3 replace light and temperature with natural conditions

Line 4 replace were with was

Line 5 remove s standards

Emergence parameter

Line 7 replace were with was

Growth and biochemical analysis

Line 2 replace at seedling age of with ‘’after’’

Line 3 seedlings

Statistical analysis is in italic

Result

Rearrange the line 3

Line 7 moringa seed not seedling

Paragraph 2 line 2 remove different

Line 9 only write showed

Paragraph 3 replace pigment with contends

Paragraph 4 line 7 remove decreased

Discussion

Discussion is very simple. Add some recent literature to support your findings in a comparative way.

Line 2 changes replace with attributes, recent references may be cited to improve the section with scientific approach.

Conclusion

Line 2 vigor or growth use one thing or write seedling emergence and vigor, growth…

Reference

Recheck and cross match all the reference. Format the reference according to journal requirement.

Table and figures

All the tables and figures may be added in the page after first citation.

Reviewer #2: The present study was conducted to evaluate four different levels of soil salinity on emergence, growth and biochemical attributes of four moringa oleifera landraces. The authors write the MS appreciably well however.

Suggestions/comments

Abstract

Line 07: Add the line regarding design of experiment.

Line 10: Add unit with salinity level of ‘7.5’ and ‘11.5’.

Line 13: Replace ‘Room volume’ with ‘root volume’.

Line 13: Add unit with salinity level of ‘7.5’.

Introduction

Line 01-02: Reference ‘Shahbaz and Ashraf, 2013’ paste after the industrialization.

Line 06: Remove the world ‘also’.

Paragraph 2, Line 03: replace ‘abiotic and abiotic’ with ‘biotic and abiotic’.

Paragraph 2, Line 03: Add names of biotic stresses.

Paragraph 3, Line 8-9: confused, rewrite it.

Paragraph 3, Line 11: Remove ‘is’ and replace expose with ‘exposes’.

Paragraph 3, Line 12-13: confusing, rewrite it.

Paragraph 4, Line 05: Add the word ‘different’ before the moringa landraces.

Material and methods

Experimental particulates:

Line 09: Rewrite the four levels of soil salinity

Emergence Parameter:

Line 04-06: Confusing and missing sentence, rewrite it

Line 09: write it just start of the parameters in line 3.

Statistical analysis

Line 1-2: Rewrite it

Result

Line 03: Rewrite it

Line 07: Confusing, rewrite it.

Paragraph 02, loin 11: Add ‘as’ after the word noted.

Paragraph 03, loin 09: Replace ‘was’ with ‘were’.

Paragraph 03, loin 10: Remove the word ‘all’.

Paragraph 03, loin 15: Remove the word ‘salinity level’.

Paragraph 04, loin 01: Rewrite it.

Paragraph 04, loin 07: confusing, reconsider it.

Discussion:

Line 1, 2, 3: Justify results with previous work and give possible reasons of correlate or contrary of your results with previous work.

Paragraph 03, line 02: Give reference of your data (fig 3)

Conclusion

Line 04: Replace ‘can be, with ‘is’.

Reference

Reference 07: Recheck and rewrite it

Reference EL Sabagh is mixing with other ref. and rewrite it.

Reference Ellis RA is missing volume, page and format is also different. Correct it.

Figure

Fig 3: Units format

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Talha Javed

Reviewer #2: Yes: Rubab Shabbir

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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26 Jan 2022

Response Sheet

Respected Editor-in-Chief,

PLOS ONE.

Subject: Response to Comments

Title: Impact of Varying Levels of Soil Salinity on Emergence, Growth and Biochemical Attributes of Four Moringa oleifera Landraces.

We are very much thankful to reviewers for sparing time to review our manuscript and provided valuable comments and suggestions for the improvement of the manuscript.

I, Shahbaz Khan, corresponding author of the manuscript, am enclosing herewith a revised manuscript entitled “Impact of Varying Levels of Soil Salinity on Emergence, Growth and Biochemical Attributes of Four Moringa oleifera Landraces” for publication in “PLOS ONE” after possible improvements.

All the comments and suggestions are addressed accordingly and incorporated in the revised manuscript. Details of individual comments are given below.

REVIEWER 1

General Comments

Reviewer #1: Article entitled “Impact of Varying Levels of Soil Salinity on Emergence, Growth and Biochemical Attributes of Four Moringa Oleifera Landraces” is of worth reading for farmers and scientific community. It can be considered for publication after changes because some suggestion and comments are given below.

Response: We are very much thankful to you for sparing time to review our manuscript and provided valuable comments and suggestions for the improvement of the manuscript.

Comment: Abstract: Line 1 replace “or” with “and”.

Response: Suggestions are incorporated and highlighted.

Comment: Line 1 replace “major adversely” with “adverse”.

Response: Suggestions are incorporated and highlighted.

Comment: Line 2 replace semiarid with semi-arid.

Response: Suggestions are incorporated and highlighted.

Comment: Line 3 replace explore with investigate/inspect.

Response: Suggestions are incorporated and highlighted.

Comment: Line 11 replace reduced with minimized.

Response: Suggestions are incorporated and highlighted.

Comment: Line 11 misspelled room change with root.

Response: Suggestions are incorporated and highlighted.

Comment: Introduction: Line 1 use one word urbanization or over population.

Response: Suggestions are incorporated and highlighted.

Comment: Line 2 replace industrialization with commercial purposes.

Response: Suggestions are incorporated and highlighted.

Comment: Line 8 remove of roots.

Response: Suggestions are incorporated and highlighted.

Comment: 2nd paragraph line 1 remove reached.

Response: Suggestions are incorporated and highlighted.

Comment: 3rd paragraph line 1 remove herb.

Response: Suggestions are incorporated and highlighted.

Comment: Line 2 it’s a source of herbal medicine because it is rich in antioxidants like….. and also used as vegetable.

Response: Suggestions are incorporated and highlighted.

Comment: Line 6 remove frequently.

Response: Suggestions are incorporated and highlighted.

Comment: Line 8 mistake remove line root extract of moringa is also.

Response: Suggestions are incorporated and highlighted.

Comment: Line 9 misspelled trees not tress.

Response: Suggestions are incorporated and highlighted.

Comment: Line 11 it behaves as a.

Response: Suggestions are incorporated and highlighted.

Comment: Line 13 replace Ca2+ with Ca2+.

Response: Suggestions are incorporated and highlighted.

Comment: Last paragraph last line saline conditions.

Response: Suggestions are incorporated and highlighted.

Comment: Italic “et al’’ in whole intro.

Response: Suggestions are incorporated and highlighted.

Comment: Material and methods, Line 6 Experiment not experiments.

Response: Suggestions are incorporated and highlighted.

Comment: Sowing and treatment imposition; Line 2 completely randomized block design.

Response: Suggestions are incorporated and highlighted.

Comment: Line 3 replace light and temperature with natural conditions.

Response: Suggestions are incorporated and highlighted.

Comment: Line 4 replace were with was.

Response: Suggestions are incorporated and highlighted.

Comment: Line 5 remove s standards.

Response: Suggestions are incorporated and highlighted.

Comment: Emergence parameter; Line 7 replace were with was.

Response: Suggestions are incorporated and highlighted.

Comment: Growth and biochemical analysis; Line 2 replace at seedling age of with ‘’after’’ Line 3 seedlings.

Response: Suggestions are incorporated and highlighted.

Comment: Statistical analysis is in italic.

Response: Suggestions are incorporated and highlighted.

Comment: Result; Rearrange the line 3.

Response: Suggestions are incorporated and highlighted.

Comment: Line 7 moringa seed not seedling.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 2 line 2 remove different.

Response: Suggestions are incorporated and highlighted.

Comment: Line 9 only write showed.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 3 replace pigment with contends.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 4 line 7 remove decreased.

Response: Suggestions are incorporated and highlighted.

Comment: Discussion; Discussion is very simple. Add some recent literature to support your findings in a comparative way.

Response: Suggestions are incorporated and highlighted.

Comment: Line 2 changes replace with attributes, recent references may be cited to improve the section with scientific approach.

Response: Suggestions are incorporated and highlighted.

Comment: Conclusion; Line 2 vigor or growth use one thing or write seedling emergence and vigor, growth….

Response: Suggestions are incorporated and highlighted.

Comment: Reference; Recheck and cross match all the reference. Format the reference according to journal requirement.

Response: Suggestions are incorporated and highlighted.

Comment: Table and figures; All the tables and figures may be added in the page after first citation.

Response: Suggestions are incorporated and highlighted.

REVIEWER 2

General comments

The present study was conducted to evaluate four different levels of soil salinity on emergence, growth and biochemical attributes of four moringa oleifera landraces. The authors write the MS appreciably well however.

Comment: Abstract; Line 07: Add the line regarding design of experiment.

Response: Suggestions are incorporated and highlighted.

Comment: Line 10: Add unit with salinity level of ‘7.5’ and ‘11.5’.

Response: Suggestions are incorporated and highlighted.

Comment: Line 13: Replace ‘Room volume’ with ‘root volume’.

Response: Suggestions are incorporated and highlighted.

Comment: Line 13: Add unit with salinity level of ‘7.5’.

Response: Suggestions are incorporated and highlighted.

Comment: Introduction; Line 01-02: Reference ‘Shahbaz and Ashraf, 2013’ paste after the industrialization.

Response: Suggestions are incorporated and highlighted.

Comment: Line 06: Remove the world ‘also’.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 2, Line 03: replace ‘abiotic and abiotic’ with ‘biotic and abiotic’.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 2, Line 03: Add names of biotic stresses.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 3, Line 8-9: confused, rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 3, Line 11: Remove ‘is’ and replace expose with ‘exposes’.

Response: It is passive voice sentence.

Comment: Paragraph 3, Line 12-13: confusing, rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 4, Line 05: Add the word ‘different’ before the moringa landraces.

Response: Suggestions are incorporated and highlighted.

Comment: Material and methods, Experimental particulates; Line 09: Rewrite the four levels of soil salinity.

Response: Suggestions are incorporated and highlighted.

Comment: Emergence Parameter; Line 04-06: Confusing and missing sentence, rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Line 09: write it just start of the parameters in line 3.

Response: Suggestions are incorporated and highlighted.

Comment: Statistical analysis; Line 1-2: Rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Result; Line 03: Rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Line 07: Confusing, rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 02, loin 11: Add ‘as’ after the word noted.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 03, loin 09: Replace ‘was’ with ‘were’.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 03, loin 10: Remove the word ‘all’.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 03, loin 15: Remove the word ‘salinity level’.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 04, loin 01: Rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 04, loin 07: confusing, reconsider it.

Response: Suggestions are incorporated and highlighted.

Comment: Discussion; Line 1, 2, 3: Justify results with previous work and give possible reasons of correlate or contrary of your results with previous work.

Response: Suggestions are incorporated and highlighted.

Comment: Paragraph 03, line 02: Give reference of your data (fig 3).

Response: Suggestions are incorporated and highlighted.

Comment: Conclusion; Line 04: Replace ‘can be, with ‘is’.

Response: Suggestions are incorporated and highlighted.

Comment: Reference; Reference 07: Recheck and rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Reference EL Sabagh is mixing with other ref. and rewrite it.

Response: Suggestions are incorporated and highlighted.

Comment: Reference Ellis RA is missing volume, page and format is also different. Correct it.

Response: Suggestions are incorporated and highlighted.

Comment: Figure; Fig 3: Units format.

Response: Suggestions are incorporated and highlighted.

Submitted filename: Response to Reviewers.doc

Click here for additional data file.

2 Feb 2022

Impact of Varying Levels of Soil Salinity on Emergence, Growth and Biochemical Attributes of Four Moringa oleifera Landraces

PONE-D-21-40489R1

Dear Dr. Khan,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Adnan Noor Shah, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Talha Javed

Reviewer #2: Yes: Rubab Shabbir