335 Research report Fluoride 53(2 Pt 2):335-355 of Pisum ...Madeeha Ansari,a Asma Zulfiqar,a Abudllah Jalal,d Aleena Nizame Lahore and Peshawar, Pakistan ABSTRACT: Fluoride in our

Research reportFluoride 53(2 Pt 2):335-355

Ameliorative effects of salicyclic acid on dry biomass and growthof Pisum sativum L under sodium fluoride stress

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[Now published in full after the initial publication as an Epub ahead of print on June 9, 2020, at www.fluorideresearch.online/epub/files/082.pdf]

April-June 2020 Ahmed, Karamat, Haider, Jabeen, Ahmad, Ansari, Zulfiqar, Jalal, Nizam

AMELIORATIVE EFFECTS OF SALICYLIC ACID ON DRY BIOMASS AND GROWTH OF PISUM SATIVUM L. UNDER SODIUM FLUORIDE STRESS

Shakil Ahmed,a,* Maryam Karamat,a Azeem Haider,b Fareeha Jabeen,a M Nauman Ahmad,c Madeeha Ansari,a Asma Zulfiqar,a Abudllah Jalal,d Aleena Nizame

Lahore and Peshawar, Pakistan

ABSTRACT: Fluoride in our physical environment causes several health hazards, notonly to humans but to plants as well. Its high concentration in soil, water, and vegetationis a major problem for today’s world. The present study was conducted to assess theimpact of salicylic acid on the growth and biomass production of Pisum sativum L.grown under sodium fluoride (NaF) stress. Pisum sativum L. (pea) is an edible plantused to nourish humans as its seeds are rich in protein and carbohydrates. Twovarieties of Pisum sativum, RKS-510 and Classic, were grown during the 2015-16season with various concentrations of NaF, i.e., 50, 100, 150, and 200 ppm, being appliedas a soil drench on a biweekly basis to create stress for the plants. The gradualaccumulation of the fluoride ion caused toxic effects on the biochemical processes ofthe plants. A foliar application of 100, 200, and 300 ppm salicylic acid was also givenexogenously to examine its effects on the plants under NaF stress. It was observed thatthe plant height and biomass production of both the varieties of pea plant decreasedunder NaF stress while this decline was overcome by the exogenous application ofsalicylic acid at the 100 and 200 ppm levels. One hundred and 200 ppm salicylic acidenhanced the growth of the plants while 300 ppm salicylic acid had a reducing effectsimilar to that of NaF. As salicylic acid at 100 and 200 ppm had ameliorative effects in onplants under NaF stress, we concluded that the foliar application of salicylic acidpromotes salt tolerance in two different cultivars of Pisum sativum L. Keywords: Fluoride stress; Pakistan; Pisum sativum L.; Salicylic acid; Yield.

INTRODUCTION

Fluoride occurs naturally in earth’s crust and acts as a pollutant in theenvironment.1 Fluorine is the lightest member of the halogen group, is the thirteenthmost common element in the earth’s crust, and is one of the most reactive of allchemical elements.2,3 It is not therefore usually found as fluorine in the environmentbut rather as fluorides which together represent about 0.06–0.09% of the earth’scrust.3 Fluorides are found at significant levels in a wide variety of mineralsincluding fluorspar (fluorite) [CaF2], rock phosphate (phosphorite) which ranges incomposition from tricalcium phosphate [Ca3(PO4)2] to hydroxyapatite andfluorapatite, fluorapatite (apatite) [Ca5(PO4)3F], cryolite [Na3AlF6], mica[KAl3Si3O10(OH)2], and hornblende [Ca2(Mg, Fe, Al)5(Al,Si)8O22(OH)2] wherefluorine often substitutes for hydroxyl in the crystalline structure.3 Difluorine hasbeen found by in situ with NMR spectroscopy as an inclusion in antozonite, a variantof fluorite, which contains uranium and it has been proposed that beta radiation fromuranium and its daughter nuclides strip fluoride anions of their extra electron with theresulting fluorine atom quickly forming a difluoride anion which is highly mobileand it loses an electron at a defect and forms difluorine, in bubbles up to 200 nm in

aDepartment of Botany, University of the Punjab, Lahore 54590, Pakistan; bInstitute of AgriculturalSciences, University of the Punjab, Lahore 54590, Pakistan; cAgricultural Chemistry Department,University of Agriculture, Peshawar, Pakistan; dIBGE, University of Agriculture, Peshawar,Pakistan; eDepartment of Zoology, University of Peshawar, Peshawar, Pakistan; Forcorrespondence: Dr Shakil Ahmed, Applied Environmental Biology & Environmental BiotechnologyResearch Lab, Department of Botany, University of the Punjab, Lahore 54590, Pakistan; E-mail:[email protected]

http://www.fluorideresearch.online/epub/files/082.pdf



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size.4 Different types of soil have 20 to 1000 µg/g of soil fluoride content.5Compounds of fluorine and many fluorides are extremely poisonous and more toxicthan potassium and sodium. They are present in contaminated acidic soil along withother pollutants which come from different sources. The main source of fluoride inirrigation water is from the high use of phosphate fertilizers, and deposition fromceramic industries and brick kilns.6 Soil, water, and vegetation may be contaminatedby the presence of fluoride from various sources and adverse effects of this on plantbiology have been reported.7

Fluoride significantly affects the various physiological processes in plantsincluding interference with enzymatic activities and the division and expansion ofcells resulting in the slow growth of plants and a reduction in various growthparameters such as percentage germination, plant height, leaf number and area, andbiomass.8 It also inhibits photosynthesis as well as other processes while movingthrough the transpiration stream from roots or by the stomata. It accumulates in plantparts especially in leaf margins causing chlorosis and necrosis and it is thereforeknown as an accumulative poison.9 Other physical responses that are the result offluoride stress are the necrosis and abscission of leaves as well as of flowers andreduced seed production.10

Salicylic acid (SA), a growth regulating plant hormone that is naturally produced inplants, is an important signalling molecule known to have diverse effects on bioticand abiotic stress tolerance. It is a phenolic compound which is antioxidant in nature.It acts as a signalling molecule which helps in maintaining various physio-biochemical processes in plants.11 It is reported that, when it is applied exogenously,it helps in improving the salt tolerance by enhancing plant growth and biomassproduction.12 Scientists believe that it lowers the lethal effects of salinity byincreasing the relative water content and thus having an ameliorative role in nutrientuptake.13

Pisum sativum L. (pea) is an edible plant used to nourish humans and belongs to thefamily Leguminaceae. Its seeds are rich in protein and carbohydrates whichconstitute 18–20% of its dry matter. It is grown annually as a fresh vegetable in thecool season. It is stored for later use by using canning and freezing. In the presentexperiment, SA was applied exogenously to two different varieties of Pisum sativumL. plants to study whether it enhanced tolerance to salt stress produced by a biweeklydrench of sodium fluoride.

MATERIALS AND METHODS

Two hybrid varieties of pea plant (Pisum sativum L.) named as RKS-510 andClassic were raised from certified seeds purchased from Punjab SeedsCorporation and Arain Seed Corporation, Lahore, Pakistan, respectively. Healthyseeds, without any damage or fungal infection, were selected for sowing andstored in paper bags for future use. The chemicals sodium fluoride (NaF) andsalicylic acid (SA), of analytical grade, were purchased from the market. Thebotanical garden of the botany department in University of the Punjab, Lahore,was chosen as the experimental site to conduct a pot experiment. The duration ofthe experimental work was from November 2015 to March 2016. Earthen potswere used to carry out the experiment. The holes present at the bottom of the pots




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were partially closed with pebbles to inhibit water loss due to extreme drainage. Eachpot was filled with 5 kg of soil and properly labelled. The soil was prepared bymixing sandy and loamy soil in a 1:3 ratio along with farmyard and leaf manure. Arandomized complete block design (RCBD) was used to design the experiment withthree replicates of all the treatments. Solutions of NaF and SA were prepared invarious concentrations by mixing the required amount in distilled water. Then,dilutions of sodium fluoride and salicylic acid were made in ppm to monitor theeffect of salicylic acid on the pea cultivars under sodium fluoride stress during thegrowth season 2015 to 2016.

The selected seeds were sown after being soaked for 24 hours in clean tap water.Four seeds were sown in each pot. After one week of germination, two equal sizedand healthy seedlings were selected in each pot for further experiment with the othersbeing removed by manual thinning. The pots were carefully examined regularly toremove weeds and to protect the plants from pathogen attack. Prepared dilutions ofsodium fluoride with different concentrations, i.e., 50, 100, 150, and 200 ppm, wereapplied as a soil drench to create stress conditions. To each pot, 150 mL of NaFsolution was applied. Six mL of salicylic acid of different concentrations (100, 200,and 300 ppm) was applied exogenously as foliar spray by using a shower bottle. Tapwater only was applied to the control plants.

The 20 different treatments used during the investigation were:

The treatments were applied on a biweekly basis throughout the experiment withthe first treatment being given 25 days after the sowing (DAS) on 22nd December,2015. The biomass estimation and the different growth parameters were measured atthree destructive harvests, at 44 DAS, 67 DAS, and 105 DAS. The plants werecarried in properly labeled bags to the laboratory for the measurement of the growthparameters, i.e., shoot length (cm), root length (cm), number of leaves, and numberof branches. For this purpose, three pots per treatment were selected from the threedestructive harvests (at 44, 67, and 105 DAS). The plants were dried in an electricoven (Gallenkamp, Model OV-455 England) at 70ºC for 72 hr after measuring thefresh weight of the root, shoots, and leaves on an electric balance (Sartorius GMBH,

Control (NaF-0 ppm + SA-0 ppm) NaF-100 ppm + SA-100 ppmNaF-100 ppm + SA-200 ppm

NaF-50 ppm NaF-100 ppm + SA-300 ppmNaF-100 ppmNaF-150 ppm NaF-150 ppm + SA-100 ppmNaF-200 ppm NaF-150 ppm + SA-200 ppm

NaF-150 ppm + SA-300 ppmSA-100 ppmSA-200 ppm NaF-200 ppm + SA-100 ppmSA-300 ppm NaF-200 ppm + SA-200 ppm

NaF-200 ppm + SA-300 ppmNaF-50 ppm + SA-100 ppmNaF-50 ppm + SA-200 ppmNaF-50 ppm + SA-300 ppm




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Type 1216MP 6E, Gottingen, Germany). The leaf area was calculated by theformula:14

The data collected from the experiment was statistically analyzed. Costat (version3.03) was used to calculate the treatment mean, standard error, and Duncan’sMultiple Range Test.15

RESULTS

The morphological characters of Pisum sativum L. were improved by usingsalicylic acid at 100 and 200 ppm and progressively more impaired as the NaFconcentration increased from 50 to 200 ppm. With the SA treatment, at 100 and 200ppm, the Pisum sativum L. variety RKS-510 showed a significant increase in manymorphological parameters including, shoot length, root length, number of leaves, andnumber of branches while with the Classic variety significant increases occurred infewer morphological characters.

Figure 1 shows the pattern of shoot length in the pea variety RKS-510 grown underthe field conditions, at 44, 67, and 105 DAS, with different treatments of NaF andSA, both individually and in combination, during the growing season 2015–2016.

In the pea variety RKS-510, at 44 DAS, the shoot length decreased with theincreases in the sodium fluoride level and the maximum reduction in plant shootlength was recorded at the highest level of sodium fluoride, i.e., 200 ppm ascompared to 50, 100, and 150 ppm NaF. However, the foliar spray of SA significantlyimproved the shoot length under the NaF stress condition. One hundred and 200 ppmof SA enhanced the growth of the plants while SA 300 has a similar reducing effectto that of NaF. SA 100 and 200 ppm caused a 15.2% and 17.5% increase,respectively, in the shoot length of the plants at 44 DAS while SA 300 ppm reducedthe shoot length of plants up to 14.4%. Fifty, 100, 150, and 200 ppm NaF reduced the

Leaf area = Length × Maximum width × 0.75 (correction factor)

Figure 1. The shoot length in the pea variety RKS-510 under different treatments of sodiumfluoride and salicylic acid during the growing season 2015–2016.




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shoot length, compared to the control, by 11.2%, 23.2%, 29.6%, and 52.7%,respectively.

When used in combination with the NaF, SA, at concentrations of 100 and 200 ppmbut not 300 ppm, lowered the NaF-induced stress. At 44 DAS, NaF-50 + SA 100showed an increase in shoot length as compared to NaF-50 only. NaF-50 + SA-100,NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 + SA-100 showed a 0%, 6.4%,18.4%, and 31.2% reduction, respectively, while NaF-50 + SA-200, NaF-100 + SA-200, NaF-150 + SA-200, and NaF-200 + SA-200 showed reductions under NaFstress of 7.2%, 22.5%, 16%, and 49.6%, respectively. NaF-50 + SA-300, NaF-100 +SA-300, NaF-150 + SA-300, and NaF-200 + SA-300 showed reductions of 22.4%,30.4%, 35.2%, and 59.2%, respectively, in shoot length due the stress of both NaFand SA.

Figure 2 shows the pattern of shoot length in the pea variety Classic grown underthe field conditions, at 44, 67, and 105 DAS, with different treatments of NaF andSA, both individually and in combination, during the growing season 2015–2016.

In the pea variety Classic, at 44 DAS, the NaF concentrations of 50 and 100 ppmwere less effective in reducing the shoot length (14 and 26.3%) than the NaFconcentrations of 150 and 200 ppm (35.6 and 43.9%). The maximum shoot lengthwas recorded in the SA-100 group and minimum level was observed in the 200 ppmNaF group. For the plants treated with NaF, the shoot length of the plants treated withNaF-50 ppm was higher than that of the rest of the NaF treatments (100, 150, and 200ppm) from the beginning to the end of the experiment. The plants treated with 50,100, 150, and 200 ppm NaF showed a progressive reduction (14%, 26.3%, 35.6%,and 43.9%, respectively) in their shoot lengths compared to the control group. Theapplication of SA alleviated the NaF-induced reduction in shoot length. The foliarapplication of 100 ppm of SA was more effective in increasing shoot length (11.2%

Figure 2. The shoot length in the pea variety Classic under different treatments of sodium fluorideand salicylic acid during the growing season 2015–2016.




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increase in shoot length compared to the control) than the applications of SA-200 andSA-300.

At 44 DAS, the shoot length of the NaF-50 + SA-100 group was greater than that ofthe NaF-50 and NaF-50 + SA-200 groups while the treatment with NaF-50 + SA-300showed a reduction in shoot length as compared to that of the NaF-50 group. Thesame pattern of growth was observed with the NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 + SA-100 groups. When the NaF-stressed plants were treated withSA in concentrations of 50, 100, and 150 ppm, they showed an increase in shootlength. NaF-50 + SA-100, NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 +SA-100 showed reductions in NaF stress, (6.7%, 8.6%, 24.3%, and 35.5% increasesin shoot lengths, respectively), as compared to the treatments with NaF only (50, 100,150, and 200 ppm, respectively) while NaF-50 + SA-200, NaF-100 + SA-200, NaF-150 + SA-200, and NaF-200 + SA-200 showed reductions in shoot length with NaFstress, compared to the relevant NaF alone groups, (10.3%, 15.9%, 30.2%, and41.2% increases in shoot lengths, respectively). The NaF-50 + SA-300, NaF-100 +SA-300, NaF-150 + SA-300, and NaF-200 + SA-300 groups showed reductions inshoot length compared to the control (30%, 29.9%, 40.9%, and 54.6% respectively)due the stress of both NaF and SA applied in combination.

For both pea plant varieties (RKS-510 and Classic), at the other two growth stages,67 and 105 DAS, similar patterns were observed in shoot length. Compared to 44DAS, the shoot length was increased at 67 and 105 DAS with the maximum valuesbeing recorded at 105 DAS. In a manner similar to the pattern at 44 DAS, at 67 and105 DAS, NaF reduced shoot length while SA, at 100 and 200 ppm, enhanced it.

Figure 3 shows the pattern of root length in the pea variety RKS-510 grown underthe field conditions, at 44, 67, and 105 DAS, with different treatments of NaF andSA, both individually and in combination, during the growing season 2015–2016.

Figure 3. The root length in the pea variety RKS-510 under different treatments of sodium fluorideand salicylic acid during the growing season 2015–2016.




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The maximum reduction in plant root length was recorded at the highest level ofNaF, i.e., 200 ppm (38%) as compared to 50, 100, and 150 ppm NaF (15.2%, 24%,and 34.2%, respectively). However, the foliar spray of SA, at 100 and 200 ppm,significantly improved the root length under the NaF stress condition. One hundredand 200 ppm SA enhanced the growth of the plant (10.2% and 1.14% increases,respectively) while SA-300 ppm reduced the root length of the plants by 15.9%.

NaF-50 + SA 100 resulted an increase in root length as compared to NaF-50. NaF-50 + SA-100, NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 + SA-100showed reductions of 3.8%, 10.6%, 17.6%, and 16.3%, respectively, while NaF-50 +SA-200, NaF-100 + SA-200, NaF-150 + SA-200, and NaF-200 + SA-200 showedreductions of root length with NaF stress of 11.4%, 16.4%, 20%, and 27.7%,respectively. NaF-50 + SA-300, NaF-100 + SA-300, NaF-150 + SA-300 and NaF-200 + SA-300 showed the largest reductions in the root length value (26.6%, 20.1%,35.3%, and 44.2%, respectively) due the stress of both NaF and SA.

Figure 4 shows the pattern of root length in the pea variety Classic grown under thefield conditions, at 44, 67, and 105 DAS, with different treatments of NaF and SA,both individually and in combination, during the growing season 2015–2016.

At 44 DAS, NaF concentrations of 50 and 100 ppm were less effective in reducingroot length (11.6% and 18%, respectively) compared to the effects of 150 and 200ppm (27.7% and 38.5%). The groups with the highest values for root length were thecontrol and SA-100 group (12.5% increase). The root length of the plants with NaF-50 ppm was higher than that of the rest of the NaF treatment complements from thebeginning to the end of the experiment. The plants treated with 100, 150, and 200ppm NaF showed progressive reductions in their root length with increased NaFconcentrations compared to the control group.

Figure 4. The root length in the pea variety Classic under different treatments of sodium fluorideand salicylic acid during the growing season 2015–2016.




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At 44 DAS, the foliar application of 100 ppm salicylic acid was more effective instimulating root growth compared to 200 and 300 ppm SA. The root length withNaF-50 + SA-100 was much greater than that with NaF-50 and NaF-50 + SA-200while the treatment with NaF-50 + SA-300 resulted in a reduction in root length. Thesame pattern of growth was observed for treatments with 100, 150, and 200 ppm NaFwith salicylic acid 100 ppm. When the NaF stressed plants were treated with 100 and200 ppm of SA, the NaF-induced reduction in root length was ameliorated. NaF-50 +SA-100, NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 + SA-100 resulted inreductions in NaF-induced stress of 0.8%, 12.8%, 20.4%, and 31.4%, respectively,while NaF-50 + SA-200, NaF-100 + SA-200, NaF-150 + SA-200, and NaF-200 +SA-200 resulted in reductions in NaF-induced salt stress 10.3%, 15.9%, 30.2%, and41.2%, respectively. NaF-50 + SA-300, NaF-100 + SA-300, NaF-150 + SA-300 andNaF-200 + SA-300 resulted in the largest reductions in root length with values of30%, 29.9%, 40.9%, and 54.6%, due the stress of both NaF and SA.

The root length was also detected at 67 and 105 DAS for both varieties of pea plant(RKS-510 and Classic) and showed a similar pattern to that at 44 DAS. At 67 and105 DAS, the overall root length was increased as compared to 44 DAS and highestvalues of root length reduction were recorded at 105 DAS, thus showing a similarreducing effect of NaF, enhancing effect of SA at 100 and 200 ppm, and reducingeffect of SA at 300 ppm.

Figure 5 shows the pattern of the number of leaves in the pea variety RKS-510grown under the field conditions, at 44, 67, and 105 DAS, with different treatmentsof NaF and SA, both individually and in combination, during the growing season2015–2016.

At 44 DAS, the number of leaves of the pea variety RKS-510 was recorded for thetreated (NaF, SA, and NaF-SA combination) and non-treated plants grown under the

Figure 5. The number of leaves in the pea variety RKS-510 under different treatments of sodiumfluoride and salicylic acid during the growing season 2015–2016.




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field conditions. Fifty and 100 ppm NaF was less effective in reducing the leafnumber (16.5% and 24%, respectively) as compared to the 150 and 200 ppm NaFgroups (29.2% and 40.7%, respectively). The leaf number was highest in the controland SA-100 groups (8.9% increase). The plants treated with NaF-50 ppm had moreleaves than the rest of the NaF treatment complements from the beginning to the endof the experiment. The groups with 100, 150, and 200 ppm NaF showed progressivereductions in their leaf number compared to the control group.

The foliar application of 100 ppm SA was more effective in increasing leaf numberthan SA-200 and SA-300 which resulted in reductions in leaf number of 1.3% and13.9%, respectively. The leaf number with the NaF-50 + SA-100 group was greaterthan with the NaF-50 and NaF-50 + SA-200 groups while the treatment with NaF-50+ SA-300 showed a reduction in leaf number. The same pattern was observed withthe groups of 100, 150, and 200 ppm NaF with 100 ppm SA. NaF-50 + SA-100, NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 + SA-100 showed an increase of12%, 22.3%, 26.7%, and 34.9%, respectively, while NaF-50 + SA-200, NaF-100 +SA-200, NaF-150 + SA-200 and NaF-200 + SA-200 showed an increment of 16.6%,24.4%, 27.9%, and 35.6%, respectively. NaF-50 + SA-300, NaF-100 + SA-300, NaF-150 + SA-300, and NaF-200 + SA-300 showed a decrease in leaf number of 21%,34.4%, 33%, and 49.7%, respectively, due the stress of both NaF and SA.

Figure 6 shows the pattern of leaf number in the pea variety Classic grown underthe field conditions, at 44, 67, and 105 DAS, with different treatments of NaF andSA, both individually and in combination, during the growing season 2015–2016.

At 44 DAS, the leaf number decreased as the NaF concentration increased in thevariety Classic with the maximum reduction in leaf number (43%) being recorded atthe highest level of NaF, i.e., 200 ppm, as compared to the reductions with 50, 100,and 150 ppm NaF of 5.8%, 10.9%, and 21.6%, respectively. The foliar spray of

Figure 6. The number of leaves in the pea variety Classic under different treatments of sodiumfluoride and salicylic acid during the growing season 2015–2016.




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salicylic acid (SA) at concentrations of 100 and 200 ppm significantly improved thenumber of leaves under the NaF stress condition (increases in leaf number of 7.3%and 2.7%, respectively) while the application of the foliar spray with SA 300 reducedthe leaf number (5.8%).

Treatments with NaF-50 + SA-100, NaF-100 + SA-100, NaF-150 + SA-100, andNaF-200 + SA-100 resulted in increases in leaf number, compared to the treatmentwith NaF only, of 12%, 22.3%, 26.7%, and 34.9%, respectively, while the treatmentswith NaF-50 + SA-200, NaF-100 + SA-200, NaF-150 + SA-200, and NaF-200 + SA-200 showed increases in the leaf number, compared to that induced by the NaF stress,of 16.6%, 24.4%, 27.9%, and 35.6%, respectively. NaF-50 + SA-300, NaF-100 + SA-300, NaF-150 + SA-300, and NaF-200 + SA-300 resulted in reductions in the leafnumber of 21%, 34.4%, 33%, and 49.7% due the stress of both NaF and SA.

The leaf number was also measured at the other two growth stages of 67 and 105DAS for both the pea plant varieties (RKS-510 and Classic). At 67 and 105 DAS, theoverall number of leaves was increased as compared to 44 DAS. The maximumvalues were recorded at 105 DAS and showed a similar reducing effect of NaF andenhancing effect of SA.

Figure 7 shows the pattern of the number of branches in the pea variety RKS-510grown under the field conditions, at 44, 67, and 105 DAS, with different treatmentsof NaF and SA, both individually and in combination, during the growing season2015–2016.

At 44 DAS, in the pea plant variety RKS-510, as the NaF level increased thenumber of branches decreased with the maximum reduction in the number ofbranches (34.5%) being recorded at the highest level of NaF, i.e., 200 ppm ascompared to the reductions with 50, 100, and 150 ppm NaF (9%, 18%, and 27.2%,respectively). The foliar spray of salicylic acid (SA), at concentrations of 100 and200 ppm, significantly improved the number of branches under the NaF stress

Figure 7. The number of branches in the pea variety RKS-510 under different treatments ofsodium fluoride and salicylic acid during the growing season 2015–2016.




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condition. One hundred and 200 ppm SA enhanced the number of branches (27.2%and 9% increases respectively) while SA 300 had a reducing effect (15.4% decrease).NaF-50 + SA 100 showed an increase in the number of branches compared to NaF-50and NaF-50 + SA-200.

At 44 DAS, compared to the treatments with NaF only, NaF-50 + SA-100 resultedin an increase of 2.7% while NaF-100 + SA-100, NaF-150 + SA-100, and NaF-200 +SA-100 showed decreases of 3.6%, 13.6%, and 17.9%, respectively, and NaF-50 +SA-200, NaF-100 + SA-200, NaF-150 + SA-200, and NaF-200 + SA-200 showed adecrease in the number of branches due to NaF stress of 0%, 9%, 15.4%, and 20%,respectively. NaF-50 + SA-300, NaF-100 + SA-300, NaF-150 + SA-300, and NaF-200 + SA-300 showed reductions in the number of branches due the stress of bothNaF and SA of 12.7%, 27.2%, 30%, and 45.7%, respectively,

Figure 8 shows the pattern of the number of branches in the pea variety Classicgrown under the field conditions, at 44, 67, and 105 DAS, with different treatmentsof NaF and SA, both individually and in combination, during the growing season2015–2016.

At 44 DAS, NaF concentrations of 50 and 100 ppm decreased the number ofbranches by 6.1% and 14.9%, respectively while 150 and 200 ppm caused decreasesof 26.3% and 32.4%, respectively. The number of branches was highest in the controland SA-100 groups (46.5% increase). The number of branches of the plants treatedwith NaF-50 ppm was higher than that of the others treated with NaF from thebeginning to the end of the experiment.

At 44 DAS, the number of branches with treatment by NaF-50 + SA-100 was muchgreater than with NaF-50 and NaF-50 + SA-200 while the treatment with NaF-50 +SA-300 showed a reduction in number of branches. The same pattern of growth wasobserved in case of NaF 100, 150, and 200 ppm with salicylic acid 100 ppm. NaF-50+ SA-100 resulted in an 8.2% increase in the number of branches while NaF-100 +

Figure 8. The number of branches in the pea variety Classic under different treatments ofsodium fluoride and salicylic acid during the growing season 2015–2016.




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SA-100, NaF-150 + SA-100, and NaF-200 + SA-100 showed reductions in stress of8.2%, 0.8%, 9.6%, and 12.4%, respectively. NaF-50 + SA-200, NaF-100 + SA-200,NaF-150 + SA-200, and NaF-200 + SA-200 showed reductions in the number ofbranches of 3.5%, 12.3%, 14%, and 21%, respectively. NaF-50 + SA-300, NaF-100 +SA-300, NaF-150 + SA-300 and NaF-200 + SA-300 showed the greatest reduction inthe number of branches due the stress of both NaF and SA with reductions of 17.5%,18.4%, 30%, and 44.7%. respectively.

At 67 and 105 DAS, in both varieties of pea plant (RKS-510 and Classic). therewas a similar pattern, of a reducing effect on the number of branches with NaF and anenhancing effect of SA, to that found at 44 DAS. At 67 and 105 DAS, the overallnumber of branches was increased as compared to 44 DAS with the highestmaximum values being recorded at 105 DAS.

The biomass assessment of the pea crop harvested at 44, 67, and 105 DAS areshown in Tables 1, 2, and 3 for variety RKS-570 and in Tables 4, 5 and 6 for varietyClassic. NaF adversely affected the fresh shoot and root weight which decreased withincreasing NaF concentrations.

For variety RKS-570, at 44 DAS, compared to the control, the shoot and root freshweights of the plants treated with 50, 100, 150, and 200 ppm NaF showed reductionsfor shoots of 24.8%, 35.5%, 39.3%, and 64.6%, respectively, and for roots of 6.6%,22.8%, 35.5%, and 51.6%, respectively. At 44 DAS, compared to the control plants,the plants treated with SA 100 ppm were much healthier and heavier in weight(12.7% and 21.1% increases for shoot weight and root weight, respectively) (Tables1–3).

For variety Classic, at 44 DAS, compared to the control, the shoot and root freshweights of the plants treated with 50, 100, 150, and 200 ppm NaF showed reductionsfor shoots of 30.7%, 41%, 43.1%, and 69.4%, respectively, and for roots of 8.6%,25.8%, 37.5%, and 55.6%, respectively (Tables 4–6). The reductions in the freshweight of shoot and root were more pronounced in the Classic variety than in theRKS-510 variety (Tables 1–6).

A similar pattern of a reducing effect of NaF and an enhancing effect of SA on theshoot and root fresh weights was observed when these were measured at 67 and 105DAS for both varieties of pea plant (RKS-510 and classic). At 67 and 105 DAS, theoverall shoot and root fresh weights were increased as compared to 44 DAS and themaximum values were recorded at 105 DAS.

The shoot and root dry weights followed a pattern similar to that found with thefresh weights. For variety RKS-510, at 44 DAS, for NaF concentrations of 50, 100,150, and 200 ppm, the dry weights of the shoots showed reductions 32%, 44.4%,50.8%, and 65.5% and the dry weights of the roots showed reductions of 10.8%,12.7%, 15.2%, and 36.3% (Tables 1– 3). For variety RKS-510. at 44 DAS, comparedto the control group, the dry weight of the shoot and root in the plants treated with100 ppm salicylic acid was increased by 17.8% and 44.6%, respectively. Theincreases with less with SA-200. For variety Classic, at 44 DAS, for NaFconcentrations of 50, 100, 150, and 200 ppm, the dry weights of the shoots showedreductions of 35%, 47.4%, 55.8%, and 69.3% and the dry weights of the rootsshowed reductions of 12.8%, 15.7%, 17.2%, and 39.5% (Tables 4–6). These




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reductions in dry weight of the shoots and roots with NaF treatment were morepronounced in the Classic variety than in the RKS-510 variety (Tables 1–6).

For variety Classic at 44 DAS, compared to the control group, the dry weight of theshoot and root in the plants treated with 100 ppm salicylic acid was increased by19.2% and 41.2%, respectively.

The shoot and root dry weight showed a similar pattern when measured at 67 and105 DAS for both varieties (RKS-510 and Classic) of pea plant. At 67 and 105 DAS,the overall shoot and root dry weight were increased as compared to 44 DAS and the

Table 1: Growth parameters of Pisum sativum var. RKS-510 harvested at 44 days after sowing by using different sodium fluoride and salicylic acid concentrations during growth season 2015-2016

Treatments

Growth parameters

Shoot fresh

weight (g)

Shoot dry weight

(g)

Root fresh

weight (g)

Root dry weight

(g)

Leaf fresh

weight (g)

Leaf dry weight

(g)

Leaf area (cm2)

Control 13.33b ± 0.88

4.27b ± 0.35

6.00a-d ± 0.58

1.57a-e ± 0.26

7.63a-c ± 0.35

2.43ab ± 0.15

11.61a ± 1.08

NaF-50 10.00d-f ± 0.58

2.97d-f ± 0.32

5.60c-e ± 0.65

1.40b-e ± 0.06

6.20d-g ± 0.26

1.90c-f ± 0.06

9.96bc ± 0.33

NaF-100 8.57f-h ± 0.62

2.37g-i ± 0.32

4.63d-g ± 0.32

1.37b-e ± 0.19

5.83e-h ± 1.01

1.70e-g ± 0.06

8.00de ± 0.58

NaF-150 8.07g-i ± 0.52

2.10h-j ± 0.17

3.87f-i ± 0.15

1.33b-e ± 0.33

4.83h-j ± 0.58

1.53f-h ± 0.12

7.63d-f ± 0.20

NaF-200 4.70jk ± 0.12

1.47k ± 0.09

2.90i ± 0.06

1.00de ± 0.00

3.77jk ± 0.09

1.20hi ± 0.06

6.25fg ± 0.16

SA-100 15.00a ± 0.58

5.03a ± 0.09

7.27a ± 0.32

2.27a ± 0.15

8.73a ± 0.12

2.63a ± 0.18

12.10a ± 0.44

SA-200 13.00b ± 0.58

5.00a ± 0.21

7.00ab ± 0.58

2.17a ± 0.17

8.10ab ± 0.10

2.30a-c ± 0.15

11.82a ± 1.13

SA-300 11.00c-e ± 0.58

4.53ab ± 0.20

5.80b-d ± 0.15

1.33b-e ± 0.33

6.57c-f ± 0.19

1.57f-h ± 0.22

9.17b-d ± 0.44

NaF-50+ SA-100 12.00bc ± 0.58

3.73c ± 0.09

6.50a-c ±0.12

1.93a-c ± 0.03

7.30b-d ± 0.12

2.30a-c ± 0.06

10.63ab ± 0.32

NaF-50+ SA-200 11.67b-d

± 0.88 3.50cd ± 0.06

6.00a-d ± 0.58

1.67a-d ± 0.33

7.03b-e ± 0.54

2.10b-e ± 0.06

9.93bc ± 0.03

NaF-50+ SA-300 8.83f-h ± 0.41

2.53f-h ± 0.27

5.50c-e ± 0.29

1.33b-e ± 0.33

5.23g-i ± 0.20

1.93c-f ± 0.13

8.97cd ± 0.61

NaF-100+ SA100 12.00bc ± 0.58

3.20c-e ± 0.17

5.63c-e ± 0.32

2.00ab ± 0.00

6.20d-g ± 0.21

2.37a-c ± 0.09

9.79bc ± 0.61

NaF-100+ SA-200 11.00c-e ± 0.58

3.00d-f ± 0.06

5.40c-e ± 0.33

1.73a-d ± 0.15

5.80e-h ± 0.15

2.23a-d ± 0.38

8.83cd ± 0.12

NaF-100+ SA-300 7.80g-i ± 0.67

2.10h-j ± 0.12

4.33e-h ± 0.33

1.27b-e ± 0.18

4.33i-k ± 0.69

1.30g-i ± 0.12

7.45d-f ± 0.73

NaF-150+ SA-100 10.67c-e ± 0.88

2.63e-h ± 0.18

5.13c-f 0.59

1.63a-d ± 0.32

5.90e-h ± 0.35

1.90c-f ± 0.12

8.73cd ± 0.43

NaF-150+ SA-200 9.37e-g ± 0.32

2.50f-h ± 0.12

4.00f-i ± 0.00

1.33b-e ± 0.33

5.60f-h ± 0.06

1.80d-f ± 0.20

8.57cd ± 0.30

NaF-150+ SA-300 7.20hi ± 0.76

1.90i-k ± 0.06

3.17hi ± 0.44

1.13b-e ± 0.13

4.23i-k ± 0.55

1.23g-i ± 0.15

7.55d-f ± 0.42

NaF-200+ SA-100 7.23hi ± 0.28

2.80d-f ± 0.06

3.80f-i ± 0.15

1.53a-d ± 0.27

4.90h-j ± 0.23

1.60f-h ± 0.12

6.52e-g ± 0.31

NaF-200+ SA-200 6.23ij ± 0.19

2.10h-j ± 0.12

3.33g-i ± 0.88

1.20b-e ± 0.20

3.77jk ± 0.20

1.27g-i ± 0.12

6.20fg ± 0.15

NaF-200+ SA-300 4.03k ± 0.09

1.77jk ± 0.09

1.33j ± 0.33

0.80e ± 0.20

3.13k ± 0.09

0.93i ± 0.09

4.73g ± 0.09

multiple range test and P= 0.05; NaF; Sodium fluoride; SA: Salicylic acid; NaF-SA: Sodium fluoride-Salicylic acid




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maximum values were recorded at 105 DAS. The results for 67 and 105 DAS weresimilar to those at 44 DAS and showed that the shoot and root dry weights werereduced by NaF and enhanced by SA.

For variety RKS-510. at 44 DAS, compared to the control group, the dry weight ofthe shoot and root in the plants treated with 100 ppm salicylic acid were increased by19.2% and 41.2%, respectively.

Table 2: Growth parameters of Pisum sativum var. RKS-510 harvested at reproductive phase 67 days after sowing by using different sodium fluoride and salicylic acid concentrations during growth season 2015-2016

Treatments

Growth Parameters

Shoot fresh weight (g)

Shoot dry weight

(g)

Root fresh

weight (g)

Root dry weight

(g)

Leaf fresh weight (g)

Leaf dry weight

(g)

Leaf area cm2

Control 20.03cd ± 0.66

8.17b ± 0.41

8.00b ± 0.06

3.07bc ± 0.12

12.20b ± 0.17

5.37b-d ± 0.24

12.79b ± 0.91

NaF-50 17.30de ± 1.23

6.53cd ± 0.27

6.87de ± 0.09

2.40e-g ± 0.23

11.80b ± 0.15

4.47ef ± 0.26

11.68bc ± 0.58

NaF-100 16.97ef ± 1.13

6.23c-e ± 0.15

6.43e-g ± 0.27

2.20f-j ± 0.15

10.57cd ± 0.52

4.13fg ± 0.33

9.93de ± 0.45

NaF-150 13.23gh ± 1.76

5.30d-g ± 0.25

5.67ij ± 0.24

1.90h-l ± 0.06

9.77de ± 0.03

3.43hi ± 0.23

8.10gh ± 0.21

NaF-200 10.00ij ± 0.58

3.30h ± 0.35

4.60m ± 0.21

1.53lm ± 0.12

7.47gh ± 0.24

3.03ij ± 0.09

14.27a ± 0.38

SA-100 24.67a ± 1.45

10.33a ± 0.88

8.90a ± 0.06

3.57a ± 0.09

14.23a ± 0.28

6.10a ± 0.21

13.94a ± 0.20

SA-200 22.70ab ± 0.53

8.43b ± 0.52

7.97b ± 0.22

3.23ab ± 0.15

13.63a ± 0.47

5.80ab ± 0.36

9.76d-f ± 0.62

SA-300 19.13c-e ± 0.35

6.13c-e ± 0.49

6.60ef ± 0.31

2.87b-d ± 0.09

11.50bc ± 0.75

4.87de ± 0.09

12.10bc ± 0.15

NaF-50+ SA-100 20.63bc ± 0.24

8.30b ± 0.15

7.53bc ± 0.09

2.67c-e ± 0.09

12.33b ± 0.44

5.53a-c ± 0.27

11.30c ± 0.35

NaF-50+ SA-200 18.53c-e ± 0.49

6.00d-f ± 0.12

7.23cd ± 0.15

2.33e-h ± 0.09

10.33d ± 0.20

5.27b-d ± 0.23

10.13de ± 0.19

NaF-50+ SA-300 12.70g-i ± 0.36

4.83fg ± 0.73

6.53e-g ± 0.12

2.03g-k ± 0.09

7.83fg ± 0.18

3.73gh ± 0.18

11.53c ± 0.29

NaF-100+ SA100 19.33c-e ± 0.34

7.30bc ± 0.35

6.60ef ± 0.31

2.50d-f ± 0.06

11.67b ± 0.15

4.93c-e ± 0.23

10.95cd ± 0.35

NaF-100+ SA-200 17.33de ± 0.54

6.43cd ± 0.23

6.27f-h ± 0.15

2.17f-j ± 0.09

10.07d ± 0.65

4.47ef ± 0.09

9.56ef ± 0.10

NaF-100+ SA-300 12.53g-i ± 0.38

4.73g ± 0.09

5.50i-k ± 0.26

1.87i-l ± 0.09

7.40gh ± 0.26

3.50hi ± 0.23

8.40g ± 0.49

NaF-150+ SA-100 14.57fg ± 0.41

6.57cd ± 0.30

6.00g-i ± 0.06

2.23e-i ± 0.18

10.30d ± 0.35

4.37ef ± 0.12

9.63ef ± 0.32

NaF-150+ SA-200 13.83gh ± 0.18

6.17c-e ± 0.12

5.80hi ± 0.06

1.97g-l ± 0.12

9.43de ± 0.26

3.47hi ± 0.27

8.30g ± 0.21

NaF-150+ SA-300 11.33h-j ± 0.63

4.10gh ± 0.55

5.00k-m ± 0.06

1.77j-l ± 0.09

6.93gh ± 0.52

2.90ij ± 0.10

8.07gh ± 0.33

NaF-200+ SA-100 12.30g-i ± 1.48

5.07e-g ± 0.07

5.20j-l ± 0.12

2.03g-k ± 0.09

8.77ef ± 0.15

3.73gh ± 0.15

8.64fg ± 0.13

NaF-200+ SA-200 9.30j

± 0.89 4.53gh ± 0.20

4.77lm ± 0.09

1.70kl ± 0.35

7.87fg ± 0.15

3.37hi ± 0.15

7.10hi ± 0.21

NaF-200+ SA-300 8.73j

± 0.90 3.33h ± 0.24

3.73n ± 0.09

1.20m ± 0.09

6.23h ± 0.28

2.43j ± 0.15

6.15i ± 0.23





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Tables 1 and 4 show the leaf fresh and dry weights for both varieties of pea at 44DAS. For the RKS-510 variety, at 44 DAS, for treatments with NaF at concentrationsof 50, 100, 150, and 200 ppm, the leaf fresh weight followed a pattern of successivereductions of 18.7%, 23.6%, 36.8%, and 51.3%, respectively, and the correspondingreductions in dry weight were 16.8%, 24%, 32.4%, and 44.5%. The fresh and dry leafweights were highest in the RKS-510 variety plants treated with 100 ppm salicylicacid with values of 8.73 g and 2.63 g, respectively (Table 1). Similar reductions in theleaf fresh and dry weights were also observed in Classic variety at 44 DAS with

Table 3: Growth parameters of Pisum sativum var. RKS-510 harvested at 105 days after sowing by using different sodium fluoride and salicylic acid concentrations during growth season 2015-2016

Treatments

Growth Parameters

Shoot fresh

weight (g)

Shoot dry weight (g)

Root fresh

weight (g)

Root dry weight (g)


Leaf dry weight (g)

Leaf area (cm2)

Control 25.43bc ± 0.32

10.90b ± 0.58

9.37b ± 0.19

3.50ab ± 0.26

15.00bc ± 1.15

7.70b-d ± 0.38

14.00bc ± 0.44

NaF-50 21.50e ± 0.29

8.97cd ± 0.55

7.67ef ± 0.09

3.03b-d ± 0.09

13.63c-f ± 0.32

6.90c-g ± 0.17

12.16c-e ± 0.69

NaF-100 19.00g ± 0.58

7.63e-g ± 0.09

6.77i-l ± 0.19

2.83cd ± 0.18

12.30e-h ± 0.91

6.47d-i ± 0.33

10.99d-f ± 1.01

NaF-150 16.60h ± 0.38

6.47h-j ± 0.12

6.23k-n ± 0.12

2.47d-f ± 0.26

11.00h-j ± 0.58

5.80g-j ± 0.44

9.57fg ± 0.30

NaF-200 12.63j ± 0.32

4.53l ± 0.12

5.70n ± 0.17

1.83g ± 0.12

9.63jk ± 0.32

4.67jk ± 0.44

7.79gh ± 0.07

SA-100 28.00a ± 0.58

12.13a ± 0.19

9.97a ± 0.58

4.03a ± 0.09

16.63a ± 0.68

8.90a ± 0.21

16.79a ± 0.54

SA-200 26.47ab ± 0.74

11.80a ± 0.15

9.23bc ± 0.15

3.53ab ± 0.29

15.27ab ± 0.37

8.63ab ± 0.35

15.33ab ± 0.88

SA-300 24.00cd ± 0.58

10.70b ± 0.21

7.50fg ± 0.29

2.93b-d ± 0.09

13.90b-e ± 0.06

7.33c-f ± 0.38

12.97cd ± 1.18

NaF-50+ SA-100 23.50d ± 0.26

9.50c ± 0.25

8.70cd ± 0.15

3.37bc ± 0.32

14.63b-d ± 0.32

7.83a-c ± 0.22

13.53bc ± 0.37

NaF-50+ SA-200 21.00ef ± 0.58

9.13cd ± 0.09

8.20de ± 0.15

3.00b-d ± 0.06

13.90b-e ± 0.10

7.53b-e ± 0.48

13.00cd ± 1.15

NaF-50+ SA-300 19.33fg ± 1.45

7.90ef ± 0.06

7.40f-h ± 0.23

2.67de ± 0.24

13.13d-g ± 0.07

6.67c-h ± 0.38

12.30c-e ± 0.15

NaF-100+ SA100 24.00cd ± 0.58

8.40de ± 0.26

7.10f-i ± 0.06

3.03b-d ± 0.24

13.57c-f ± 0.26

6.80c-h ± 0.62

12.83cd ± 1.01

NaF-100+ SA-200 23.23d ± 0.28

7.97ef ± 0.09

6.90g-j ± 0.06

2.90b-d ± 0.06

13.13d-g ± 0.09

6.43e-i ± 0.23

12.58c-e ± 0.29

NaF-100+ SA-300 17.53gh ± 0.48

6.87g-i ± 0.09

5.90mn ± 0.06

2.53de ± 0.26

11.80g-i ± 0.06

5.90g-j ± 0.31

10.41ef ± 0.44

NaF-150+ SA-100 19.40fg ± 0.26

7.43fg ± 0.27

6.83h-k ± 0.12

2.80c-e ± 0.12

12.07f-i ± 0.12

6.10f-i ± 0.44

12.60c-e ± 0.83

NaF-150+ SA-200 18.00gh ± 0.06

7.23f-h ± 0.39

6.40j-m ± 0.12

2.63de ± 0.22

11.57g-i ± 0.30

5.83g-j ± 0.09

11.96c-e ± 0.13

NaF-150+ SA-300 16.40h ± 0.21

6.03jk ± 0.09

5.83mn ± 0.12

1.93fg ± 0.18

9.73jk ± 0.15

5.23ij ± 0.65

9.67fg ± 0.88

NaF-200+ SA-100 14.67i ± 0.88

6.40ij ± 0.23

6.20l-n ± 0.12

2.20e-g ± 0.17

10.63i-k ± 0.32

5.60h-j ± 0.31

9.26fg ± 0.81

NaF-200+ SA-200 13.00ij ± 0.58

5.53k ± 0.24

5.73n ± 0.12

1.90fg ± 0.06

9.47jk ± 1.01

4.77jk ± 0.54

8.20gh ± 0.40

NaF-200+ SA-300 9.67k ± 0.88

4.50l ± 0.25

4.36o ± 0.13

1.57g ± 0.02

8.97k ± 0.50

3.90k ± 0.06

6.42h ± 0.53





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reductions in the fresh leaf weight of 21.7%, 25.6%, 39.8%, and 56.3%, respectively,and in dry leaf weight of 19.8%, 27%, 35.2%, and 46.6%, respectively (Table 4).

At 44 DAS, for the Classic variety, compared to the control, the root and shoot dryweights in the SA-100 group were increased by 41.2% and 19.2%, respectively.

At 44 DAS, compared to the controls, the reductions in the shoot and root freshweights with the NaF treatments were more pronounced in the Classic variety than inthe RKS-510 variety (Tables 1 and 4).

Table 4: Growth parameters of Pisum sativum var. Classic harvested at 44 days after sowing by using different sodium fluoride and salicylic acid concentrations during growth season 2015-2016

Treatments

Growth Parameters

Shoot fresh

weight (g)


Root fresh

weight (g)

Root dry weight (g)

Leaf fresh

weight (g)

Leaf dry weight (g)

Leaf area (cm2)

Control 12.73b ± 0.27

3.80a-c ± 0.25

5.70b ± 0.12

1.70bc ± 0.15

6.90c ± 0.12

2.30bc ± 0.12

10.93b ± 0.15

NaF-50 10.20d-f ± 0.17

3.17b-e ± 0.15

4.87b-d ± 1.07

1.47b-f ± 0.03

6.13de ± 0.19

1.90de ± 0.06

8.80de ± 0.06

NaF-100 8.80f-h ± 0.76

2.90c-f ± 0.23

3.63e-g ± 0.07

1.37b-f ± 0.19

5.43f ± 0.15

1.57fg ± 0.20

7.47hi ± 0.23

NaF-150 7.17hi ± 0.42

2.23e-g ± 0.18

3.00f-h ± 0.06

1.10d-g ± 0.10

4.33i ± 0.09

1.30i-k ± 0.12

6.90j ± 0.06

NaF-200 4.83j

± 0.52 1.90fg ± 0.06

2.70gh ± 0.12

0.94fg ± 0.09

3.43k ± 0.12

1.03g-i ± 0.09

5.37l ± 0.33

SA-100 15.33a ± 1.20

4.53a ± 0.20

6.90a ± 0.15

2.40a ± 0.26

8.23a ± 0.15

3.03a ± 0.15

11.90a ± 0.06

SA-200 12.67bc ± 0.88

4.10ab ± 0.40

5.93ab ± 0.15

2.33a ± 0.33

7.60b ± 0.15

2.53b ± 0.09

11.03b ± 0.20

SA-300 11.00b-e ± 0.58

3.23b-e ± 0.38

5.33bc ± 0.20

1.53b-e ± 0.22

6.37d ± 0.20

1.90de ± 0.06

10.70b ± 0.15

NaF-50+ SA-100 12.10b-d

± 1.07 4.07ab ± 0.97

5.40bc ± 0.15

1.80b ± 0.06

7.27b ± 0.18

2.30bc ± 0.12

9.63c ± 0.15

NaF-50+ SA-200 11.00b-e ± 1.00

3.33b-e ± 0.20

5.07bc ± 0.43

1.67b-d ± 0.17

6.90c ± 0.06

2.00de ± 0.06

9.13d ± 0.09

NaF-50+ SA-300 8.67f-h ± 0.20

2.93c-f ± 0.20

3.93d-f ± 0.07

1.27b-f ± 0.09

5.90e ± 0.06

1.70ef ± 0.06

8.40ef ± 0.12

NaF-100+ SA100 10.63c-f ± 1.33

3.47b-d ± 0.15

4.37c-e ± 0.23

1.77bc ± 0.15

6.37d ± 0.18

2.03cd ± 0.03

8.13fg ± 0.07

NaF-100+ SA-200 9.87e-g ± 0.55

3.17b-e ± 0.33

3.87d-g ± 0.55

1.50b-f ± 0.25

5.90e ± 0.06

1.77d-f ± 0.09

7.73gh ± 0.18

NaF-100+ SA-300 7.93g-i ± 0.58

2.67d-f ± 0.19

3.27e-h ± 0.37

1.23b-g ± 0.15

4.97gh ± 0.09

1.33gh ± 0.15

7.10ij ± 0.15

NaF-150+ SA-100 9.37e-g ± 0.18

3.07b-e ± 0.43

3.50e-g ± 0.36

1.53b-e ± 0.19

5.23fg ± 0.09

1.90de ± 0.06

7.20ij ± 0.21

NaF-150+ SA-200 8.73f-h ± 0.09

2.63d-f ± 0.38

3.37e-h ± 0.19

1.30b-f ± 0.10

4.87h ± 0.09

1.50fg ± 0.06

6.93j ± 0.09

NaF-150+ SA-300 6.90hi ± 0.21

1.90fg ± 0.06

2.70gh ± 0.12

1.07e-g ± 0.15

3.90j ± 0.06

1.20h-j ± 0.06

6.30k ± 0.12

NaF-200+ SA-100 7.97g-i ± 0.09

2.53d-g ± 0.37

3.20f-h ± 0.12

1.20c-g ± 0.06

4.00ij ± 0.06

1.57fg ± 0.03

6.00k ± 0.12

NaF-200+ SA-200 6.00ij ± 0.06

1.90fg ± 0.10

2.73gh ± 0.43

1.00e-g ± 0.15

3.70jk ± 0.06

1.00jk ± 0.06

5.53l ± 0.29

NaF-200+ SA-300 3.97j

± 0.12 1.43g ± 0.15

2.20h ± 0.31

0.67g ± 0.18

2.60l ± 0.12

0.73k ± 0.09

4.03m ± 0.09

Mean ± S.E; within each parameter values not followed by same letter are significantly multiple range test and P= 0.05; NaF; Sodium fluoride; SA: Salicylic acid; NaF-SA: Sodium fluoride-Salicylic acid




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The leaf fresh and dry weights showed a similar pattern when measured at 67 and105 DAS for both varieties (RKS-510 and Classic) of pea plant. At 67 and 105 DAS,the overall leaf fresh and dry weight were increased as compared to 44 DAS and themaximum values were recorded at 105 DAS. The results for 67 and 105 DAS weresimilar to those at 44 DAS and showed that the leaf fresh and dry weights werereduced by NaF and enhanced by SA.

Tables 1 and 4 show the data for both pea plant varieties, RKS-510 and Classic, at44 DAS, for the leaf area. NaF, at concentrations of 50, 100, 150, and 200 ppm


Treatments

Growth Parameters

Shoot fresh

weight (g)


Root fresh

weight (g)

Root dry weight (g)


Leaf dry weight (g)

Leaf area cm2

Control 18.73c ± 0.41

7.93a-c ± 0.52

7.63bc ± 0.09

2.43bc ± 0.18

12.83b ± 0.12

5.10b ± 0.20

12.37b ± 0.23

NaF-50 16.93d ± 0.38

6.80de ± 0.12

6.47ef ± 0.15

2.17b-e ± 0.09

11.20de ± 0.12

3.87d-f ± 0.15

11.43cd ± 0.27

NaF-100 14.87f ± 0.27

5.97fg ± 0.07

5.40g ± 0.06

1.87e-g ± 0.09

9.90f ± 0.06

3.60e-g ± 0.15

9.37fg ± 0.26

NaF-150 12.70i ± 0.17

4.83hi ± 0.09

4.63h ± 0.38

1.53gh ± 0.07

8.83g ± 0.09

3.20gh ± 0.15

7.90ij ± 0.06

NaF-200 9.63k ± 0.15

2.73j ± 0.09

3.93i ± 0.09

1.20hi ± 0.21

6.87i ± 0.09

2.73hi ± 0.32

6.87l ± 0.13

SA-100 21.00a ± 0.58

8.30a ± 0.12

8.37a ± 0.09

3.23a ± 0.15

15.20a ± 0.17

5.80a ± 0.12

13.87a ± 0.09

SA-200 20.03b ± 0.09

8.00ab ± 0.58

8.03ab ± 0.20

3.07a ± 0.15

14.73a ± 0.28

5.47ab ± 0.18

13.27a ± 0.29

SA-300 16.03e ± 0.09

7.60bc ± 0.21

7.13cd ± 0.12

2.30b-d ± 0.21

12.23c ± 0.34

4.50c ± 0.12

11.90bc ± 0.06

NaF-50+ SA-100 18.17c ± 0.27

7.27cd ± 0.15

7.43cd ± 0.19

2.53b ± 0.07

12.93b ± 0.15

4.23cd ± 0.19

12.53b ± 0.29

NaF-50+ SA-200 17.03d ± 0.15

6.90de ± 0.06

6.97de ± 0.18

2.23b-e ± 0.12

12.30c ± 0.23

4.00d-f ± 0.06

12.00bc ± 0.58

NaF-50+ SA-300 15.93e ± 0.20

6.43ef ± 0.30

6.17f ± 0.09

1.90d-g ± 0.06

10.87e ± 0.20

3.63e-g ± 0.12

10.90bc ± 0.06

NaF-100+ SA100 16.43de ± 0.20

6.60d-f ± 0.21

6.50ef ± 0.35

2.20b-c ± 0.17

11.43d ± 0.12

4.30cd ± 0.15

10.05e ± 0.10

NaF-100+ SA-200 15.03f ± 0.26

6.23ef ± 0.09

5.97f ± 0.23

2.03c-e ± 0.09

10.70e ± 0.12

4.07c-e ± 0.07

9.80ef ± 0.06

NaF-100+ SA-300 13.53gh ± 0.32

5.40gh ± 0.17

4.83gh ± 0.12

1.60f-h ± 0.17

9.57f ± 0.20

3.57e-g ± 0.23

8.60h ± 0.12

NaF-150+ SA-100 13.97g ± 0.07

5.20hi ± 0.12

5.23g ± 0.28

2.00d-f ± 0.06

9.93f ± 0.09

3.90d-f ± 0.06

9.03gh ± 0.15

NaF-150+ SA-200 13.00hi ± 0.15

5.00hi ± 0.12

4.90gh ± 0.06

1.90d-g ± 0.06

8.90g ± 0.06

3.53fg ± 0.09

8.53hi ± 0.20

NaF-150+ SA-300 12.53i ± 0.19

4.53i ± 0.22

4.00i ± 0.06

1.27hi ± 0.09

8.57g ± 0.20

2.93h ± 0.23

7.80jk ± 0.06

NaF-200+ SA-100 11.30j ± 0.15

3.33j ± 0.09

4.40hi ± 0.29

1.90d-g ± 0.06

7.57h ± 0.19

3.67e-g ± 0.19

7.57jk ± 0.28

NaF-200+ SA-200 10.07k ± 0.12

2.90j ± 0.06

3.90i ± 0.12

1.53gh ± 0.12

6.47i ± 0.28

3.03h ± 0.09

7.13kl ± 0.09

NaF-200+ SA-300 8.43l

± 0.09 1.87k ± 0.09

3.00j ± 0.06

0.83i ± 0.18

5.27j ± 0.15

2.23i ± 0.15

5.99m ± 0.31





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significantly reduced the leaf area in the RKS-510 variety by 14.7%, 32.7%, 34.2%,and 46.2%, respectively, and in the Classic variety by 17.4%, 36.5%, 39.1%, and50.4%, respectively. In contrast, the application of SA, at concentrations of 100 and200 ppm significantly enhanced the leaf area of the plant under NaF stress. SA at theconcentration of 100 ppm was more effective against NaF stress at all growth stagescompared to SA at concentrations of 200 ppm and 300 ppm. SA-100 ppm resulted inan increase in leaf area in both the RKS-510 and Classic varieties of 4.8% and 3.5%,respectively.


Treatments

Growth Parameters

Shoot fresh

weight (g)


Root fresh

weight (g)

Root dry weight

(g)


Leaf dry

weight (g)

Leaf area (cm2)

Control 23.33bc ± 0.88

10.00c ± 0.09

8.93c-e ± 0.09

3.80de ± 0.12

14.77c ± 0.19

6.97bc ± 0.29

13.90b ± 0.06

NaF-50 21.00d ± 1.15

8.40de ± 0.18

8.50d-g ± 0.17

3.47e-h ± 0.15

13.23e ± 0.18

6.57b-d ± 0.23

12.50d-f ± 0.12

NaF-100 18.30ef ± 0.65

7.50fg ± 0.27

7.40h-j ± 0.12

3.03g-i ± 0.09

11.83hi ± 0.09

5.93e-g ± 0.12

11.10g-I ± 0.06

NaF-150 15.03i ± 0.19

6.90gh ± 0.09

6.97i-k ± 0.29

2.87i-k ± 0.09

10.57kl ± 0.12

5.27hi ± 0.15

10.40i ± 0.12

NaF-200 11.37k ± 0.42

4.07j ± 0.20

5.73mn ± 0.29

1.90l ± 0.06

9.70m ± 0.12

4.50j ± 0.12

8.83j ± 0.23

SA-100 26.63a ± 0.32

13.23a ± 0.15

11.10a ± 0.21

5.03a ± 0.15

16.10a ± 0.15

7.80a ± 0.06

15.73a ± 0.12

SA-200 24.30b ± 0.65

11.80b ± 0.15

9.70bc ± 0.25

4.50bc ± 0.15

15.47b ± 0.28

7.00bc ± 0.12

15.00a ± 0.58

SA-300 21.80cd ± 0.15

9.70c ± 0.26

8.53d-f ± 0.15

3.53e-g ± 0.19

14.30cd ± 0.15

6.53b-d ± 0.20

13.50bc ± 0.17

NaF-50+ SA-100 22.90bc ± 0.06

11.30b ± 0.09

9.97b ± 0.12

4.73ab ± 0.09

14.60c ± 0.31

7.70a ± 0.12

13.07bc ± 0.09

NaF-50+ SA-200 22.10cd ± 0.06

9.90c ± 0.18

9.10cd ± 0.15

4.20cd ± 0.17

13.93d ± 0.15

7.03b ± 0.20

12.80c-e ± 0.15

NaF-50+ SA-300 19.43e ± 0.30

7.77ef ± 0.09

8.23e-g ± 0.18

3.27f-i ± 0.37

12.70ef ± 0.06

6.30de ± 0.12

12.30d-f ± 0.17

NaF-100+ SA100 22.07cd ± 0.12

9.00d ± 0.12

8.50d-g ± 0.21

4.20cd ± 0.21

13.03ef ± 0.19

6.90bc ± 0.06

12.00ef ± 0.06

NaF-100+ SA-200 17.97e-g ± 0.55

8.37de ± 0.34

7.90f-h ± 0.17

3.73d-f ± 0.09

12.60fg ± 0.12

6.50cd ± 0.06

11.70f-h ± 0.12

NaF-100+ SA-300 16.60gh ± 0.31

6.97gh ± 0.09

6.87jk ± 0.59

2.80i-k ± 0.15

11.50ij ± 0.17

5.47gh ± 0.23

10.77i ± 0.15

NaF-150+ SA-100 19.07e ± 0.12

7.77ef ± 0.17

7.70g-i ± 0.21

3.77d-f ± 0.12

12.13gh ± 0.28

6.23d-f ± 0.12

11.85fg ± 0.48

NaF-150+ SA-200 17.17fg ± 0.37

7.20fg ± 0.21

7.03i-k ± 0.21

2.97h-j ± 0.20

11.00jk ± 0.26

5.80fg ± 0.06

11.01hi ± 0.58

NaF-150+ SA-300 13.43j ± 0.34

6.43hi ± 0.59

6.43k-m ± 0.28

2.50jk ± 0.12

9.97m ± 0.12

4.93ij ± 0.09

10.30i ± 0.40

NaF-200+ SA-100 15.23hi ± 0.34

6.13i ± 0.22

6.70j-l ± 0.12

3.10g-i ± 0.12

10.87k ± 0.09

5.10hi ± 0.06

9.40j ± 0.26

NaF-200+ SA-200 13.30j ± 0.91

5.97i ± 0.12

6.00lm ± 0.58

2.40k ± 0.15

10.07lm ± 0.23

4.60j ± 0.31

9.03j ± 0.19

NaF-200+ SA-300 8.63l

± 0.32 3.80j

± 0.15 4.90n ± 0.12

1.67l ± 0.15

8.50n ± 0.21

3.23k ± 0.15

6.67k ± 0.22

multiple range test and P= 0.05; NaF; Sodium fluoride; SA: Salicylic acid; NaF-SA: Sodium fluoride Salicylic acid




353353



At 44 DAS, compared to the control group, the SA-100 group had increases in theleaf area in the RK-510 and Classic varieties of 4.22% and 8.87%, respectively(Tables 1 and 4).

Similarly, the leaf area was recorded at 67 and 105 DAS for both varieties (RKS-510 and classic) of pea plant. At 67 and 105 DAS, the overall leaf area was increasedas compared to 44 DAS and the maximum values were recorded at 105 DAS.

DISCUSSION

Fluorine is the 13th most abundant element in nature and contamination by it of thenatural environment has become a global issue.16 Fluorides are present in acidic soilsand are usually poisonous. It has been reported that a high soil fluoride content hasadverse effects on plant growth and development,17 including by causing leafdamage18 and decreasing the content of chlorophyll in leaves.19 These create stressconditions for plants. Pisum sativum L., known as the pea plant, is very importanteconomically and used as a fresh vegetable and a dry pulse. In the present study theimpact of fluoride stress on the growth and development of the pea plant wasdetermined by providing various concentrations of NaF as a biweekly soil drenchbefore harvesting at 44, 67, and 105 DAS.

The growth of pea plants was affected due to stress created by applying NaF. As theconcentration of NaF was increased, there was decrease in the growth parameterssuch as height of plant, number of leaves, leaf area, and the number of branches.Sabal and Saxena reported that fluoride stress from increasing NaF concentrationscaused progressively greater decreases in root and shoot length.2 With a 5 µMconcentration of NaF, the average root and shoot length inhibition, compared to thecontrol, was 23% and 51.4%, respectively, while with a NaF concentration of 25 µMthe corresponding values were 66% and 73%.20

In the present experiment, the exposure of pea plants to various concentrations ofNaF, i.e., 50, 100, 150, and 200 ppm, resulted in reductions in various growthparameters. SA, applied exogenously in low concentrations, was able to overcomethe reduction in growth. A low concentration of SA, i.e., 100 ppm, enhanced thegrowth and development of the plants under NaF stress, while a high concentration,i.e., 300 ppm, caused a decrease in growth and proved to be harmful. A SAconcentration of 200 ppm had a variable effect and, although usually enhancinggrowth, sometimes growth was reduced. It has been proposed by scientists that SA isa signalling molecule which is produced naturally in plants and is responsible forinducing abiotic stress tolerance in plants.21 A significant increase in the growth ofmung bean plants under salt stress was observed after applying 100 mg.L-1 salicylicacid.12 Another, study found that 100 mg.L-1 of SA enhanced both the overall growthand the biomass of sunflower under water stress conditions.22

The present study investigated the effects of NaF stress on pea plants and observeda gradual decrease in growth as well as plant biomass with increased concentrationsof NaF. We found that the fresh and dry weights of root and shoot were higher in thecontrol and SA-100 groups than in the other groups, due to the ameliorative effects ofsalicylic acid, and that increased concentrations of NaF caused a decrease in the rootand shoot dry weight. Gupta et al. found that at 30 mg NaF.L-1 the average shoot androot dry and fresh weights were reduced to 50%, 27%, 62%, and 29%, respectively.6




354354



The exogenous foliar application of SA has been found to increase the growth, leafarea, and photosynthetic capacity of sunflowers, which may be due to a SA-inducedincrease in the activity of peroxidase.23 In wheat plants, the presence of fluoride inhigh concentrations had pronounced detrimental effects on the growth of the plantswith decreases in leaf area, root length, and shoot length.24

In the present study, NaF stress was created by providing NaF solution as a soildrench on a biweekly basis and it resulted in deleterious effects on plant growth andbiomass production. Pakistan has areas with salt stress and, as an agriculturalcountry, this is a matter of concern. The increase in NaF in soil results from a numberof sources. Plants suffer with this stress silently but show their response in the formof a diminished growth pattern and reduced yield, as observed in presentinvestigation. The exogenous application of SA, in a concentration of 100 ppm, is aneffective tool against NaF-induced stress. Its application resulted in enhanced growthand biomass production of Pisum sativum when applied exogenously as a foliarspray. However, at higher concentrations, such as 300 ppm, SA itself may be a causeof a high level of stress in plants and at this dosage it was not effective in combatingNaF-induced stress.

CONCLUSION

As salicylic acid at 100 and 200 ppm had ameliorative effects on plants under NaFstress, we concluded that the foliar application of salicylic acid promotes salttolerance in two different cultivars of the pea plant Pisum sativum L.

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335 Research report Fluoride 53(2 Pt 2):335-355 of Pisum ...Madeeha Ansari,a Asma Zulfiqar,a Abudllah Jalal,d Aleena Nizame Lahore and Peshawar, Pakistan ABSTRACT: Fluoride in our