An attempt towards reducing salt stress on Flame Seedless ...enhancing their tolerance capacity and thereby enduring the salt stress (Carretero et al., 2008). Arbuscular Mycorrhizal

Middle East Journal of Applied Sciences ISSN 2077-4613

Volume : 09 | Issue :02 |April-June| 2019 Pages: 575-590

Corresponding Author: Abd El-Rahman, A.S., Viticulture Res. Dept., Hort. Res. Instit., Agric. Res. Center, Giza, Egypt

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An attempt towards reducing salt stress on Flame Seedless grape rootings through the inoculation of Arbuscular Mycorrhizae fungi and Pseudomonas fluorescens

Abd El-Rahman, A.S.1, Sh.M.M. El-Mogy1, S.F.S. Al-Sharnouby2 and H.A. Abo-Kora2

1Viticulture Res. Dept., Hort. Res. Instit., Agric. Res. Center, Giza, Egypt 2Microbiology Res. Dept., SWE Res. Instit., Agric. Res. Center, Giza, Egypt

Received: 14 March 2019 / Accepted 20 May 2019 / Publication date: 30 June 2019 ABSTRACT

This study was conducted during 2017 and 2018 seasons to study the effect of soil inoculation with Arbuscular Mycorrhizal fungi (AM) and Pseudomonas fluorescens (Ps.) under different water salinity levels (1000, 2000 and 3000 ppm) in an attempt to improve vegetative growth traits, nutritional acquisition and microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings.

The results indicated that irrigation with increasing levels of water salinity was accompanied with decreasing survival percentage and depressing all vegetative growth traits including namely main shoot length, shoot diameter, number of leaves/plant, average leaf area, coefficient of wood ripening, shoot and root biomass, total biomass, leaf total chlorophyll, nitrogen, phosphorus, potassium, calcium, magnesium and sulfur content and shoot total carbohydrate content. On the contrary, leaf proline amino acid, sodium, and chloride content increased with increasing levels of salinity. Concerning the microbial count and enzyme activity in the rhizosphere of Flame Seedless grape rootings, it was noticed that populations of total microbial count, spore numbers of AM fungi, AMF root colonization and dehydrogenase activity in the rhizosphere were also decreased with increasing levels of water salinity.

Flame Seedless grape rootings strategy for salt stress tolerance could be achieved by inoculation with AM fungi and Ps. fluorescens. Dual inoculation of AM fungi and Ps. fluorescens had the best results for alleviating the harmful effects of irrigation water salinity by improving vegetative growth traits, nutritional acquisition and microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings. Keywords: salt stress, grape, Flame Seedless, inoculation, Arbuscular Mycorrhizae fungi and

Pseudomonas fluorescens

Introduction

Plantation of the grape cultivars in Egypt has been progressively developed in the last few years. However, a great acreage is located at the new reclaimed soils which have many problems including salinity. The concentration and composition of dissolved constituents in water determine its quality for irrigation (Miller et al., 1990).

Salinity is considered as one of the most important a biotic stresses that limits crop productivity, affecting several aspects of plant metabolism that generally results in the reduction of plants growth (Singh and Chatrath 2001). The direct effects of salt on plant growth may involve: (a) reduction in the osmotic potential of the soil solution that reduces the amount of water available to the plant causing physiological drought; (b) toxicity of excessive ions, mainly Na and Cl, results in the disruption of enzyme activity, photosynthesis, respiration, as well as protein synthesis, and damaging of plasma membrane including cell organelles; and (c) nutrient imbalance caused by depression in uptake and/or transport, all of which lead to reduced plant growth (Feng et al., 2002). To cope with this stress, the interaction between plant roots and salt-tolerant microorganisms such as bacteria and fungi play a key role in alleviating the toxicity was induced by salt stress, thus normalizing the uptake mechanism in plants by supplying the essential nutrients. In this way, the plant recovers the water balance machinery, enhancing their tolerance capacity and thereby enduring the salt stress (Carretero et al., 2008).

Arbuscular Mycorrhizal (AM) fungi form symbiotic associations with most of the plants and enhance the tolerance capacity to withstand the abiotic stresses including salinity besides increasing the uptake of inorganic nutrients (Hajbagheri and Enteshari, 2011). The alleviation of salt stress by AMF has been reported through improved osmotic balance, increased activity of anti-oxidant enzymes and

Middle East J. Appl. Sci., 9(2): 575-590, 2019 ISSN 2077-4613

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photosynthetic rate, enhanced nutrient uptake and water use efficiency and increased osmoregulator compounds in plants (Jahromiet al., 2008; Sheng et al., 2008 and Cekicet al., 2012).

The application of Plant Growth Promoting Rhizobacteria (PGPR) bacteria is one of the most promising alternative approaches to improve crop production in saline soils (Yang et al., 2009). Various salt-tolerant PGPR bacteria including Azospirillum, Burkholderia, Rhizobium, Pseudomonas, Acetobacter and Bacillus have been successfully applied or tested for plant growth promotion under salt stress (Egamberdieva et al., 2015). Pseudomonas fluorescens is considered an important model to assess beneficial plant–bacteria interactions, including plant growth promotion under a biotic stress (Sitaraman, 2015). Inoculation of plants with Pseudomonas bacteria was found to alleviate salinity effects on plant development by reducing the uptake of toxic ions, inducing systemic resistance, producing phytohormones, increasing nutrient uptake and establishing root colonization (Chu et al., 2019).

The interactions between AMF and rhizobacteria have been shown to improve mutualistic fungus-host interaction (Artursson et al., 2006) and plant growth (Gamalero et al., 2008). Some studies have reported the positive effects of co-inoculation of AMF and plant growth promoting bacteria on plant growth and nutrient uptake under saline stress conditions (Lee et al., 2015).

Therefore, the main objective of this study is to achieve the possibility of alleviating the harmful effects of irrigation water salinity on vegetative growth traits, nutritional acquisition and microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings through the soil inoculation with Arbuscular Mycorrhizal fungi (AM) and Pseudomonas fluorescens (Ps.) under different water salinity levels.

Materials and Method

This study was conducted during 2017 and 2018 seasons in a shade house located at Aga, Dakahlia governorate to study the possibility of alleviating the harmful effects of irrigation water salinity on vegetative growth traits, nutritional acquisition and microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings through the soil inoculation with Arbuscular Mycorrhizal fungi (AM) and Pseudomonas fluorescens (Ps.) under different water salinity levels. Uniform and healthy 480 own rooted one-year-old Flame Seedless grape rootings were chosen. The rootings were planted through the first week of March in polyethylene bags filled with 5 kg of a medium containing clean sand carefully washed with tap water several times to remove any soluble salts. All bags had bottom holes to allow excess water drainage. Field capacity and wilting point of the sand medium were: 6.5% and 2.3%, respectively, while the Electric Conductivity (E.C.) of irrigation tap water was 0.85 m mhos/cm (544 ppm). The plants were irrigated with saline water treatments twice a week to keep moisture content of the planting medium about 70% of the field capacity throughout the period of the experiment from the first of May till the end of October. Leaching of accumulated salts was done every 15 days by tap water up to the end of the experiment. Sixteen treatments were conducted as follows: 1) Irrigation with tap water at 544 ppm salinity (control) 2) Inoculation with Arbuscular Mycorrhizal fungi (AM) 3) Inoculation with Pseudomonas fluorescens (Ps) 4) Inoculation with AM +Ps 5) Irrigation with saline water at 1000 ppm 6) Irrigation with saline water at 1000 ppm +AM 7) Irrigation with saline water at 1000 ppm +Ps 8) Irrigation with saline water at 1000 ppm + AM +Ps 9) Irrigation with saline water at 2000ppm 10) Irrigation with saline water at 2000 ppm + AM 11) Irrigation with saline water at 2000 ppm + Ps 12) Irrigation with saline water at 2000 ppm + AM + Ps 13) Irrigation with saline water at 3000 ppm 14) Irrigation with saline water at 3000 ppm +AM


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15) Irrigation with saline water at 3000 ppm +Ps 16) Irrigation with saline water at 3000 ppm + AM +Ps

Salinity in the irrigation water was Strogonov stock solution chloride consisting of: 78g NaCl,

10g MgSO4, 9g CaCO3, 2g MgCl2, 1g CaSO4 mixture dissolved in one liter (Strogonov, 1964) to yield a balance of cations and anions with a value of sodium absorption ratio (SAR) reaching 6.0 and for preparing 1000, 2000 and 3000 ppm concentrations.

Arbuscular Mycorrhizal spores were originally extracted from the Egyptian soil. Spores of AMF including Genera Glomus, Gigaspora and Acaulospora were added before planting. Extraction and counting of identified mycorrhizal spores were carried out according to the method described by (Massoud, 1999). Fifty grams per bag of mixed spores (250 spores/gram) of AM fungi genera were prepared and mixed with soil, then the rooting were planted (Massoud, 2005).

Pseudomonas fluorescence bacteria promising strain as alleviating the effects of salinity (Chu et al., 2019). It was maintained refrigerated at 40C on nutrient agar medium (Jacobs and Gerstein, 1960). The strain provided by department of microbiology /SWERI/ARC, Egypt.

All treatments were fertilized with a nutrient solution (Hoagland and Arnon, 1950) at half weekly intervals till the end of the growing season.

Each treatment was comprised of 30 plants distributed in 3 replicates (10 plants/ replicate) in completely randomized design. The following parameters were determined.

1. Morphological studies:

Survival percentage Number of survived plants was counted in the end of experimental season.

Vegetative growth parameters

Shoot length (cm), shoot diameter (cm), number of leaves/plant, average leaf area (cm2) of the apical 5th and 6th leaves using a CI-203- Laser Area-meter made by CID, Inc., Vancouver, USA. were recorded in both seasons. Coefficient of wood ripening was calculated by dividing length of the ripened part of the shoot by total length of the shoot according to Bouard (1966).

Plant biomass

Shoot biomass (g dry weight), root biomass (g dry weight) and total biomass (g dry weight) were recorded.

2. Chemical studies: Leaf total chlorophyll content (SPAD). This was measured by using nondestructive Minolta

chlorophyll meter SPAD 502 (Wood et al., 1992).

Leaf proline content (mg/g) was colorimetrically estimated on fresh weight basis according to the method of Batels et al. (1973).

Shoot total carbohydrate content (%) (Smith et al., 1956). Leaf mineral content: N (%) (Pregl, 1945), P (%) (Snell and Snell ,1967) K (%) (Jackson, 1967) and

Ca, Mg, S, Cl and Na percentages were estimated according to Evenhuis (1978).

3. Microbiological studies:- Samples were taken for carrying out the following determinations:

Total microbial count (-x105 colony forming unit (cfu)/g soil) according to (Esher and Jensen, 1972). Number of AM (spore/g soil) after each season was counted according to (Massoud, 2005). AMF root colonization (%) according to (Trouvelot et al., 1986). Dehydrogenase activity (μgTPF.g-1 dry soil day-1) was determined according to Thalmann (1967).


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Experimental design and statistical analysis The completely randomized design was adopted for the experiment. The statistical analysis of

the present data was carried out according to Snedecor and Cochran (1980). Averages were compared using the new L.S.D. values at 5% level (Steel and Torrie, 1980).

Results and Discussion 1. Morphological studies:

Survival percentage

As shown in Table (1), data reveal that survival percentage was significantly declined with increasing salinity level, irrigation with high saline water at 3000 ppm significantly resulted in the least percentages of survival compared to non-saline plants. Soil inoculation with AM fungi or Ps. fluoresence significantly improved in survival percentage at all salt levels. A significantly higher survival percentage was attained by dual inoculation of AM fungi and Ps. fluoresence in both seasons.

These results are in harmony with Kilany et al., (2006) who found that water stress due to salinity by raising salt concentration in the irrigation water effectively depressed the percentage of survival.

As for the effect of AMF, Yang et al., (2014) pointed out that AM fungi inoculation improved survival percentage of apple seedlings under salt stress.

With respect to Ps. fluorescens, Chu et al., (2019) found that inoculation with Pseudomonas sp. improved survival percentage of Arabidopsis thaliana plants under salt stress up to 225 mM NaCl.

Vegetative growth parameters

Data presented in (Table 1) show that all of the studied vegetative growth parameters namely main shoot length, shoot diameter, number of leaves/plant, average leaf area and coefficient of wood ripening were significantly decreased with increasing levels of salinity and a more prominent effect was evident at the highest salt concentration of 3000 ppm. Both inoculation with AM fungi or Ps. fluorescens inoculation significantly reduced the adverse effects of salinity on vegetative growth parameters. The highest significant values for these parameters were obtained from dual inoculation of AM fungi and Ps. fluorescens compared to individual inoculation in both seasons.

Plant biomass

As shown in Table (2), data reveal that plant biomass production; i.e. shoot biomass, root biomass and total biomass was significantly declined with increasing salinity level. Irrigation with high saline water at 3000 ppm significantly had the least values of plant biomass production compared to non-saline plants. Soil inoculation with AM fungi or Ps. fluorescens significantly improved in plant biomass production at all salt levels. A significantly higher plant biomass production was attained by dual inoculation of AMF and Ps. fluorescens in both seasons.

The reduction observed on growth parameters with increasing salinity levels can, in some instances, be attributed to salinity-induced adverse change in leaf water relations reducing photosynthesis, dehydration of proteins and protoplasm to a lower extent (Tozlu et al., 2000) and this may also be because of osmotic effect of salt on root and toxic effect of accumulated ions on the plant tissues (Lambers 2003).

Several mechanisms for the explanation of AMF role have been proposed: AM plants have an improved ability for growth and tolerance to salt stress. Ruiz-Lozano et al., (1996) concluded that the mechanisms underlying AM plant growth improvement under saline conditions were based on physiological processes (increased carbon dioxide exchange rate, transpiration, stomatal conductance and water use efficiency) rather than on nutrient uptake. In addition, Feng et al., (2002) showed that AM fungus improved the resistance capacity to osmotic stress by increasing soluble sugar and electrolyte concentrations in plants roots. Moreover, Plenchette and Duponnis, (2005) mentioned that AM fungi contribute to plant growth via enhancement of mineral nutrient uptake.

Inoculation of plants with Ps. fluorescens was found to alleviate salinity effects on plant development by reducing the uptake of toxic ions, inducing systemic resistance, producing phytohormones, increasing nutrient uptake and establishing root colonization (Chu et al., 2019).


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Table 1: Effect of soil inoculation with Arbuscular Mycorrhizal fungi (AM) and Pseudomonas fluorescens bacteria (Ps) under different water salinity levels

on survival (%) and some vegetative growth traits of Flame Seedless grape rootings during 2017 and 2018 seasons

Survival

(%) Average shoot

length (cm) Average shoot diameter (cm)

No. of leaves/shoot Average leaf

area/shoot (cm2) Coefficient of wood ripening

Water salinity Soil inoculation 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018

control

Non- inoculation 92.1 91.3 73.3 77.7 0.77 0.80 23.6 24.3 93.6 97.8 0.62 0.67

AM 95.2 94.1 88.7 94.1 0.93 0.96 28.5 29.4 113.2 118.3 0.76 0.82

Ps 93.6 92.2 79.9 84.7 0.83 0.87 25.7 26.5 102.0 106.6 0.68 0.74

AM+ Ps 98.6 95.9 100.2 106.3 1.05 1.09 32.2 33.2 127.9 133.7 0.85 0.92

1000 ppm

Non- inoculation 81.7 80.4 69.2 73.3 0.75 0.77 20.8 21.4 88.0 93.0 0.59 0.65

AM 84.4 82.7 83.7 88.7 0.90 0.93 25.2 26.0 106.5 112.6 0.72 0.79

Ps 82.8 81.9 75.4 79.9 0.81 0.83 22.7 23.4 95.9 101.4 0.65 0.71

AM+ Ps 85.3 84.1 94.6 100.2 1.02 1.05 28.5 29.3 120.3 127.2 0.81 0.89

2000 ppm

Non- inoculation 68.3 66.7 61.5 62.9 0.65 0.63 17.6 18.4 84.7 89.3 0.57 0.62

AM 70.4 69.7 74.4 76.1 0.79 0.77 21.3 22.3 102.5 108.1 0.69 0.76

Ps 69.6 67.6 67.1 68.6 0.71 0.69 19.2 20.1 92.3 97.4 0.63 0.68

AM+ Ps 72.7 70.9 84.1 86.0 0.89 0.87 24.1 25.2 115.8 122.1 0.78 0.85

3000 ppm

Non- inoculation 49.4 48.6 58.0 58.7 0.57 0.54 15.9 17.3 83.3 86.8 0.54 0.58

AM 52.5 51.9 70.2 71.0 0.69 0.66 19.3 21.0 100.8 105.0 0.66 0.71

Ps 51.8 51.2 63.2 64.0 0.63 0.59 17.3 18.9 90.8 94.6 0.59 0.64

AM+ Ps 54.2 53.8 79.3 80.3 0.78 0.74 21.8 23.7 113.9 118.7 0.74 0.80

New L.S.D. at (0.05) = 2.1 1.7 8.7 9.1 0.07 0.09 2.7 2.4 10.7 11.3 0.05 0.04


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Table 2: Effect of soil inoculation with Arbuscular Mycorrhizal fungi (AM) and Pseudomonas fluorescens bacteria (Ps) under different water salinity levels on shoot biomass, root biomass and total biomass of Flame Seedless grape rootings during 2017 and 2018 seasons

Shoot biomass (g dry weight)

Root biomass (g dry weight)

Total biomass (g dry weight)

Water salinity Soil inoculation 2017 2018 2017 2018 2017 2018

Control

Non- inoculation 13.8 14.1 18.1 18.5 31.9 32.6

AM 16.7 17.3 21.7 22.4 38.4 39.7

Ps 14.9 15.2 19.6 20.2 34.5 35.4

AM+ Ps 18.6 18.9 24.5 24.9 43.1 43.8

1000 ppm

Non- inoculation 12.5 12.7 17.6 18.1 30.1 30.8

AM 15.2 15.5 19.2 19.7 34.4 35.2

Ps 13.7 13.8 18.3 18.9 32.0 32.7

AM+ Ps 17.1 17.4 20.1 20.4 37.2 37.8

2000 ppm

Non- inoculation 11.2 11.5 16.1 16.4 27.3 27.9

AM 13.5 13.9 17.5 18.1 31.0 32.0

Ps 12.2 12.6 16.9 17.2 29.1 29.8

AM+ Ps 15.3 15.7 18.2 18.7 33.5 34.4

3000 ppm

Non- inoculation 9.8 10.2 13.5 13.9 23.3 24.1

AM 11.9 12.4 14.7 15.2 26.6 27.6

Ps 10.7 10.9 14.1 14.5 24.8 25.4

AM+ Ps 13.5 13.8 15.2 15.7 28.7 29.5

New L.S.D. at (0.05) = 1.4 1.3 1.9 1.6 3.8 3.2


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The results are in agreement with those obtained by Khalil (2013) who found that vegetative growth parameters were significantly decreased with increasing salinity levels of Dogridge, 1103 Paulsen and Harmoney grape rootstocks. As for the effect of AM fungi, Yang et al., (2014) pointed out inoculation with AM fungi improved vegetative growth of apple seedlings under salt stress. With respect to Ps. fluorescens, Pirhada et al., (2017) showed that application of Pseudomonas sp. bacteria significantly increased growth of wheat. Moreover, Selvakumar et al., (2018) mentioned that the co-inoculation of AMF and Ps. fluorescens bacteria significantly improved plant growth of maize plants under saline stress.

2. Chemical studies:

Leaf total chlorophyll content: Data presented in (Table 3) show that leaf total chlorophyll content was significantly decreased

with increasing levels of salinity and a more prominent effect was evident at the highest salt concentration of 3000 ppm. Soil inoculation with AM fungi or Ps. fluorescens significantly reduced the adverse effects of salinity on leaf total chlorophyll content. The highest significant values of leaf total chlorophyll content were obtained from dual inoculation of AM fungi and Ps. fluorescens compared to individual inoculation in both seasons.

The adverse effects of water salinity on total chlorophyll content in the leaves can be attributed to its negative action on interrupting and reducing the availability of water and nutrients particularly magnesium, destroying the building and conductance tissue and decreasing the biosynthesis of pigments and photosynthesis (Nijjar, 1985). In addition to, Murkute et al., (2006) stated that leaf chlorophyll content decreased under stress due to the suppression of specific enzymes that are responsible for the synthesis of photosynthetic pigments.

However, the previous increase of total chlorophyll content in the leaves in mycorrhizal plants could be ascribed due to the cytokinin-like substances secreted by fungi, which enhance the chloroplast development (Nawar et al., 1988). In addition, Sheng et al. (2008) mentioned that AM fungi enhances leaf chlorophyll content can be attributed to increase the absorption of mineral elements particularly magnesium.

The results are in harmony with those obtained by Rizk-Alla et al., (2006) who found that leaf chlorophyll content were significantly decreased with increasing levels of salinity of Thompson Seedless grape rootings.

As for the effect of AM fungi, Khalil (2013) pointed out inoculation with AM fungi increased leaf chlorophyll content of Dogridge, 1103 Paulsen and Harmoney grape rootstocks under salt stress.

With respect to Ps. fluorescens, Pirhada et al., (2017) showed that the maximum chlorophyll index was obtained by application of Pseudomonas sp.

Leaf proline content

As shown in Table (3), data reveal that leaf proline content was significantly increased with increasing salinity level; irrigation with high saline water at 3000 ppm significantly had the highest values of leaf proline content compared to non-saline plants. Soil inoculation with AM fungi or Ps. fluorescens significantly reduced in proline accumulation in leaf tissues under saline conditions. Significantly lower leaf proline content was attained by dual inoculation of AM fungi and Ps. fluorescens under non-salinity conditions in both seasons.

The accumulation of proline in the leaves under salt stress might be attributed to protect the cell by balancing the osmotic pressure of cytosol with external environment (Giridara et al., 2003).

The results are in agreement with those obtained by Rizk-Alla et al., (2006) who found that proline accumulation were consistently increased with gradual salinization of Thompson Seedless grape rootings.

Shoot total carbohydrate content

Data presented in (Table 3) show that shoot total carbohydrate content was significantly decreased with increasing levels of salinity and a more prominent effect was evident at the highest salt concentration of 3000 ppm. Soil inoculation with AM fungi or Ps. fluorescens significantly reduced the


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Table 3: Effect of soil inoculation with Arbuscular Mycorrhizal fungi (AM) and Ps. fluorescens bacteria (Ps) under different water salinity levels on leaf

content of total chlorophyll and proline and shoot content of total carbohydrate of Flame Seedless grape rootings during 2017 and 2018 seasons

Leaf

total chlorophyll content (SPAD)

Leaf proline content

(mg/g F.W.)

Shoot total carbohydrate

content (%)

Water salinity Soil inoculation 2017 2018 2017 2018 2017 2018

Control

Non- inoculation 35.7 25.8 0.09 0.11 23.2 24.6

AM 43.3 31.2 0.04 0.06 26.3 27.6

Ps 39.0 28.1 0.07 0.09 25.2 26.8

AM+ Ps 48.9 35.2 0.02 0.04 28.7 29.1

1000 ppm

Non- inoculation 33.4 24.8 0.10 0.12 22.7 23.4

AM 40.4 30.0 0.05 0.07 24.8 25.3

Ps 36.4 27.0 0.08 0.10 23.1 24.5

AM+ Ps 45.7 33.9 0.03 0.05 25.6 26.2

2000 ppm

Non- inoculation 31.8 23.6 0.12 0.13 22.1 22.9

AM 38.5 28.5 0.07 0.08 24.6 24.1

Ps 34.7 25.7 0.10 0.11 23.9 23.4

AM+ Ps 43.5 32.2 0.05 0.06 25.2 25.7

3000 ppm

Non- inoculation 30.4 23.3 0.14 0.15 21.5 22.1

AM 36.8 28.1 0.11 0.12 23.1 24.5

Ps 33.1 25.4 0.13 0.14 22.4 23.8

AM+ Ps 41.6 31.8 0.10 0.11 24.2 25.1

New L.S.D. at (0.05) = 1.7 1.3 0.02 0.01 1.1 1.4


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adverse effects of salinity on shoot total carbohydrate content. The highest significant values of shoot total carbohydrate were obtained from dual inoculation of AM fungi and Ps. fluorescens compared to individual inoculation in both seasons.

The reduction observed on growth parameters with increasing salinity levels can be attributed to salinity-induced effectively depressed the synthesis of carbohydrates (Kilany et al., 2006).

However, AM fungi improve physiological processes, like water absorption capacity of plants by increasing root hydraulic conductivity and favourably adjusting the osmotic balance and composition of carbohydrates (Rosendahl and Rosendahl 1991).

The results are in harmony with those obtained by Goicoechea et al. (2005) who found that shoot total carbohydrate content were significantly decreased with increasing levels of salinity.

As for the effect of AM fungi, Khalil (2013) pointed out inoculation with AM fungi increased leaf total carbohydrate content of Dogridge, 1103 Paulsen and Harmoney grape rootstocks under salt stress.

Leaf mineral content

As shown in Table (4), data reveal that leaf mineral content; i.e. nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, sodium and chloride were significantly affected with increasing salinity level, irrigation with high saline water at 3000 ppm significantly had the least values of leaf N, P, K, Ca, Mg and S content and the highest values of leaf Na and Cl content compared to non-saline plants. Soil inoculation with AM fungi or Ps. fluorescens significantly improved in leaf N, P, K, Ca, Mg and S content at all salt levels. A significantly higher N, P, K, Ca, Mg and S uptake were attained by dual inoculation of AM fungi and pseudomonas bacteria, while it significantly attain the least values of Cl and Na in both seasons.

The reduction occurring in N, P, K, Ca, Mg and S content of the leaves under salt stress might be attributed to the increase in osmotic pressure, thereby reducing the water and nutrients uptake (Stevens and Walker 2002).

Some mechanisms have been suggested to explain the role of AM inoculation: AM can improve salt tolerance through inducing osmotic adjustment (Duke et al., 1986), AM capability of dissolving weakly soluble soil minerals by releasing acids (Leyval and Berthelin, 1989), improve and balance nutrition in plants could also increase salt tolerance (Marschner, 1995), reduce the negative effects of Na and Cl by maintaining membrane integrity (Rinaldelli and Mancuso, 1996) that would facilitate compartmentalization within vacuoles, and selective ion intake. In this respect, Cantrell and Linderman (2001) suggested that improved mineral nutrient absorption by AM fungi in plants grown under saline conditions might reduce the negative effects of Na and Cl and retain them in roots without being translocated to the shoots by maintaining vacuolar membrane integrity and retaining in intracellular AM fungal hyphae or was compartmentalized in the root cell vacuoles which prevented these ions from interfering in the metabolic pathways of growth. Moreover, Al- Karaki, (2006) who explained that the higher mineral nutrient acquisition in AM compared to non-AM plants likely occurred because of increased availabilities or transport (absorption and/or translocation) by AM fungi hyphae.

Nutrient uptake was enhanced by inoculation of Ps. fluorescens may attribute to its production of plant growth regulators at the root interface, which stimulated root development and resulted in better absorption of water and nutrients from the soil (Abbasi et al., 2011).

The results are in harmony with those obtained by Rizk-Alla et al., (2006) who found that leaf mineral content were significantly decreased with increasing levels of salinity of Thompson Seedless grape rootings. As for the effect of AM fungi, Khalil (2013) pointed out inoculation with AM fungi improved leaf mineral content of Dogridge, 1103 Paulsen and Harmony grape rootstocks under salt stress. With respect to pseudomonas bacteria, Pirhada et al., (2017) showed that application of Pseudomonas sp. bacteria significantly increased leaf mineral content of wheat. Moreover, Selvakumar et al., (2018) mentioned that the co- inoculation of AMF and Ps. fluorescens significantly improved leaf mineral content of Dogridge, 1103 Paulsen and Harmoney grape rootstocks under salt stress.

3. Microbiological studies

Data concerning the effect of saline water and soil inoculation with AM fungi and P. fluoresence on microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings during 2017and 2018 seasons are shown in Table (5).


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on leaf mineral content of Flame Seedless grape rootings during 2017 and 2018 seasons

Nitrogen

(%) Phosphorus

(%) Potassium

(%) Calcium

(%) Magnesium

(%) Sulfur

(%) Chloride

(%) Sodium

(%)

Water salinity Soil inoculation 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018 2017 2018

control

Non- inoculation 1.99 2.04 0.29 0.34 1.37 1.46 2.47 2.55 0.53 0.60 0.24 0.26 1.11 1.05 0.39 0.43

AM 2.41 2.47 0.35 0.41 1.66 1.77 2.99 3.08 0.65 0.73 0.29 0.32 1.05 0.99 0.31 0.35

Ps 2.17 2.23 0.32 0.37 1.49 1.59 2.69 2.78 0.58 0.66 0.26 0.29 1.08 1.02 0.35 0.39

AM+ Ps 2.73 2.79 0.40 0.47 1.87 2.00 3.37 3.48 0.73 0.83 0.33 0.36 1.01 0.95 0.28 0.32

1000 ppm

Non- inoculation 1.96 1.98 0.27 0.31 1.34 1.42 2.45 2.50 0.49 0.57 0.20 0.23 1.19 1.13 0.44 0.49

AM 2.38 2.40 0.33 0.38 1.62 1.72 2.96 3.02 0.60 0.69 0.24 0.28 1.12 1.06 0.35 0.40

Ps 2.14 2.16 0.30 0.34 1.46 1.55 2.67 2.72 0.54 0.63 0.22 0.25 1.15 1.10 0.39 0.45

AM+ Ps 2.68 2.71 0.37 0.43 1.83 1.94 3.35 3.41 0.67 0.78 0.28 0.32 1.07 1.02 0.31 0.37

2000 ppm

Non- inoculation 1.92 1.95 0.26 0.29 1.31 1.40 2.25 2.35 0.46 0.53 0.17 0.21 1.22 1.17 0.49 0.53

AM 2.33 2.36 0.32 0.35 1.58 1.69 2.72 2.84 0.56 0.65 0.21 0.26 1.13 1.08 0.38 0.42

Ps 2.10 2.13 0.29 0.32 1.43 1.53 2.45 2.56 0.50 0.58 0.19 0.23 1.18 1.12 0.44 0.47

AM+ Ps 2.63 2.67 0.36 0.40 1.79 1.91 3.07 3.21 0.63 0.73 0.23 0.29 1.08 1.02 0.34 0.37

3000 ppm

Non- inoculation 1.85 1.91 0.23 0.25 1.27 1.36 2.21 2.29 0.42 0.48 0.15 0.18 1.26 1.21 0.53 0.56

AM 2.24 2.31 0.28 0.30 1.54 1.64 2.67 2.77 0.51 0.58 0.18 0.22 1.14 1.09 0.39 0.42

Ps 2.02 2.09 0.25 0.27 1.38 1.48 2.40 2.49 0.46 0.53 0.16 0.20 1.21 1.14 0.47 0.48

AM+ Ps 2.53 2.62 0.32 0.34 1.73 1.86 3.02 3.13 0.58 0.66 0.21 0.25 1.08 1.01 0.34 0.35

New L.S.D. at (0.05) = 0.03 0.05 0.02 0.03 0.02 0.03 0.02 0.04 0.05 0.03 0.02 0.03 0.03 0.02 0.01 0.02


585

Total microbial count As can be seen, tabulated data in Table (5) all treatments significantly affected with increasing

salinity level, irrigation with the lowest concentration of saline water 1000ppm improved the total microbial count especially in the application of dual inoculation with AMF and the tested Ps. fluorescens, while irrigation with high saline water at 3000 ppm had the least value in the two seasons. These findings are in consistence with those reported by Abd El- Wahab et al., (2011).

A significant interaction was observed between saline water and soil inoculation with AM fungi and Ps. fluorescens, the results revealed that total microbial count of non-inoculants plant significantly decreased with increasing salinity level, particularly in case of the high salinity concentration (3000 ppm), while the opposite trend was shown for dual inoculation of AMF and Ps. fluorescens plants under low and medium salinity concentrations (1000-2000 ppm). The highest significant values of total microbial count were obtained from the inoculation AM and Ps. fluorescens plants under salinity levels (1000-2000 ppm) recording (76.98, 78.00 & 67.13, 68.00 x105cfu/g soil) for both seasons respectively, and resulting in an increase over control by (1.27, 1.22 & 1.11, 1.07) fold for both seasons respectively, while non-inoculants plant grown under high saline conditions (3000 ppm) recorded the lowest values in both seasons of The study.The results are in agreement with those obtained by Godeas et al., (1999) who explained that the increase in populations of rhizospheric microorganisms in the roots of most plants are influenced by a combined inoculation of microorganism and AM fungi.

Number of AM spores

The results concerning number of AM spores/soil in Flame Seedlings grape rootings in response to irrigate water salinity and dual inoculation AMF and Ps. fluorescens are presented in Table (5). There is a significant difference in all treatments with irrigate saline water at in Flame Seedling rootings, there was a decline in number of AM spores in soil with increased salinity level. Number of AM spores in soil recorded the lowest values in plants grown under high salinity (3000ppm) compared to control regardless of AM fungi inoculation. Concerning the effect of dual inoculation with AM fungi and Ps. fluorescens, it is clear that number of AM spores in soil was higher in inoculated plants than those of without inoculation with AM plants grown under both irrigate with salinity and non-salinity water in both seasons.

The interaction effect was significant. It can be shown from the results that AMF inoculation benefits the plants under low to medium levels of salt concentrations (1000-2000 ppm). However, AMF didn’t protect the plants at the highest salt concentration (3000ppm). The highest significant values of number of AM spores in soil were obtained from the dual inoculation AM fungi and Ps. fluorescens plants under salinity levels (1000-2000 ppm) recording (974, 968 & 712, 744 spores/g soil) for both seasons respectively, while dual inoculated AM and Ps. fluorescens plants grown under high saline conditions (3000 ppm) recorded the lowest values (534 and 615 spores/g soil) in both seasons of this study. These finding are consistence with those reported by Selvakumar et al. (2018) who pointed out that the co-inoculation of AMF and Ps. fluorescens bacteria significantly improved number of AM spores in soil of maize plants under saline stress. Obviously in the results number of AM spore increase in the inoculation with AMF and helper bacteria (MHB) Ps. fluorescens this is in agreement with Hrynkiewicz et al., (2009) and Dominguez-Nez et al., (2013) who suggested that the effect of MHB is not always limited to mycorrhizae formation and/or functioning. Some MHB also behave as plant growth promoting rhizobacteria (PGPR), through either direct or indirect effects on the plant development. Several paramaters can be affected by MHB: strain Pseudomonas sp. GM41 stimulated the formation of secondary roots of populous tricochocarpa and P. deltoids (Labbe et al., 2014), while strain Ps. fluorescens CECT5281modified the response of roots of Pinus halepensis to drought.

AMF root colonization (%)

Data in Table (5) obvious that all salinity treatments caused a significant decrease in AMF root colonization in comparison to control, while there is significant increase in dual inoculation AMF and Ps. fluorescens exhibited the most performance in AM root colonization % at different concentration of salinity 1000, 2000 ppm (92, 95 & 73, 76%) at the 1st and 2nd growing seasons, respectively followed by single inoculation, Ps. fluorescens (53, 50, 45 and 48%) at the 1stand 2nd growing seasons, respectively without significant difference between them, whereas the higher concentration salinity of water irrigates plant 3000ppm gave the percentage of AMF root colonization. The range of AM


586


on microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings during 2017 and 2018 seasons

Total microbial count

(-x105cfu/g soil) No. of AM

(spore/g soil) AM root colonization

(%)

Dehydrogenase enzyme activity

(μgTPF/g soil/day)

Water salinity Soil inoculation 2017 2018 2017 2018 2017 2018 2017 2018

control

Non- inoculation 60.75 63.80 345.0 367.0 35.0 38.0 58.55 60.21

AM 80.81 81.90 890.0 895.0 89.0 90.0 136.13 138.91

Ps 65.41 65.55 698.0 718.0 70.0 75.0 118.15 120.33

AM+ Ps 82.60 93.82 998.0 1010.0 95.0 100.0 139.87 141.33

1000 ppm

Non- inoculation 58.00 60.00 325.0 334.0 33.0 35.0 49.16 55.18

AM 70.00 70.55 834.0 816.0 83.0 85.0 113.78 115.99

Ps 66.70 68.18 511.0 504.0 53.0 50.0 85.13 90.05

AM+ Ps 76.98 78.00 974.0 968.0 92.0 95.0 126.55 128.11

2000 ppm

Non- inoculation 55.18 57.33 228.0 233.0 24.0 24.8 48.89 50.23

AM 62.28 63.23 578.0 620.0 58.0 63.0 78.89 81.55

Ps 60.97 62.50 437.0 472.0 45.0 48.0 69.18 57.00

AM+ Ps 67.13 68.00 712.0 744.0 73.0 76.0 88.15 90.78

3000 ppm

Non- inoculation 40.00 40.66 214.0 225.0 23.0 24.6 40.11 43.67

AM 63.18 64.51 375.0 412.0 35.0 40.0 63.00 65.18

Ps 61.11 63.18 318.0 365.0 33.0 38.0 49.87 55.23

AM+ Ps 63.22 67.11 534.0 615.0 55.0 63.0 68.76 75.61

New L.S.D. at (0.05) = 1.83 2.07 29.0 34.0 4.0 7.0 3.81 3.54


587

colonization observed in Flame Seedless grape rootings of this study during two seasons 2017 and 2018 at salt concentration 1000 & 2000ppm (83, 85 & 58, 63%), respectively was similar to other studies conducted in soil under salinity condition but considerably lower than dual inoculation AM and Ps. fluorescens trials. These findings are in consistence with those reported by Schreiner (2003) who confirms that colonization by AM fungi may be related to the growth potential of the scion on different rootstocks and that the crop load carried on grapevines may influence nutrient exchange between plant and fungus. Moreover, Alves et al. (2010) and Lee et al., (2015) who pointed out that AM fungi colonize plant roots and mainly in the surrounding soil extending the roots depletion zone around the root system and consequently completed its life cycle and obtained plenty of resting spores in soil. As expected Mycorrhizal colonization of Flame Seedling grape rootings was significantly affected by increasing in salinity. In the contrary, the dual inoculation by AM fungi and Ps. fluorescens increase AM root colonization % at different concentration of salinity. These data its explanation goes back to (Rigamonte et al., 2010) the concept of Mycorrhization Helper Bacteria (MHB) was created. Five main actions of MHB on mycorrhizae were proposed: in the receptivity of root to the mycobiont, in root-fungus recognition, in fungal growth, in the modification of rhizospheric soil and in the germination of fungal propagates.

Dehydrogenase activity

Data shown in Table (5) revealed the existence of dehydrogenase enzyme activity among treatments giving an indication of microbial activity in the soil inoculated with AM fungi and Ps. fluorescens at different concentrations of salinity. In the salinity treatments; there was a decline in activity of dehydrogenase enzyme with increasing salinity level. Activity of dehydrogenase enzyme recorded the lowest values in plants grown under high salinity (3000 ppm) compared to control regardless of AM fungi inoculation. As regards the effect of inoculation with AM fungi, it is clear that the activity of dehydrogenase enzyme was higher in AM plants than that of non-AM plants grown under both salinity and non-salinity conditions in both seasons. The highest dehydrogenase activity was observed at the dual inoculation by AMF and Ps. fluorescens at 1000, 2000ppm irrigate saline water concentration (126.55,128.11 & 88.15, 90.78 µgTPF/g/D.W.soil/day) at the 1stand 2nd growing seasons, respectively as compared to individual inoculants, the lowest dehydrogenase activity was recorded for Ps. fluorescens (85.13, 90.05 & 69.18, 57.00 µgTPF/g/D.W.soil/day), respectively and AMF (113.78,115.99 & 78.89, 81.55 µgTPF/g/D.W.soil/day) at the 1stand 2nd growing seasons, respectively. The increase in dehydrogenase activity due to the inoculation of PGPR which colonize plant roots and produce IAA may be a good means of alleviating salt stress (Egamberdieva et al., 2015). Moreover, the increase in dehydrogenase enzyme activity was attributed to the intense activity of microflora as a mixture of biomass than each individual one. The highest increase in microbial respiration was recorded with the mixture of microorganism in the soil (Massoud, 2005).

In conclusion, it can be concluded that there is a possibility of alleviating the harmful effects of irrigation water salinity by improving survival percentage, vegetative growth traits, nutritional acquisition and microbial and enzyme activity in the rhizosphere of Flame Seedless grape rootings through the dual inoculation with AM fungi and Ps. Fluorescens.

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Documents

An attempt towards reducing salt stress on Flame Seedless ...enhancing their tolerance capacity and thereby enduring the salt stress (Carretero et al., 2008). Arbuscular Mycorrhizal