Potential Application of Glomus Intraradices (AMF) and Different Isolates of PGPR (Biotol) to Enhance the Yield and Quality of Wheat Grown in The Field in Calcareous Soil Under Different Salinity Levels

Introduction

Wheat (Triticum aestivum L.) is the most important cereal crop in Egypt.  Increasing wheat production is an essential national target to fill the gap between production and consumption (Tawfik et al. 2006).Salinity is one of the most brutal environmental factors limiting the productivity of crop plants because most of them are sensitive to salinity caused by high concentrations of salts in the soil (Shrivastava and Kumar, 2015). Salinity affects almost all aspects of plant development including: germination, vegetative growth and reproductive development. Soil salinity imposes ion toxicity, osmotic stress, nutrient (N, Ca, K, P, Fe, and Zn) deficiency and oxidative stress on plants, and thus limits water uptake from soil, some elements, such as sodium, chlorine, and boron besides having specific toxic effects on plants. Excessive accumulation of sodium in cell walls can rapidly lead to osmotic stress and cell death (Munns et al. 2002). Salinity and drought stresses inhibit the production of auxins, gibberellins, and zeatin in the roots and leaves of plants (Sakhabutdinova et al. 2003; Figueiredo et al. 2008; Perez-Alfocea et al., 2010).Calcareous soils occupy wide areas in the North African countries such as Egypt. These soils have a high percentage of calcium carbonate and normally basic in their reaction. Low soil fertility and nutrients deficiency in calcareous soils are very common and could be considered the main constraints for agricultural production in some cases (Hilal et al. 1990; Awad et al. 1996).Several strategies have been developed in order to decrease the toxic effects caused by high salinity on plant growth, such as mycorrhizal fungi (Cho et al. 2006 and Kohler et al. 2009) and plant growth-promoting bacteria (PGPB) (Kohler et al. 2006 and Dimkpa et al. 2009).

Under salt stress conditions, plant tolerance and production are complicated mechanisms. Arbuscular mycorrhizal fungi employ different mechanisms to enhance salt tolerance of host plants such as enhancing nutrient acquisition (P, N, Mg and Ca) (Azcon and El-Atrash 1997; Giri and Mukerji 2004 and Sheng et al., 2009), inhibiting high uptake of Na and Cl and their transport to plant shoots (Dai et al. 2009), improving water uptake (Ruiz-Lozano and Azcon 2000), accumulating of proline and polyamines (Evelin et al. 2009) and increasing some of enzymatic antioxidant defense system (SOD and CAT) (Wu et al. 2010). Other arbuscular mycorrhizal mechanisms may include an osmotic adjustment, which assist in maintaining the leaf turgor pressure, and effects on the photosynthesis, transpiration, stomatal conductance and water use efficiency (Juniper and Abbott, 1993).

Tank and Saraf (2010) showed that PGPRs which are able to solubilize phosphate, produce phytohormones and siderophores in salt condition promote growth of tomato plants under 2% NaCl stress. PGPR are able to increase AM fungal development by affecting root colonization as well as by enhancing plant N and P uptake (Artursson et al. 2006 and Richardson et al. 2009). There are different examples of enhanced associations between different bacterial strains including Bacillus, Paenibacillus, Pseudomonas and Rhizobia and different AM species including G. clarum, G. intraradices, G. mosseae, and G. versiforme (Artursson et al. 2006). These stimulating effects include the growth of fungi and germination of then spores, respectively, root colonization of the host plant by AM fungi, the solubilization of phosphate, and the suppression of pathogens (Artursson et al. 2006).

The external hyphae of mycorrhizal fungi, which were about 100 times finer than wheat roots and 10 times finer than root hairs, access sites normally not permeable by roots or root hairs, thus reducing the P diffusion distances and increasing the surface area for nutrient absorption. Also, the length of external hyphae of mycorrhizal fungi can be a good predictor of its relative ability to take up P (Manske et al. 2000).Proline levels were found to be increased significantly with salinity stress in mycorrhizal plants when compared to non-mycorrhizal plants. Marked increase in proline occurs in many plants during moderate or severe salt stress and this accumulation, mainly as a result of increased proline biosynthesis, is usually the most outstanding change among free amino acids (Hurkman et al. 1989). Salicylic acid (SA), a plant phenolic compound is considered as a hormone like endogenous regulator, and its role in the defence mechanisms against biotic and abiotic stresses has been well characterized (Szalai et al. 2009).The aim of this investigation is to study the effect of inoculation with Glomus intraradices and/or with different isolates of plant growth promoting rhizobacteria (Biotol) on growth, yield and chemical contents of two wheat cultivars grown under four levels of soil salinity in calcareous soil.

Materials and methods

Soil physicochemical characteristic

of the surface layers (0-30 cm) of the experimental field were as follows pH: 8.28-8.39, CaCO₃ %_: 23.29-24.34, O.M. %: 0.30-.045, available N: 50.48-40.36 mg/kg, available P: 3.59-3.00 mg/kg and available K: 107.13-85.96 mg/kg. Soil texture was sandy loam (Page et al. 1982 and Klute, 1986).

Wheat seeds:

Two wheat (Triticum aestivum, L.) cultivars, Sakha 93 and Gemmeza 9, were provided from the Agricultural Research Center, Ministry of Agriculture, Giza, Egypt.

Isolation of microorganisms and inoculums preparation

1. The mycorrhizal strain Glomus intraradices, isolated from the Experimental Station of Alexandria University at Abies, (Aboul- Nasr, 1993), was used in both experiments. The inoculum consists of expanded clay aggregates (2-4 mm in diameter, leca), containing chlamydospores and fungus mycelium, which had been produced on Tagetes erecta L. (Aboul-Nasr, 2004). Inoculant was thrown at the rate of 100 g   per plot under wheat grains. The control plants received the same amount of heat sterilized expanded clay.

2. Biotol was used as plant growth promoting rhizobacteria (PGPR). Biotol contains a mixture of Bacillus megaterium, B. thuringiensis, B. mycoides, Paenibacillus graminis and P. borealis. It was obtained from the Soil, Water and Environment Research Institute – Agricultural Research Center, Giza, Egypt. It has added to the ground with the first irrigation after 25 days from sowing.

NPK fertilizers:

Four different rates of NPK fertilizers were used in this study (NPK_zero, NPK_50%, NPK_75% and NPK_100% of the recommended dose). The recommended doses of N, P₂O₅ and K₂O fertilizers are 240, 108 and 57.6 kg/ha, respectively. Nitrogen fertilizer (Ammonium nitrate 33.5 % N) was added twice in equal doses, at 25 and 45 days after sowing. Mono-calcium phosphate (15.5 % P₂O₅) was added at the time of soil preparation at one dose. Potassium sulphate (48 % K₂O) was added at 45 days after sowing.

Soil salinity levels:

Four places with different salinity levels (EC dSm^-1: average 2.8, 5.3, 7.6 and 10.5) were used in these experiments during the two growing seasons.

Field experiment:

Two field experiments were carried out during two winter seasons of 2012/2013 and 2013/2014 at the Agricultural Research Station of Nubaria. The field experiments were laid out in a split–split –plot design with three replicates.

The following parameters were measured:

The percentage of mycorrhizal root length colonization

was estimated when plants were 45, 90 and 120 days old, according to Koske and Gemma (1989). The percentage of AM root colonization was estimated according to Giovannetti and Mosse (1980).

1000 grains weight (g).

1000-grain weight was expressed as the weight of 1000 clean grains in grams.

 Grain yield t/ha.

Grain yield was obtained by harvesting one square meter from each sub-sub plot. Plots were bundled, threshed, and then the grain were cleaned and weighted.

Chemical analysis

Plant samples were taken from each plot, at the suitable age, washed with running tap water, then distilled water. Samples were dried at 65ºC till the weight constant. After dryness, the plant samples were milled well and stored for analysis. 0.5g of plant powder was wet-digested with H₂SO₄ – H₂O₂ digest (Lowther, 1980) and the following determinations were carried out in the digested solution.

1. Shoot Na content

It was carried out according to the method described by (Jackson, 1973) using Beckman flame photometer.

1. Nitrogen uptake (kg/ha) and N % in grains

Total nitrogen was determined in digested wheat leaves colormeterically by  Nessler's method (Chapman and Pratt, 1978) using 1 ml of nessler solution (35g KI/100 ml d.w + 20g HgCl₂/500 ml d.w) +120g NaOH/250 ml d.w. Reading was achieved using wave length at 420 nm by spectrophotometer (Model 390, Agricultural Microbiology Lab at the Faculty of Agricultural Saba-Basha). The percentage of total nitrogen was calculated as follows:

% N = NH₄% × 0.7764857

Nitrogen uptake was calculated by multiplication of the N content × plant dry wt. (g).

The same method was use in case of determination N% in grains.

1. Phosphorus uptake (kg/ha)

It was determined in shoots during both seasons by a mixture of sulphuric, nitric and perchloric acids (1: 10: 40 v: v: v) to determine the total phosphorus in wet ash. Phosphorus was determined by the Vanadomolybdate yellow method (Jackson, 1958) using Millton Ray spectronic 21 D. Phosphorus uptake was calculated by multiplication the P content × plant dry wt. (g).

1. Potassium uptake (kg/ha)

Total potassium content in plant shoots and grains was determined using a mixture of sulphuric, nitric and perchloric acids (1: 10: 40 v: v: v) according to the method described by (Jackson, 1973) using Beckman flame photometer. Potassium uptake was calculated by multiplication the K content × plant dry wt. (g).

Determination of chlorophyll index (SPAD)

Chlorophyll indexwas measured by chlorophyll meter device (SPAD 502) Ganji Arjenaki et al. (2012).

Determination of protein content in grains (%)

Protein was determined as percentage as follows: protein % = N % x 6.24

Determination of proline (mg/g dry wt.)

The content of proline was determined according to Umbreit et al. (1972) using the same extract prepared previously for the determination of total proteins and total soluble carbohydrates. 0.5 ml of extract, 1 ml citrate buffer (pH 5), 0.5 ml ninhydrine and 3.5 ml isopropanol solution were added. The optical density was measured spectrophotometerically at 450nm for proline, 492 nm for phenylalanine and 515 nm for arginine. In addition, 0.5 ml of distilled water was used instead of extract in reference cuvette. The concentration of each amino acid was determined according to the prepared standard curves of each corresponding amino acids.

Determination of salicylic acids (mg/100g root dry wt.)

Determination was implemented according to the method of Iqbal and Vaid (2009) and Malamy et al. (1992) as follows;

1. One gram of frozen root tissue is ground in 3.0 ml methanol 90% and centrifuged at 6000 r.p.m. for 15 min.

2. The pellet is re-extracted with 3.0 ml 90% methanol and centrifuged for 10.0 min at 4000 r.p.m.

Assay of salicylic acid was carried out using spectrophotometer according to Iqbal and Vaid (2009).

The supernatant from the both extractions in combined and 2.5 ml of these extractions is diluted to 25.0 ml A.d. in volumetric flask

                       2.5 ml extraction + 0.5 ml FeCl3 5% + 22.0 ml A.d.

Absorbance of the sample was determined using a spectrophotometer set at 360 nm.

Statistical analysis

Data were statistically analyzed by ANOVA, the analysis of variance to test the treatments effect on different measured parameters. Data were analysed using an ANOVA split split design, the differences between the different treatments combinations were tested using the Duncan's Multiple range method outlined by (Snedecor and Cochran, 1982).

Results

Mycorrhizal root length colonization

The percentage of AM colonization was estimated after 45, 90 and 120 days old. Records of wheat plants, inoculated either with G. intraradices alone or with G. intraradices and Biotol significantly increased under all the tested levels of soil salinity, compared to un-inoculated plants. The highest percentages of AM colonization were attained after 90 days under NPK_75% and normal soil salinity being, 65.57 and 65.49 for cv. Sakha 93 and 58.56 and 60.39 for cv. Gemmeza 9, respectively. By increasing soil salinity, the percentage of AM colonization significantly decreased (Tables 1, 2 and 3).

Shoot Na content

Results presented in Table (4) showed that, the lowest values of Na contents (mg/kg) were observed under EC ≤4 dSm^-1 for plants inoculated with G. intraradices and Biotol (9.79 and 18.94 mg/kg) under NPK_100% for Sakha 93 and Gemmeza 9, respectively. Un-inoculated plants recorded 18.75 and 26.86 mg/kg Na for both cultivars, respectively, under the same treatments. The same trends were noticed by increasing soil salinity levels.

Chlorophyll index

Chlorophyll index was significantly affected with soil salinity and levels of mineral fertilizers. Under soil salinity level ≤4 dSm^-1 the highest values of chlorophyll were 55.75 and 49.96 for plants inoculated with AM+Biotol under NPK_100% for the tested wheat cultivars; representing increase percentages 26.73 and 26.31 % over uninoculated ones. No significant differences were observed between NPK₇₅ and NPK_100% of the recommended dose of mineral fertilizers. The same trends were observed with increasing the soil salinity levels.Significant differences in chlorophyll contents were found between the wheat cultivars at soil salinity level 8–12 dSm^-1. Sakha 93 recorded higher values of chlorophyll, compared to the Gemmeza 9 (Table 5).

NPK uptake (kg/ha)

Data in Tables (6, 7 and 8) reveal that inoculation with the AM fungus and Biotol, significantly increased NPK uptake (kg/ha) when compared to uninoculated ones. Under salinity level ≤4 dSm^-1 the highest uptake values of N (Table 6) P (Table 7) K (Table 8) were recorded in case of plants inoculated with AM+Biotol under NPK_100% for both the tested cultivars. No significant differences were observed between NPK₇₅ and NPK_100% mineral fertilizers. The same trends were observed with increasing the soil salinity levels. The NPK uptake values decreased under soil salinity level 8-12 dSm^-1.

Table (1). Effect of wheatinoculation with Glomus intraradices and Biotol on the percentage of mycorrhizal root colonization after 45 days from planting in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014).

Table (2). Effect of wheatinoculation with Glomus intraradices and Biotol the percentage of mycorrhizal root colonization after 90 days from planting in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014).

Table (3). Effect of wheatinoculation with Glomus intraradices and Biotol on the percentage of mycorrhizal root colonization after 120 days from planting in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)

Table (4). Effect of wheatinoculation with Glomus intraradices and Biotol on Shoot Na content (mg/kg) in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)

± % Increase or decrease to uninoculated (control) plants

Table (5). Effect of wheatinoculation with Glomus intraradices and Biotol on chlorophyll index (SPAD) in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)

± % Increase or decrease to uninoculated (control) plants

Table (6). Effect of wheatinoculation with Glomus intraradices and Biotol on N uptake (kg/ha) in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)

± % Increase or decrease to uninoculated (control) plants

Table (7). Effect of wheatinoculation with Glomus intraradices and Biotol on P uptake (kg/ha) in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)

± % Increase or decrease to uninoculated (control) plants

Table (8). Effect of wheatinoculation with Glomus intraradices and Biotol on K uptake (kg/ha) in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)

± % Increase or decrease to uninoculated (control) plants

1000 grain weight

Results presented in Table (9) showed that the highest value of 1000 grain weight (g) was obtained from Gemmeza 9 plants inoculated with G. intraradices (54.59 g) under soil salinity level ≤4 dSm^-1 and NPK_75%. No significant differences were observed between NPK₇₅ and NPK_100% mineral fertilizers. At soil salinity 8 – 12 dSm^-1, the 1000 grain weight were 40.92 and 35.98 g for plants inoculated with AM+Biotol under NPK_100% for Sakha 93  and Gemmeza 9, respectively.

Grain yield (t/ha)

Results presented in Table (10) indicated that, grain yield due to dual inoculation with Glomus intraradices and Biotol resulted the maximum yield of grain (6.723 t/ha) under soil salinity level ≤4 dSm^-1 and NPK_100% in case of Gemmeza 9. No significant differences were observed between NPK₇₅ and NPK_100% mineral fertilizers. When the soil salinity level increased to 8-12 dSm^-1, the wheat grain yield decreased. The grain yield was 1.991 t/ha in plants inoculated with AM+Biotol under NPK_100% for Sakha 93, while it was 1.710 t/ha for Gemmeza 9. Significant differences in the grain yield (t/ha) were found between the two wheat cultivars. Sakha 93 recorded highest value of grain yield, compared to Gemmeza 9 under high level of soil salinity.

Grain protein

Wheat plants Inoculated with G. intraradices alone or G. intraradices + Biotol resulted high values of protein content of wheat grains for both cultivars. Under normal salinity levels ≤4 dSm^-1, the highest grain protein content was obtained in case of plants inoculated with mycorrhizal fungus and Biotol (1.39 %) for Gemmeza variety at NPK_75%. Under salinity level 8-12 dSm^-1 the highest protein content was obtained from both cultivars in the presence of NPK_100% with percentage increases 36.31 and 43.96% more than un-inoculated plants, for Sakha 93 and Gemmeza 9, respectively. Significant differences in protein contents were found between the two cultivars, Gemmeza 9 recorded the highest value compared to Sakha 93 (Table 11).

Proline content

Significant differences in shoot proline contents among the two wheat cultivars were recorded by increasing soil salinity levels. Sakha 93 recorded higher values of proline than Gemmeza 9 (Table 12). Data clearly show positive effect of AM inoculation on proline content under the tested levels of soil salinity.

Salicylic acid

Dual inoculation with G. intraradices and Biotol significantly increased the salicylic acid concentration at all the tested levels of soil salinity. The percentage increases, as compared to uninoculated control was reached 192.57 and 135.42 for Sakha 93 and Gemmeza 9, respectively, in the presence of NPK_75% and soil salinity 8-12 dSm^-1 (Table 13).

Discussion

Salinity represents one of the most important environmental stresses since it limits crop plant production which is contrary to the increased demand for food all over the world. Therefore, the studies of salinity tolerance in plants consider a special importance. From the above results we concluded that, wheat inoculated with AM fungus showed significant increases in the percentage of AMF colonization and growth yield parameters compared to un-inoculated plants under different levels of soil salinity. It was clear that, by increasing soil salinity, the percentage of AMF colonization and growth yield parameters significantly decreased. Aroca et al. (2013) found that, increasing soil salinity levels lowered the percentage of mycorrhizal root colonization in lettuce plants. Miransari et al. (2007) observed that, Zea mays plant inoculated with AM fungi (Glomus mosseae and Glomus etunicatum)

Table (9). Effect of wheatinoculation with Glomus intraradices and Biotol on 1000 grains weight (g) in the presence of four levels of soil salinity (averages of the two seasons 2012/2013 and 2013/2014)