Impaginato 39 1. Introduction Biological and non-biological stresses, which are mostly due to adverse weather conditions, are main factors in yield reduction (Wu et al., 2006). There is much evidence that mycorrhizal fungi cause varia- tions in plant-water relations and improve drought tolerance. Improvement in plant-water relations is affected by direct and indirect mechanisms (Davies et al., 1993). In general, plants that have mycorrhizal symbiosis grow and perform better as they absorb more nutrients and water from the soil. These plants are also more tolerant towards environmental stress- es including biotic and abiotic stresses (Porcel and Ruiz-Lozano, 2004). Most varieties of citrus, like orange, trifoliate orange, Cleopatra mandarins, Swingle citrumelo, and Citrange, are very dependent, because of their hairy roots, on Glomus species (Davies et al., 1993). Plant adaptations to arid cli- mate conditions, morphological and physiological changes, and concentration of novel metabolites along with structural variations, increase their effi- ciencies in stress conditions (Wu et al., 2006). When plants are under drought stress, osmotic adjustments occur to reduce potential water loss. This phenomenon leads to good water flow mainte- nance from the soil to plant roots (Porcel and Ruiz- Lozano, 2004). G. versiforme fungus increased leaf water potentials of trifoliate orange and mandarin seedlings under both drought stress and enough- water-supply conditions (Wu et al., 2006, 2008). Moreover, when trifoliate orange seedlings were under drought stress, the leaf relative water content (RWC) significantly increased compared to plants with no fungus (Wu et al., 2006). In mandarin seedlings, plant height, leaf area and number of leaves per plant, decreased under drought stress conditions, while all those factors were improved using G. versiforme fungi (Wu and Zou, 2009). In cit- rus plants, G. versiforme fungi increased growth and Adv. Hort. Sci., 2016 30(1): 39-45 DOI: 10.13128/ahs-18700 Analysis of the effects of Glomus etunicatum fungi and Pseudomonas fluorescence bacteria symbiosis on some morphological and physiological characteristics of Mexican lime (Citrus aurantifolia L.) under drought stress conditions A.R. Shahsavar 1 (*), A. Refahi 1, M. Zarei 2, E. Aslmoshtaghi 1 1 Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran. 2 Department of Soil Science, College of Agriculture, Shiraz University, Shiraz, Iran. Key words: chlorophyll content, drought deficit, leaf water potential, Mexican lime. Abstract: To analyze the effects of Glomus etunicatum fungi and Pseudomonas fluorescence bacteria on some morpho- logical and physiological characteristics of Mexican lime plant under drought stress conditions, a factorial experiment was conducted. This experiment was based on a completely randomized design with three replicates; each replicate was composed of two pots. The factors used consisted of G. etunicatum fungi and control, Pseudomonas fluorescence bacte- ria and control, and drought stress at three levels (-0.35, -0.47, and -0.6 bars). The analyzed characteristics were leaf chlorophyll content, leaf temperature, rate of net photosynthesis, transpiration, leaf relative water content (RWC), and percentage of root colonization. Data analysis revealed that both fungi and bacteria increased leaf chlorophyll content, net photosynthesis rate, transpiration, and leaf RWC. Moreover, the presence of fungi reduced leaf temperature while inoculation of bacteria had no effects on that the parameter. In addition, with the increase of irrigation periods, leaf temperature and transpiration were also increased. Results showed that root colonization percentage dropped with increased irrigation and the highest root colonization percentage was observed in simultaneous inoculations of fungi and bacteria with a two-day irrigation period. (*) Corresponding author: shahsava@shirazu.ac.ir Received for publication 31 October 2015 Accepted for publication 4 February 2016 Copyright: © 2016 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Adv. Hort. Sci., 2016 30(1): 39-45 40 biomass while they reduced their colonization per- centage. These increases were attributed to the improvement of fungi water absorptions and increas- es in the length and volume of plant fungal roots (Faber et al., 1991; Bryla and Duniway, 1997; Wu et al., 2011). In these plants, root colonization increases with a decrease in drought stress (Augé, 2001). Under drought stress conditions, G. versiforme fungi increased fresh and dry weight of plant roots and shoots and increased the root colonization percent- age (Wu and Xia, 2006). G. intraradices fungus, under drought stress conditions, increased root growth and respiration rate of Rough lemon (Leyv and Syvestern, 2006). Many studies showed that G. etunicatum fungi could affect plant-water relations of host plants including citrus, under both drought stress and enough-water-supply conditions (Wu et al., 2006). Therefore, they cause higher water use efficiency and this water use efficiency in mycorrhizal plants becomes even more tangible in drought stress condi- tions (Davies et al., 1993). Glomus etunicatum fungus increased phosphorus, potassium, zinc, and copper in pistachio trees planted under sufficient water supply conditions and also increased nitrogen and calcium in pistachio trees planted under drought stress condi- tions. However, this fungus did not change the mag- nesium concentration (Abbaspour et al., 2011). It is reported that Pseudomonas bacteria enhances growth and yield of some plants (Rodriguez and Fraga, 1999). Construction of active metabolites such as vitamins, amino acids, and Indole acetic bacteria may have a direct effect on the growth and metabo- lite contents of Piriformospora indica and mycorrhizal fungi. As a helpful microorganism, it seems that bac- teria supports fungal performances (Vivas et al., 2003). Plant inoculations with different types of Pseudomonas bacteria in drought stress situations increased plant proline contents, thus the plants’ water levels were maintained and their protein con- tents and membranes remained safe from drought stress damage (Yoshiba et al., 1997). Inoculation with Pseudomonas species, led to moderation of drought stress effects, improvement of plant growth and increase of proline, soluble sugars and amino acids production, explaining their effectiveness in absorbing water and nutrients from the soil (Wu et al., 2008). These types of bacteria also help the plant maintain its RWC and LWL (leaf water loss) levels under drought stress conditions. Studies have shown that mycorrhizal plants absorb more CO2 in the pres- ence of light. Hence, their photosynthesis rates are also higher. The increase of CO2 absorption in mycor- rhizal plants is related to a decrease of liquid-phase resistance of mesophyll cells to CO2 transmission (Wu and Zou, 2009). Miller (2000) reported that in mycor- rhizal plants, due to the increase of photosynthesis materials and rate, water use efficiency increased per water use unit. Mycorrhiza can increase plant weight, leaf area, and plant pigments, and these increases may be attributed to the improvement of fungi water and phosphorus absorptions (Bethlenfalvay et al., 1988; Davies et al., 1993). Glomus etunicatum and Pseudomonas bacteria have positive effects on plant growth and employing them, instead of fertilizers, is considered a positive approach to reduce fertilizer use (Davies et al., 1993). Despite the lack of compre- hensive scientific investigations on the horticultural characteristics of Mexican lime (Citrus aurantifolia Swingle cv. Mexican Lime) as a rootstock, its seed availability for propagation and some its characteris- tics, such as good crop load and vigorous habit of grafted cultivars as scion, have made it a favorite in Fars province, Iran. Considering the positive effects of fungi and bac- teria in symbiosis with some plant roots, the aim of this study was to investigate the effects of G. etunica- tum fungi and Pseudomonas fluorescence bacteria and their interactions on some morphological and physiological characteristics of Mexican lime plant under drought stress conditions. 2. Materials and Methods Preparation and inoculation of plant materials Mexican lime seedlings, six months of age and disease-free, were provided in Khafr city of Fars province, Iran. They were transferred to the green- house. Planting soil mixture in ratio 1:1:1 (sand:soil: leaf compost) was sterilized and 2.7 kg were placed in plastic pots. The arbuscular mycorrhizal fungi iso- late used in this study was G. etunicatum supplied from the soil lab of the Faculty of Shiraz University. The lyophilized fungal inoculum of Pseudomonas flu- orescence was supplied from Tehran University School of Soil and Water and was prepared as fol- lows. To prepare a solution containing growth-stimu- lating bacteria, a nutrient broth (NB) medium was applied. First, 0.8 g of NB was dissolved in 100 mg of distilled water and then media were sterilized by autoclaving at 121°C and pressure of 1.1 atm for 25 Shahsavar et al. - Effects of Glomus etunicatum and Pseudomonas fluorescence on Mexican lime under drought stresss 41 min. A lyophilised pre-culture vial was first suspend- ed in 0.3 mL of nutritive medium. One drop (1 ml) of that suspension was added to 5 mL of nutritive medi- um and incubated on an orbital shaker at 28°C for 24 h. This final preparation of medium was used as the inoculum. After the incubation period, roots were placed in a solution containing bacteria for 30 min. Moreover, to ensure its effectiveness, 10 cc of the solution containing bacteria were added to each pot. For drought stress treatments, pots containing 2.7 kg soil without a seedling were selected and their mois- ture contents were equilibrated with the previously measured field capacity. The wet soils of the pots were weighed daily for 15 days, always at the time. Daily water reductions and moisture curves were graphed. Using those diagrams, irrigation periods were identified for every 2, 4 and, 6 days. For G. etu- nicatum fungus inoculation, 70 g of inoculum con- taining spores, hyphae, and root fragments were introduced 5 cm beneath the soil surface in the pots, and mixed thoroughly. Equal to the amount of added inoculum, hyphae, and mycelium to the fungal treat- ment pots, inoculum without hyphae and mycelium was added to control pots. For bacteria inoculation, seedlings were placed in a solution containing Pseudomonas fluorescence bacteria for 30 min and were then planted into pots. For fungi and bacteria treatments, bacteria-inoculated seedlings were planted in pots in which fungus was previously added. One seedling was planted per pot and, two months later, water treatments were applied. After six months, the implants were removed. The study was conducted using a factorial experiment, based on a completely randomized design with three repli- cations in two replicate pots. Factors used in the experiment were: 1) G. etunicatum fungus in two lev- els of G. etunicatum and control; 2) Growth stimulat- ing bacteria in two levels of Pseudomonas fluores- cence and control; 3) Drought stress at three levels. The Kormanik and McGraw method (Kormanik and McGraw, 1982) was used to measure coloniza- tion percentage. In this method, 2 g of roots previ- ously stored in FAA (formaldehyde - acetic acid - ethanol) were washed with water three or four times and were placed in Falcon tubes containing 10% KOH solution for 24 h at room temperature. The color of the solution was almost yellow or light yellow. The solution was then poured out and the roots were again washed with water three or four times. The samples were placed in 2% hydrochloric acid for at least 15 min for staining. The acid was poured out and a colored solution was poured over the acidic roots. Acid fuchsine stain was used in this study; the ratio of the fuchsine acid colored solution was 14 ml lactic acid, 1 ml glycerin and, 1 ml water. The roots and the solution were kept at room temperature for 24 h. The coloring solution was then removed. Besides, due to elimination of extra colors, the color- ing solution was poured on the roots. After 6-12 h, fungal organs such as arbuscules, hyphae, and vesi- cles were observed under a light microscope and col- onization was calculated as a percentage. After appli- cation of water stress treatments, leaf chlorophyll content was measured with a SPAD-502 chlorophyll- meter using three fully-expanded leaves to find an average for chlorophyll content. Leaf temperature factors, net photosynthesis and transpiration rates were measured by portable photosynthesis meter (LCi, ADC, England). Relative water content was determined by using ten 7 mm-diameter leaf discs. Leaf discs for each treatment were weighed (FW). They were hydrated until saturation (constant weight) for 48 h at 5°C in darkness (TW). The leaf discs were then dried in an oven at 105°C for 24 h (DW). Relative water content was calculated accord- ing to the following expression (Filella et al., 1998): RWC% = (FW-DW)/(TW–DW) × 100 Statistical analysis The data were analyzed for significance (P<0.050) by ANOVA (analysis of variance) with mean separa- tion by Duncan’s Multiple Range test. 3. Results and Discussion Leaf relative water content (RWC) Analysis of the effects of interaction between inoculation of G. etunicatum fungi and Pseudomonas fluorescence bacteria on Mexican lime leaf RWC, at different irrigation periods, identified that the maxi- mum leaf RWC was observed in simultaneous inocu- lation of fungi and bacteria with the two-day irriga- tion period (74.7%). The general results indicate that the leaf RWC decreased with the increase in irriga- tion period, while inoculation with fungi or bacteria significantly increased RWC in all irrigation periods (Table 1). Osmotic adjustment is one of the most important factors in plant drought tolerance and it is closely related to RWC (Haley et al., 1993). When plants are under a drought stress condition, osmotic adjust- Adv. Hort. Sci., 2016 30(1): 39-45 42 ment occurs to reduce water potential and maintain a good flow of water from the soil to the plant roots. Plants with mycorrhizal fungi have more osmotic adjustment potentials than plants without fungi (Porcel and Ruiz-Lozano, 2004). Manette et al. (1988) reported that plants which are under drought stress conditions have specific morphological and physio- logical characteristics that enable them to store more water. Clarke and Craig (1982) stated that plants under drought stress conditions loose their water content more slowly. They also indicated that there are significant relationships between water content of the loss of leaves, plant drought tolerance, and leaves ability to retain water content (Clarke and Craig, 1982). Therefore, mycorrhizal plants have high- er osmotic adjustment and are more capable of retaining their water content. Chlorophyll content Chlorophyll content decreased with the increase of irrigation periods. In addition, inoculations of G. etunicatum fungi and Pseudomonas fluorescence bacteria increased leaf chlorophyll content. Analysis of the effects of interaction between inoculation of G. etunicatum fungi and Pseudomonas fluorescence bacteria leaf chlorophyll content identified that the maximum leaf chlorophyll content was observed when both fungi and bacteria were inoculated and there was a two-day irrigation period (634.7). The lowest chlorophyll content was observed in the treat- ment without fungi and bacteria inoculations with six-day irrigation periods (Table 2). Analysis of the effects of G. etunicatum fungi and Pseudomonas fluorescence bacteria inoculations on chlorophyll content of the Mexican lime leaves in the current study revealed that the chlorophyll content decreased with an increase of drought stress periods. However, inoculations of fungi and bacteria largely reduced the deleterious effects of drought. This can be explained by the fact that in drought stress condi- tions, the chlorophyllase enzyme becomes activated while its activation results in the loss of chlorophyll content (Shaharoona et al., 2008). Under drought, oxygen free radicals, which are damaging to various cellular organelles, are formed. One of the most sen- sitive organelles to drought stress and free radicals is chloroplast (Kaya et al., 2003). G. etunicatum fungi and Pseudomonas fluorescence bacteria, by increas- ing antioxidant content and antioxidant enzyme activities, cause a loss of detrimental free radicals and consequently preserve plant chlorophyll content (Molinari et al., 2007). They also increase the absorp- tion of elements such as magnesium, iron, and nitro- gen that lead to the plant’s production of more chlorophyll (Molinari et al., 2007). Leaf temperature Our results indicate that the increase of irrigation periods led to an increase of leaf temperature. The presence of G. etunicatum fungi decreased leaf tem- perature while Pseudomonas fluorescence bacteria inoculation had no effect on it. Analysis of the effects of interaction between inoculation of G. etunicatum fungi and Pseudomonas fluorescence bacteria on leaf temperature revealed that the minimum leaf tem- perature was with simultaneous inoculation of fungi without bacteria and a two-day irrigation period (31.47°C). Likewise, the maximum temperature was observed in the treatment without fungi and bacteria inoculations and a six-day irrigation periods (Table 3). Rate of net photosynthesis Analysis of the net photosynthesis rate of Mexican lime revealed that it declined with the increase of irrigation periods: the maximum and min- imum rates were observed with two- and six-day irri- Irrigation periods (day) GE + GE - PF + PF - PF + PF - 2 634.7 a 574.3 b 565.4 b 529.6 c 4 578.2 b 512.9 cd 511.7 cd 441.7 f 6 503.8 d 484.6 e 479.3 e 320.5 g Table 2 - Effects of G. etunicatum fungus and Pseudomonas flo- rescence bacteria inoculations on Mexican lime leaf chlorophyll content with different irrigation periods (SPAD value) In each column, means followed by different letters differ significantly at P≤0.05 according to Duncan's multiple range test. GE + = G. etunicatum presence; GE - = G. etunicatum absence. PF + = Pseudomonas florescence presence; PF - = Pseudomonas flo- rescence absence. Irrigation periods (day) GE + GE - PF + PF - PF + PF - 2 74.7 a 73.5 ab 72.9 ab 72.6 ab 4 71.9 b 70.3 b 69.5 bc 67.6 c 6 70.2 b 69.5 bc 68.4 bc 66.3 c Table 1 - Effects of G. etunicatum fungus and Pseudomonas flo- rescence bacteria inoculations on Mexican lime leaf RWC with different irrigation periods (%) In each column, means followed by different letters differ significantly at P≤0.05 according to Duncan's multiple range test. GE + = G. etunicatum presence; GE - = G. etunicatum absence. PF + = Pseudomonas florescence presence; PF - = Pseudomonas flo- rescence absence. Shahsavar et al. - Effects of Glomus etunicatum and Pseudomonas fluorescence on Mexican lime under drought stress 43 gation periods, respectively. The results also indicat- ed that the presence of G. etunicatum fungi and Pseudomonas fluorescence bacteria increased the plants’ rate of net photosynthesis. Analysis of the effects of interaction between inoculation of G. etu- nicatum fungi and Pseudomonas fluorescence bacte- ria identified that the maximum rate was observed in simultaneous inoculation of both fungi and bacteria and with a two-day irrigation period (12.3 micro- mole/m2/s)(Table 4). Rate of transpiration Analysis of the effects of interaction between inoculation of G. etunicatum fungi and Pseudomonas fluorescence bacteria on Mexican lime transpiration rate in plants grown with different irrigation periods identified that the highest rate was observed in simultaneous inoculation of both fungi and bacteria and a two-day irrigation period (10.25 micro- mole/m2/s). Likewise, the minimum transpiration rate was observed in the treatment without fungi and bacteria inoculations and a six-day irrigation period. The overall results showed that the leaf tran- spiration rate increased with the increase of irrigation period (Table 5). Wu and Xia (2006) specified that under drought stress conditions, G. versiforme fungi increase leaf water potential, photosynthesis rate, respiration rate, RWC, and stomatal conductance of mandarin seedlings; however, leaf temperature is decreased compared to plants without fungi. Effects of irrigation period on leaf temperature, photosynthesis rate, and transpiration showed that with the increase of irriga- tion period, they all declined (Figueiredo, 2008). This can be explained by the fact that under drought con- dition, more stomata are closed; with a loss of evapo- ration, the leaf surface loses less heat and the leaf temperature increases (Dietz and Foyer, 1986.). Moreover, because of stomata closure, less water is lost and the transpiration rate decreases. It should be noted that stomata closure causes less carbon diox- ide to enter into the leaf, resulting in a lower rate of photosynthesis (Zhang et al., 2010). The presence of G . e t u n i c a t u m fungi and inoculation with Pseudomonas fluorescence bacteria leads to better water absorption and higher drought stress toler- ance, thus increasing the plant’s rate of photosynthe- sis. Many studies have reported the effects of G. etu- nicatum fungi on increasing photosynthesis rate (Johnson et al., 1986), increasing root hydraulic con- ductivity for water uptake (Graham and Syvertsen, 1984), and increasing transpiration rate (Leyv and Syvestern, 2006). Root colonization percentage Results of the present study showed that root col- onization occurred in the presence of G. etunicatum fungi and P s e u d o m o n a s f l u o r e s c e n c e bacteria. Moreover, an increase of irrigation period led to a decrease of root colonization percentage. Analysis of the effects of interaction between inoculation of G. Irrigation periods (day) GE + GE - PF + PF - PF + PF - 2 32.59 de 31.47 e 33.16 d 33.05 d 4 34.25 d 33.87 d 34.92 c 36.50 b 6 35.94 b 36.35 b 36.28 b 38.41 a Table 3 - Effects of G. etunicatum fungus and Pseudomonas flo- rescence bacteria inoculations on Mexican lime leaf temperature with different irrigation periods (°C) In each column, means followed by different letters differ significantly at P≤0.05 according to Duncan's multiple range test. GE + = G. etunicatum presence; GE - = G. etunicatum absence. PF + = Pseudomonas florescence presence; PF - = Pseudomonas flo- rescence absence. Table 4 - Effects of G. etunicatum fungus and Pseudomonas flo- rescence bacteria inoculations on Mexican lime photo- synthesis rate with different irrigation periods (micro- mole/m2/s) In each column, means followed by different letters differ significantly at P≤0.05 according to Duncan's multiple range test. GE + = G. etunicatum presence; GE - = G. etunicatum absence. PF + = Pseudomonas florescence presence; PF - = Pseudomonas flo- rescence absence. Irrigation periods (day) GE + GE - PF + PF - PF + PF - 2 12.3 a 11.6 ab 11.4 b 10.50 c 4 10.2 c 10.2 c 10.1 c 9.06 d 6 9.51 d 8.52 de 8.37 e 6.48 f Table 5 - Effects of G. etunicatum fungus and Pseudomonas flo- rescence bacteria inoculations on Mexican lime tran- spiration rate with different irrigation periods (micro- mole/m2/s) In each column, means followed by different letters differ significantly at P≤0.05 according to Duncan's multiple range test. GE + = G. etunicatum presence; GE - = G. etunicatum absence. PF + = Pseudomonas florescence presence; PF - = Pseudomonas flo- rescence absence. Irrigation periods (day) GE + GE - PF + PF - PF + PF - 2 10.25 a 9.96 b 10.07 ab 9.83 b 4 9.68 c 9.58 c 9.16 d 8.74 e 6 9.17 d 9.72 b 8.91 e 8.65 e Adv. Hort. Sci., 2016 30(1): 39-45 44 etunicatum fungi and Pseudomonas fluorescence bacteria on Mexican lime percentage of root colo- nization revealed that the maximum percentage was observed in simultaneous inoculation of both fungi and bacteria with a two-day irrigation period (49.66%) (Table 6). As previously mentioned, root colonization occurred only in the presence of G. etunicatum fungi and its percentage dropped with an increase in irriga- tion period. Until now, no specific reason has been proposed for the reduction of colonization in drought stress conditions. Probably water is one important element in fungi growth. The formation of secondary metabolites that prevent fungi growth in the plant roots is also a possible explanation. Wu et al. (2006) reported that, in the case of citrus roots, the highest colonization percentage of mycorrhizal fungi occurs when the roots are not under drought stress condi- tions, which is consistent with the present study results. Regarding other types of citrus, they found similar results in their subsequent studies (Wu et al., 2006, 2008). In order to utilize root colonization of fungi and bacteria capacities in sustainable agricul- ture, there must be appropriate establishment of both fungi and bacteria on the plant roots. Accordingly, observation of Mexican lime root colo- nization percentage in the current investigation was a very important and valuable factor. In addition, specification of the appropriate colonization percent- age for effective interaction between fungi and plant is an important issue. 4. Conclusions The results of the current study and other research projects in this field have shown the practi- cal and scientific advantages of G. etunicatum fungi and Pseudomonas fluorescence bacteria applications in arid or semi-arid areas. The synergistic effect, which was observed between G. etunicatum fungi and Pseudomonas fluorescence bacteria, could increase most of the plant characteristics such as leaf chlorophyll content, net photosynthesis and transpi- ration rates, leaf RWC and root colonization percent- age which provide the material energy and informa- tion for plant growth, development and reproduc- tion. Pseudomonas fluorescence bacteria could reduce the negative effects of drought stress less than G. etunicatum fungi. Using their hyphae and extra/intra root mycelia, G. etunicatum fungi expand root evacuation area for better uptakes of water and nutrients. Arbuscular mycorrhizal fungi can be inte- grated in soil management to achieve low-cost sus- tainable agricultural systems, offering a sustainable and environmentally safe treatment to improve drought tolerance. Consequently, using these fungi as well as Pseudomonas fluorescence bacteria can be very effective in achieving the goals of sustainable agriculture. References ABBASPOUR H., SAEIDSARAND S., AFSHAR H., 2011 - I m p r o v i n g d r o u g h t t o l e r a n c e o f Pistacia vera L . seedlings by arbuscular mycorrhiza under greenhouse conditions. - J. Medicinal Plants Research, 5: 7065- 7072. AUGÉ R.M., 2001 - Water relations, drought and vesicular- arbuscular mycorrhizal symbiosis. - Micorrhiza, 11: 3-42. BETHLENFALVAY G.J., BROWN M.S., AMES R.N., THOMAS R., 1988 - Effects of drought on host and endophyte development in mycorrhizal soybeans in relation to water use and phosphate uptake. - Physiol. Plant., 72: 565-571. BRYLA D.R., DUNIWAY J.M., 1997 - Effects of mycorrhizal infection on drought tolerance and recovery in saf- flower and wheat. - Plant and Soil., 197(1): 95-103. CLARKE J.M., CRAIG T.N., 1982 - Excised-leaf water reten- tion capability as an indicator of drought resistance of Triticum genotypes. - Can. J. Plant Sci., 62: 571-578. DAVIES F.T., POTTER J.R., LINDERMAN R.G., 1993 - Drought resistance of mycorrhizal pepper plants independent of leaf P concentration response in gas exchange and water relations. - Physiologia Plantarum., 87: 45-53. DIETZ K.J., FOYER C., 1986 - The relationship between phosphate status and photosynthesis in leaves. - Planta, 167(3): 376-381. FABER B.A., ZASOSKE R.J., MUNNS D.N., SHACKEL K., 1991 Table 6 - Effects of G. etunicatum fungus and Pseudomonas flo- rescence bacteria inoculations on Mexican lime root colonization percentage with different irrigation periods (%) Irrigation periods (day) GE + GE - PF + PF - PF + PF - 2 49.66 a 42.36 ab 0 d 0 d 4 38.73 b 36.87 bc 0 d 0 d 6 34.24 c 35.12 c 0 d 0 d In each column, means followed by different letters differ significantly at P≤0.05 according to Duncan's multiple range test. GE + = G. etunicatum presence; GE - = G. etunicatum absence. PF + = Pseudomonas florescence presence; PF - = Pseudomonas flo- rescence absence. Shahsavar et al. - Effects of Glomus etunicatum and Pseudomonas fluorescence on Mexican lime under drought stresss 45 - A method for measuring hyphal nutrition and water uptake in mycorrhizal plants. - Can. J. Bot., 69: 87-94. FIGUEIREDO V.B., 2008 - Alleviation of drought stress in the common bean (Phaseolus vulgaris L.) by co-inocula- tion with Paenibacillus polymyxa and Rhizobium tropi- ci. - Appl. Soil Ecol., 40: 182-188. FILELLA I., LLUSIA J., PIN J.O., PEN J.U., 1998 - Leaf gas exchange and fluorescence of Phillyrea latifolia, Pistacia lentiscus and Quercus ilex saplings in severe drought and high temperature conditions. - Environ. Exp. Bot., 39: 213-220. GRAHAM H., SYVERTSEN J.P., 1984 - Influence of vesicular- arbuscular mycorrhiza on the hydraulic conductivity of roots of two citrus rootstocks. - New Phytologist., 97: 277-284. HALEY S.D., QUICK J.S., MORGAN J.A., 1993 - Excised-leaf water status evaluation and associations in field-grown winter wheat. - Can. J. Plant Sci., 73:55-63. JOHNSON C.R., DUKE E.R., KOCH K.E., 1986 - Accumulation of phosphorus, dry matter and betaine during NaCl stress of split-root citrus seedlings colonized with vesic- ular-arbuscular mycorrhizal fungi on zero, one or two halves. - New Phytologist., 104: 583-590. KAYA C., HIGGS D., KIRNAK H., TAS I., 2003 - Mycorrhizal colonization improves fruit yield and water use efficien- cy in watermelon (Citrullus lanatus Thunb.) grown under well-watered and water-stressed conditions. - Plant Soil., 253: 287-292. KORMANIK P.P., MCGRAW A.C., 1982 - Quantification of vesicular-arbuscular mycorrhizae in plant root, pp. 37- 45. - In: SCHENK N.C. (ed.) Methods and principles of mycorrhizal research. The American Phytopathological Society, St. Paul, MN, USA, pp. 244. LEYV Y., SYVESTERN J.P., 2006 - Effect of drought stress and vesicular arbuscular mycorrhiza on citrus transpira- tion and hydraulic conductivity of roots. - J. Plant Physiol., 85: 25-31. MANETTE A.S., RICHARD C.J., CARVER B.F., MORNHINWEG D.W., 1988 - Water relations in winter wheat as drought resistance indicators. - Crop Sci., 28: 526-531. MILLER M.H., 2000 - Arbuscular mycorrhizae and the phos- phorus nutrition of maize: A review of Guelph studies. - Can. J. Plant Sci., 80: 47-52. MOLINARI H.B., MARUR C.J., DAROS E., MARILIA CAMPOS K.F., CARVALHO J.F., FILHO J.C., PEREIRA L.F., VIEIRA L.G., 2007 - Evaluation of the stress inducible produc- tion of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. - Physiol. Plant., 130: 218-229. PORCEL R., RUIZ-LOZANO J.M., 2004 - Arbuscular mycor- rhizal influence on leaf water potential, solute accumu- lation, and oxidative stress in soybean plants subjected to drought stress. - J. Exp. Bot., 55: 1743-1750. RODRIGUEZ H., FRAGA R., 1999 - Phosphate solubilizing bacteria and their role in plant growth promotion. - Biotech. Adv., 17: 319-339. SHAHAROONA B., NAVEED M., ARSHAD M., ZAHIR Z.A., 2008 - Fertilizer-dependent efficiency of Pseudomonads for improving growth, yield, and nutrient use efficiency of wheat (Triticum aestivum L.). - Appl. Microbiol. Biotechnol., 79: 147-155. VIVAS A., JUAN B., RUIZ-LOZANO M., 2003 - Influence of a Bacillus sp. on physiological activities of two arbuscular mycorrhizal fungi and on plant responses to PEG- induced drought stress. - Mycorrhiza, 13: 249-256. WU Q.S., XIA R.X., 2006 - Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosyn- thesis of citrus under well-watered and water stress conditions. - J. Plant Physiol., 163: 417-425. WU Q.S., XIA R.X., ZOU Y.N., 2006 - Reactive oxygen metabolism in nonmycorrhizal citrus (Poncirus trifolia- ta) seedlings subjected to water stress. - J. Plant Physiol., 163: 1101-1110. WU Q.S., XIA R.X., ZOU Y.N., 2008 - Improved soil structure and citrus growth after inoculation with three arbuscu- lar mycorrhizal fungi under drought stress. - Euro. J. Soil Biol., 44(1): 122-128. WU Q.S., ZOU Y.N., 2009 - The effect of dual application of arbuscular mycorrhizal fungi and polyamin upon growth and nutrient uptake of Trifolia orange seedling. - Not. Bot. Agrobot. Cluj., 37(2): 95-98. WU Q.S., ZOU Y.N., HE X., LUO P., 2011 - Arbuscular myc- orrhizal fungi can alter some root characters and physi- ological status in trifoliate orange (Poncirus trifoliata L. Raf.) seedlings. - Plant Growth Regul., 65: 273-278 YOSHIBA Y., KIYOSUE T., NAKASHIMA K., YAMAGUCHI-SHI- NOZAKI K., SHINOZAKI K., 1997 - Regulation of levels of proline as an osmolyte in plants under water stress. - Plant Cell Physiol., 38: 1095-1102. ZHANG Y., ZHONG C.L., CHEN Y., CHEN Z., JIANG Q.B., WU C., PINYOPUSAREK K., 2010 - Improving drought toler- ance of Causarina equisetifilia seedlings by arbuscular mycorrhizal under glasshouse conditions. - New For., 40(3): 261-271.