Cleopatra mandarin ( Citrus reshni Hort . Ex Tan . ) modulate physiological mechanisms to tolerate drought stress due to arbuscular mycorrhizal fungi and mycorrhizal helper bacteria

1 Division of Crop Production, Central Institute for Subtropical Horticulture, Rehmankhera, P.O. Kakori, Lucknow, Uttar Pradesh, 227 107 India. 2 Division of Fruit Science and Horticulture Technology, Indian Agricultural Research Institute, New Delhi, 110 012 India. 3 Division of Fruit Science, Bihar Agricultural University, Sabour, Bihar, 813 210 India. 4 Division of Microbiology, Indian Agricultural Research Institute, New Delhi, 110 012 India. 5 Division of Crop Improvement, Central Institute for Subtropical Horticulture, Rehmankhera, P.O. Kakori, Lucknow, Uttar Pradesh, 227 107 India.


INTRODUCTION
Drought stress is a major abiotic factor that limits plant's growth, thus becoming one of the growing concerns in agriculture management around the world (Saeidnejad et al., 2013).The mycorrhizal association with roots of most of the plants not only stimulate the plant growth, but also contribute in enhancing tolerance to drought (Navarro et al., 2011).
The free living and endo-symbiotic bacteria, known as "mycorhhizal helper bacteria" (MHB) stimulate presymbiotic fungal growth leading to an increase in rootfungus contacts and root colonization (Frey-Klett et al., 2007).The inoculation of Glomus intraradices along with PSB or Azospirillum brasilense can improve plant growth than single inoculation (Toro et al., 1997;Ruiz-Sanchez et al., 2011).Providencia sp., the bacterial strain AW5 (accession no FJ866760), used in the present investigation, was shown to have potential to stimulate plant growth through phosphorus solubilization, ammonia and indole-3-acetic acid production (Rana et al., 2011a).
Cleopatra mandarin (Citrus reshni Hort.Ex Tan.) produces good quality fruits in scion variety for most of the citrus species (Ouko and Abubaker, 1988).The potential benefit of G. intraradices has been reported in Cleopatra mandarin (Camprubi et al., 1995).However, no work has been carried out to study the physiology of citrus plant under drought stress, when co-inoculated with AMF and MHB.Hence, G. intraradices alone or with MHB was used in the study so as to determine the suitable microbial combination for improving adaptive behaviour of Cleopatra mandarin against drought stress.

Site of experimentation
The potted culture experiment was conducted in glasshouse at Indian Agricultural Research Institute (IARI), New Delhi, located at 77°12' E longitude, 28°40' N latitude and an altitude of 228.6 m above mean sea level.Climate is categorized as semi-arid, subtropical with hot dry summer and cold winter.

Source of microbial inoculants
The AMF G. intraradices was procured from Division of Agricultural Microbiology, Kittur Rani Channamma College of Horticulture, Arabhavi, Karnataka.The starter culture was multiplied in ragi (Elevusine coracana), raised in plastic pot (12 × 20 cm) filled with a mixture of soil, sand and farm-yard manure (FYM) (2:2:1) that had been autoclaved (1.05 kg cm -2 ) for 2 h.The inoculum was sealed in a polythene packet consisting of freshly collected rhizosphere soil and AMF spores along with hyphae, arbuscules, vesicles and root segments of ragi plant.
Providencia sp.(AW5) was isolated from the wheat rhizosphere and identified using 16S rDNA sequence analysis in the division of Microbiology, IARI and submitted to NCBI (Accession No. FJ866760) (Rana et al., 2011b).Inoculum of bacterial strain was prepared by growing in nutrient broth at 28 ± 1°C for 48 h at 100 rpm, such that the inoculum contained 10 11 cells ml -1 .
The other bacteria like A. brasilense and PSB (Bacilus subtilis + Bacilus megaterium) were cultured in nutrient broth and then multiplied in carrier like finely powdered and sterilized charcoal powder.The broth containing 10 9 cells ml -1 was added to 1/3 of the water holding capacity of the carrier.

Plant material, microbial inoculation and growth conditions
The seeds of Cleopatra mandarin were collected from the germplasm of Division of Fruits and Horticultural Technology, IARI and then surface sterilized by immersing in 70% alcohol for 5 min, followed by rinsing three times with sterile distilled water and then kept over wet filter paper in Petri dishes at 28°C for germination.After 7 days, the seedlings were planted in plastic containers (12 × 20 cm) containing 4.1 kg of mixture of sterilised soil : sand : FYM (2:2:1) having EC (1:2) 6.35 mS m -1 , pH (1:2) 7.92, HCO3 -1.14 g kg -1 and Cl -5910.75ppm.
During planting, the seedlings were inoculated with either AMF (5 g per kg of potting mixture consisting of 80 to 88 infective propagules per 5 g of inoculum) or bacterial species (5 g per kg of potting mixture containing 10 9 cells per g of carrier) or both.In case of Providencia sp.(AW 5), 5 ml of liquid media containing 10 11 cells per ml of media was used for each pot.The seedlings were maintained in glasshouse with day-night temperatures of 27 ± 1°C and humidity of 80-85%.
Day lengths were extended up to 16 h with cool white fluorescent lights at 630 µmol m -2 s -1 for improving the vegetative growth of the plants.Seedlings were watered on alternate days with 250 ml of autoclaved water, maintaining soil moisture content above 80% using soil moisture meter.Water used for irrigation in high density orchard had EC (1:2) 288 µS m -1 , pH (1:2) 7.48, HCO3 -1.0 milliequivalent/litre and Cl -110.76 ppm.

Imposition of differential moisture regimes
The differential water treatments were started at 270 DAI under glasshouse condition.Half of seedlings under each treatment received ample water (750 ml) at an interval of two days and the remaining half were imposed drought stress by withholding water.Daily soil relative water content was measured using soil moisture meter (FieldScout TDR 300, Spectrum Technologies, Inc.) fitted with 4.8-inch probe rods.Wet point was fixed at 90% and dry point at 8%.The soil relative water content (RWC) for well-watered (WW) seedlings was monitored at 80%.Entire plants were harvested after 20 days, when drought stressed (WS) seedlings showed visible symptoms of temporary wilting.
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Fresh and dry weight of shoot and root
Fresh weight of root and shoot of each plant was recorded by electronic balance.The samples were then put in the perforated paper bag and kept in hot air oven at 70°C until constant dry weight.

Leaf nutrient analysis
For tissue nutrient analyses, oven-dried samples were ground, sieved and digested in nitric acid : perchloric acid (9:4).Total nitrogen (N) was determined in samples of 0.5 g dry weight using the Kjeldahl method.Phosphorous (P) was analysed by a vanadatemolybdate method.The P transmittance was read at 420 nm.Potassium (K) was determined with the help of flame photometer (Systronics 128, Ahmedabad) using specific filter and LPG flame.Determination of other foliar nutrients like Ca, Mg, Zn, Fe, Cu and Mn was done by the atomic absorption spectrophotometer (GBC-Avanta PM; GBC-Advanta Scientific Equipment, Dandenong, Victoria, Australia) using nitrous oxideacetylene flame.

Reactive oxygen species
Leaf samples (1 g) were homogenised in 5 ml of pre-cooled phosphate buffer (0.2 M, pH 7.2) containing 1 mM diethyl dithiocarbamate and centrifuged at 5000 g for 5 min.Superoxide radical was estimated as per the method of Chaitanya and Naithani (1994).Hydrogen peroxide was estimated as per the method of Rao et al. (1996).

Soil microbiological parameters
Alkaline phosphatase activity was assayed as per the method of Tabatabai and Bremner (1969) and the enzymatic activity was expressed as µg of -nitrophenol g -1 soil dry weight h -1 .Dehydrogenase activity, expressed as µg of triphenyl formazon g -1 soil dry weight day -1 , was assayed as per the method of Casida et al. (1964).Microbial biomass carbon (MBC), expressed as µg g -1 soil sample, was estimated by the method of Nunan et al. (1998).Experimental data from a period of two years was pooled and then subjected to analysis of variance (ANOVA) using statistical analysis software SPSS package (SPSS 11.0) and means were evaluated by Fisher's protected least significant difference (LSD).Differences at P < 0.05 were considered significant.(42.31, 41.43 and 38.13, respectively), whereas G. intraradices in combination with A. brasilense or PSB exhibited increased SOD activity by 74.01 and 71.60%, respectively, as compared to control under WS condition.Under WW condition, activities of CAT, APX, G-POD, SOD and GR were respectively increased by 10. 58, 17.55, 22.64, 141.70 and 114.89% in seedlings coinoculated with G. intraradices and PSB, as compared to control, which was statistically at par with co-inoculation of G. intraradices and A. brasilense and single inoculation of G. intraradices for CAT (9.19 and 9.05%, respectively) and co-inoculation of G. intraradices and A. brasilense for APX (16.64%).

Experimental design and statistical analysis
The co-inoculation of G. intraradices and PSB significantly stimulated highest increase in total glutathione in leaf under WS and WW conditions (24.47 and 27.82%, respectively), as compared to control, which was statistically at par with co-inoculation of G. intraradices and A. brasilense (22.24%) under WS (Figure 3C).
The leaf nutrient content was found to be influenced by microbial inoculation, regardless of any moisture regime.The leaf N content was 58.19 and 38.13% higher in seedlings co-inoculated with G. intraradices and A. brasilense under WS and WW, respectively, as compared to the control, which was statistically at par with seedlings inoculated with Azospirillum alone (34.92%) under WS (Table 2).The synergistic effect of G. intraradices and PSB resulted in significant increase in leaf P, K, Ca and Mg content, regardless of any moisture regime, as compared to the control, which was statistically at par with inoculation of G. intraradices alone and Azospirillum brasilense alone under WS and WW, respectively, for Ca content, inoculation of G. intraradices alone and co-inoculation of G. intraradices and A. brasilense under WW condition for Mg content (Table 2).
The drought stress also reduced leaf micronutrient content.However, inoculation of G. intraradices along with PSB resulted in significantly higher level of leaf Fe, Cu, Mn and Zn content, regardless of any moisture regime, which was statistically at par with co-inoculation of G. intraradices and Providencia sp.(AW 5) under WS for Cu content, co-inoculation of G. intraradices and A. brasilense under WS condition for both Mn and Zn content (Table 3).
The analysis of rhizospheric soil inoculated with different microbial culture including control revealed the superiority of co-inoculation of AMF and MHB over single inoculation and in particular, co-inoculation of G. intraradices and PSB for alkaline phosphatase activity under WS and WW conditions (81.02 and 87.30%, respectively), as compared to the control (Figure 4A).
Drought stress had negative effect on soil dehydrogenase activity (Figure 4B).However, it had significantly lesser effect in the rhizosphere of seedlings inoculated with G. intraradices and PSB (61.55%) as compared to the control.The particular treatment also showed 49.61% more enzyme activity, as compared to the control, under WW condition.
Microbial biomass carbon (MBC) was affected by different microbial treatment, however, inoculation of AMF and MHB had significant effect than single inoculation or uninoculated control (Figure 4C).Under WS and WW conditions, the MBC content was 192.73 and 100.04% more in G. intraradices + PSB inoculated rhizosphere as compared to the control, which was   Mean ± Standard error, n = 2; values followed by same letter in a column were not significantly different (p ≤ 0.05).
statistically at par with that of rhizosphere of seedlings co-inoculated with G. intraradices and Providencia sp.(AW 5) under WW condition (86.51%).

DISCUSSION
Colonization of roots by AMF and the subsequent benefits derived by a host plant depend initially on the survival of AMF propagules, particularly spores.The higher G. intraradices colonization in roots of Cleopatra mandarin treated with PSB might be due to improved AMF interaction with plant roots due to production of active metabolites such as vitamins, amino acids and indole-3-acetic acid by the bacteria (Vivas et al., 2003), resulting in increased germination of fungal spores and rapid AMF establishment in soil (Toljander et al., 2006).
Drought had adverse effect on biomass production in Cleopatra mandarin.The synergistic effect of PSB with G. intraradices for increase in both shoot and root weight indicated its compatible interaction with AMF.This increase in plant growth has been attributed to the stimulation of activity of AM fungal mycelium in the rhizosphere by PSB (Marulanda et al., 2003), resulting in improving root-fungus interaction, thereby increasing the hyphal efficiency for exploring greater volume of soil for water and nutrient uptake (Allen, 2011).
The AMF inoculation can lower the H 2 O 2 accumulation in plants (Wu et al., 2006) and also enhance the production of SOD involved in catalyzing the conversion of free O 2 -to O 2 (Huang et al., 2010).Thus, lower ROS levels in leaves of Cleopatra mandarin co-inoculated with AMF and MHB might be due to the fact that bacteria induced a higher increase in antioxidant enzyme activities in AM plants in response to stress, resulting in alleviating the negative effect of stress (Kohler et al., 2009).
The AMF possess several special genes encoding for antioxidant enzymes, whose expression patterns can regulate the activities of antioxidant enzymes (Wu and Zou, 2009).In the present study, the enhanced antioxidant activity in AMF and MHB co-inoculated seedlings supports the view that increased enzyme activities could be involved in the beneficial effects of microbial inoculation on the performance of plants grown under semi-arid conditions (Alguacil et al., 2003), indicating better plant protection against the drought stress (Azcón et al., 2013).
The plants inoculated with AMF can accumulate antioxidants to counteract ROS under any environmental stress (Kaya et al., 2009).The synergistic effect of MHB with AMF leading to higher antioxidant level might help the host plant in dissipating the photo-synthetically produced electrons and in alleviating oxidative damage.
The higher foliar N content in Cleopatra mandarin coinoculated with G. intraradices and A. brasilense could be attributed to the increase in N-assimilating enzymes, such as nitrate reductase in the shoots of AMF colonised plants (Caravaca et al., 2005).The higher level of P in seedlings co-inoculated with G. intraradices and PSB might be due to release of phosphate ions from sparingly soluble inorganic and organic P compounds in soil, thereby contributing to increased soil phosphate pool available for the extraradical AM fungal hyphae to pass on to the plant (Artursson et al., 2006).The enhanced acquisition of other mineral nutrients (K, Ca, Mg, Fe, Cu, Mn and Zn), in co-inoculated microbial treatment, could be attributed to the greater absorption of the surface area provided by extensive fungal hyphae (Navarro et al., 2011).The acquisition of P and Ca by AM plants were recorded lesser under WW than WS condition, which could be attributed to improved efficiency of fungal hyphae in transfer of nutrients especially phosphorus and calcium which are immobile in soil and plant, respectively, from nutrient depletion zone to root cortex.Bryla and Duniway (1997) reported that AM fungal infection might be less beneficial for P transfer in plants under well watered condition than under drought condition.
Mycorrhizal symbionts are an integral part of the rhizosphere microflora, and thus contribute to the dynamic equilibrium of the rhizosphere.The increased alkaline phosphatase and dehydrogenase activities and MBC content in seedlings co-inoculated with G. intraradices and PSB could be due to increase in the rhizosphere microbial population as a consequence of the inoculation treatments (Aseri and Tarafdar, 2006).
From the above results, it could be concluded that coinoculation of G. intraradices and PSB could be done in nursery in citrus propagation, which was found to synergistically interact to improve growth and performance of Cleopatra mandarin seedlings, much better than other microbial combinations under drought stress.

Figure 1 .
Figure 1.Influence of microbial inoculants on (A) AMF colonization in root (12 root segments observed per treatment); (B) Superoxide radicals; (C) hydrogen peroxide content in fresh leaves of Cleopatra mandarin grown in pots under differential moisture regime condition.WS, water stress; WW, well-watered.The bars of treatment in particular moisture regime having same letter do not differ significantly at р ≤ 0.05, n = 2 (Web Agri Stat Package 2.0).

Figure 2 .Figure 3 .
Figure 2. Influence of microbial inoculants on (A) Catalase; (B) Ascorbate peroxidise; (C) Guaiacol peroxidase content in fresh leaves of Cleopatra mandarin grown in pots under differential moisture regime condition.WS, water stress; WW, well-watered.The bars of treatment in particular moisture regime having same letter do not differ significantly at р ≤ 0.05, n = 2 (Web Agri Stat Package 2.0).

Figure 4 .
Figure 4. Influence of microbial inoculants on (A) alkaline phosphatase; (B) dehydrogenase; (C) microbial biomass carbon (MBC) in rhizosphere of Cleopatra mandarin grown in pots under differential moisture regime condition.WS, water stress; WW, well-watered.The bars of treatment in particular moisture regime having same letter do not differ significantly at р ≤ 0.05, n = 2 (Web Agri Stat Package 2.0).

Table 2 .
Response of Cleopatra mandarin to microbial inoculants on leaf macro nutrient content (%).

Table 3 .
Response of Cleopatra mandarin to microbial inoculants on leaf micro nutrient content (ppm).