Multifaceted potential of bioinoculants on red bell pepper (F1 hybrid, Indam Mamatha) production

Abstract

The present investigation was undertaken to determine the comparative efficacy of two arbuscular mycorrhizal (AM) fungi (Funneliformis mosseae and Acaulospora laevis) with Trichoderma viride and Pseudomonas fluorescens on growth and yield of red bell pepper. The results indicate that F. mosseae colonized the plant roots better as compared to A. laevis and promoted maximum increment in AM spore number, root colonization, leaf area, acid phosphatase activity, early fruit formation along with maximum increase in fruit nitrogen, and protein content. Whereas F. mosseae+P. fluorescens promoted maximum increase in plant height, shoot weight, mycorrhizal dependency, chlorophyll a, alkaline phosphatase activity, and fruit phosphorus content. Regarding root length, root weight, leaf photosynthesis, chlorophyll b, number of fruits per plant and their fresh weight, it was found best in F. mosseae+A. laevis+P. fluorescens. Therefore, soil inoculation with suitable bioinoculant should be used at nursery stage for better yield.

Keywords:

• arbuscular mycorrhizal fungi
• Capsicum annuum L.
• fruit yield
• nutrient uptake
• P. fluorescens
• T. viride

Introduction

Sweet pepper (Capsicum annuum L.), also called bell pepper, paprika, capsicum, or shimla mirch is one of the important vegetable crops of India. In India, green pepper production is very common but over the past few years, there has been a strong upsurge in the demand for colored bell pepper varieties like red, yellow, purple, and orange from urban consumers, and therefore these peppers fetches high price in the market (Kurubetta & Patil 2009). However, the supply of peppers is inadequate due to their low productivity (Muthukrishnan et al. 1986). The brighter colored peppers tend to be sweeter than immature green peppers because the sugar content increases as the pepper matures. Peppers are high-value crop as they are rich in vitamins, particularly provitamin A, vitamin B, vitamin C, and minerals such as Ca, P, K and Fe (Malik et al. 2011). Colored bell peppers are normally grown in soilless media under green house to get high-quality colored fruits (Jovicich et al. 2004). However, in India, colored bell pepper production is often more difficult and expensive because of high production cost, high cost of green house, costly chemical fertilizers, and unavailability of authorized seeds, and longer time required for fruit maturity limits commercial production of these pepper. Any alternative method, which decreases the costs of production but maintains the yields, is desirable and urgently needed in order to fulfill the increasing demand.

Plant microbe interactions are the interesting events that contribute for the sustainable agriculture (Lambers et al. 2009). The need of producing good-quality fruits, while mitigating deleterious environmental impact makes the use of soil microbes as preferred alternative and feasible production practice (Nzanza et al. 2011). However, these microbes not yet have any impact on agricultural practices because of inconsistent results and inability of a single micro-organism to sufficiently promote plant growth (Gray & Smith 2005; Artursson et al. 2006). Arbuscular mycorrhizal (AM) fungi, Trichoderma spp. and plant growth–promoting Pseudomonas constitute an important biological resource in this respect and could be effectively used in plant production (Kaushish et al. 2012; Parkash et al. 2011). Only recently the synergistic effects of these microorganisms have been studied with respect to their combined beneficial effects on plants (Barea et al. 2002).

AM fungi are obligate biotrophs and despite their obligate nature, AM fungal symbionts remain remarkably successful and integral components of plant root systems, contributing to nutrient uptake in the large majority of land plants and stimulating plant growth (Smith & Smith 2012). Screening various strains of mycorrhizal fungi is necessary to identify the best ones for maximum benefits because not all strains are beneficial and others may even depress plant growth (Smith & Read 2008). Similarly, Trichoderma isolates have been identified as being able to act as endophytic plant symbionts. They alter plant physiology, enhance nitrogen fertilizer uptake and photosynthetic efficiency, and provide resistance to pathogens. Typically, the net result of these effects is an increase in plant growth and productivity (Hermosa et al. 2012). Many endophytic bacteria can establish themselves in plants other than their original hosts, indicating a lack of host specificity. Fluorescent Pseudomonas spp. has received special attention to influence plant growth because of its excellent root colonizing ability that increases root growth and morphology (Harman 2006). It also supports plant symbiosis with other beneficial microorganisms like AM fungi and thus increases nutrient availability in the rhizosphere (Vessey 2003; Ardebili et al. 2011).

Until now, most of the research related to bioinoculant-induced growth improvement has been focused on green bell peppers under pot as well as under field conditions (Bajaj et al. 1979; Sharma et al. 1997; Malik et al. 2011; Tanwar et al. 2011). The use of bioinoculants for red bell pepper production is not a practice, and to our knowledge there are no available local isolates for use. A careful selection of suitable bioinoculant is necessary to provide an effective formulation for better plant growth support. In the present study, we assessed the effects of two native AM fungi (Funneliformis mosseae and Acaulospora laevis) with Trichoderma viride and Pseudomonas fluorescens alone and in combination on mycorrhizal colonization, growth, nutrient acquisition, and fruit yield of red bell pepper ((F1 hybrid, Indam Mamatha). Moreover, we aimed to select specific strains that can be used as bioinoculants at the seedling stage to improve red bell pepper productivity without increasing production cost.

Materials and methods

Growth conditions

The experiment was carried out in a glass house at Botany Department, Kurukshetra University, Kurukshetra, Haryana during mid January, 2011 to mid May, 2011 at a temperature (20°C±5°C) and humidity (50%–70%). Light was provided by cool white fluorescent lamps (8000 lux) under a 16-hour photoperiod. The glasshouse also received sunlight. The soil characteristics are as follows: sand-64.2%, silt-21.81%, clay-3.90%, pH-6.8±0, EC-0.25 dS/m, organic carbon-0.40%, total N-0.042%, P-0.0018 Kg/m², K-0.022 Kg/ m², and S-14.80 ppm.

Isolation and identification of AM fungi

Dominant AM fungi were isolated from the rhizosphere soil of field-grown bell pepper plants by ‘wet sieving and decanting technique’ of Gerdemann and Nicolson (1963). The isolated spores were given a thorough microscopic examination to record their morphological features, like color, size, shape, wall structure, surface ornamentation, nature, and size of subtending hyphae, bulbous suspensor, number, and arrangement of spores in sporocarp. These spores were identified using keys of Schenck and Perez (1990) and Funneliformis mosseae (Nicol. and Gerd.) Walker and Schüβler (earlier known as Glomus mosseae) and Acaulospora laevis Gerd. and Trappe were found to be the most dominant AM fungal strains.

Bioinoculant preparation

The starter inoculum of each selected dominant AM fungus was raised by ‘funnel technique’ of Menge and Timmer (1982) using maize as host for three months. Likewise, Trichoderma viride was isolated from soil by soil dilution plate method (Johnson et al. 1959) and was mass produced on wheat bran: saw dust: water (3: 1: 4) medium for 10 days. The spore density (conidiospores) was 3×10⁹ per gram wheat bran medium. The inoculum of P. fluorescens (MTCC No. 103) was obtained from Institute of Microbial Technology, Chandigarh, India and cultured in a nutrient broth medium incubated at 32°C for 48 hours to obtain a concentration of 1×10⁹ colony forming units (cfu) ml⁻¹.

Experimental setup

Soil from experimental site was collected and mixed with sand in a proportion of 1:3 (sand:soil). This mixture was then sieved through 2-mm sieve and autoclaved at 121°C for two hours for two consecutive days. Earthen pots (25.4×25 cm) were selected and filled with 2 kg soil. Chopped AM colonized root pieces of maize having 80%–85% of colonization along with the soil having AM spores (620–650 per 100 g inoculum) were used as AM inoculum. To each pot 10% (w/w), i.e. 200g/pot inoculum of AM fungi and 2 g inoculum of T. viride alone and in combinations were added into the soil before plantation. The experiment had 16 treatments with combinations of single inoculation [F. mosseae (F), A. laevis (A), T. viride (T), P. fluorescens (P)], double inoculations (F. mosseae+A. laevis, F. mosseae+T. viride, F. mosseae+P. fluorescens, A. laevis+T. viride, A. laevis+P. fluorescens, T. viride+P. fluorescens), triple inoculations (F. mosseae+A. laevis +T. viride, F. mosseae+A. laevis+P. fluorescens, F. mosseae+T. viride+P. fluorescens, A. laevis+T. viride+P. fluorescens) and lastly consortium of all the bioinoclulants (i.e. F. mosseae+A. laevis+T. viride+P. fluorescens). In control set no inoculum was added.

Plant material

The seeds of red bell pepper (F1 hybrid, Indam Mamatha) were surface sterilized with 0.5% (v/v) sodium hypochlorite for 10 minutes, subsequently washed with sterilized deionized water, and germinated using a shallow tray containing sterilized soil: sand (3:1). Thirty days after emergence, seedlings were uprooted and roots were first washed with the running water to remove adhering soil particles and thereafter treated with P. fluorescens. The roots were first dipped in cell suspension of P. fluorescens for five minutes and single seedling was planted in each pot, while in treatment without P. fluorescens including control, plants were planted as such after washing. Plants were watered daily and 100ml/pot Hoagland nutrient solution (without KH₂PO₄) was also added to each plant after regular intervals of 15 days. Each treatment was replicated five times.

Plant harvest and analysis

Plants were harvested 120 days after transplantation (DAT), and the effect of bioinoculants on various growth parameters were recorded. Change in plant height (cm), leaf area (cm²) by using leaf area meter (Systronics 211, Ahmedabad, India), chlorophyll content (mg g⁻¹ FM) (Arnon 1949), and photosynthetic rate using a portable infrared gas analyzer (CIRCAS-I, PP Systems, Stotfold, UK) were recorded in the standing crop. Number of days taken from the date of transplanting to first flower opening was counted and recorded as number of days of flowering. Similarly, number of days of fruiting was recorded. After that all the fruits were harvested from each plant, counted and weighted to get their fresh weight (g). The fruit color was recorded by visual observations of fruits on the day of harvest using a scale of 1–5, where 1 is green: fruit surface is completely green; 2 is turning: tannish yellow, red color on over 10% but not more than 20%; 3 is pink: red color on over 30% but less than 60%; 4 is light red: pinkish red, red color on over 60% but not more than 90%; and 5 is red: more than 90% fruit surface is red.

After that plants were harvested, separated into roots and shoots, weighted separately for their fresh weight (g), and oven dried at 70°C until a constant weight was obtained to determine the dry weight (g). Percentage of root colonization was assessed using the method of Phillips and Hayman (1970) and the quantification of root colonization was done by the following formula:

Mycorrhizal dependency was calculated according to the method of Plenchette et al. (1983):

Fresh roots were used for extraction of alkaline and acid phosphatases, assayed by using p-nitrophenyl phosphate as substrate, which is hydrolyzed by the enzyme to p-nitrophenol. For this 1 g of fresh, washed roots were homogenized in 5 ml of ice cold sodium acetate buffer (0.05 M with pH 4.8) for acid phosphatase and sodium carbonate-bicarbonate buffer (0.05 M with pH 10) for alkaline phosphatase activity using a pre-chilled mortar and pestle. The resulting homogenate was centrifuged at 10,000 rpm for 15 minutes, and supernatant thus obtained is referred to as crude enzyme extract used for the assay of acid phosphatase activity and measured in terms of IU/g FW. The AM spore quantification was also done by following the procedure of Gerdemann and Nicolson (1963). Phosphorous content (P) of shoot, root, and fruits were determined by vanadomolybdate phosphoric yellow color method (Jackson 1973). Total nitrogen (N) was calculated by Kjeldahl method (Kelplus nitrogen estimation system, supra-LX, Pelican Equipments, Chennai, India).

Statistical analysis

Data were subjected to analysis of variance and means separated with least significant difference test using the Statistical Package for Social Sciences (ver. 11.5, Chicago, IL, USA).

Results

AM spore number, colonization, and mycorrhizal dependency

It is apparent from Table 1 that AM spore number in the soil and root colonization varied among different treatments. Maximum spore numbers as well as colonized roots were recorded in single inoculation of F, followed by A, FA, and FP as compared to control in which AM spores and root colonization was absent. There was a positive interaction between AM spores and root colonization. With regard to mycorrhizal dependency among all the AM-inoculated plants, FP, FAP, and F showed maximum response compared to uninoculated control, T, P, and TP in which AM inoculum was not provided.

Table 1. Effect of bioinoculants on Mycorrhization of bell pepper plants.

Treatment	AMF spore number/10 g of soil	AMF root colonization (%)	Mycorrhizal dependency (%)
Control	0g	0g	0f
F	221.6±18.6a	100.0±0a	62.84±2.16a
A	206.8±18.0a	92.13±5.41b	45.40±6.75b
T	0g	0g	0f
P	0g	0g	0f
FA	182.8±11.8b	93.40±4.45b	25.65±2.00e
FT	146.6±10.9d	58.18±7.83e	47.86±1.50b
FP	182.8±10.5b	69.60±7.83d	64.96±5.47a
AT	162.2±10.0cd	59.50±7.00e	47.00±1.28b
AP	169.0±16.0bc	80.67±1.71c	47.97±1.97b
TP	0g	0g	0f
FAT	145.8±13.3d	59.00±3.52e	23.88±4.04e
FAP	159.8±13.3cd	63.19±5.06e	63.27±1.92a
FTP	128.6±12.3e	50.73±6.31f	31.41±6.43d
ATP	75.00±9.60f	63.01±4.74e	39.63±2.78c
FATP	156.8±20.5cd	77.25±4.01c	47.39±2.10b
L.S.D (p≤0.05)	3.1	5.4583	4.3096
ANOVA (F 15,32)	207.511	330.948	119.657

Notes: F: Funneliformis mosseae, A: Acaulospora laevis, T: Trichoderma viride, P: Pseudomonas fluorescens, ±: standard deviation, AMF: Arbuscular mycorrhizal fungi.

Values in columns followed by the same letter are not significantly different, p ≤ 0.05, least significant difference test.

Plant photosynthetic activity

It is depicted from Table 2 that after 120 DAT leaf area, plant photosynthesis and chlorophyll content increased in all the inoculated plants over control. In bell pepper plants, maximum increase in leaf area was observed in plants in which F was applied alone, followed by FAP and FATP. There was also an increase in the leaf photosynthesis, which was again found maximum in FAP, followed by FATP, F, and FTP respectively. Regarding chlorophyll content, chlorophyll a was found highest in plants supplemented with FP, whereas chlorophyll b was found maximum in FAP as compared to control that showed least chlorophyll content.

Table 2. Effect of inoculation with AM fungi, T. viride and P. fluorescens on leaf area, photosynthesis, chlorophyll content and phosphatase activity of bell pepper plants.

Treatment	Leaf area (cm^2)	Photosynthesis (mg CO2/m^2·s^−1)	Chlorophyll a (mg·g^−1 FM)	Chlorophyll b (mg·g^−1 FM)	Alkaline phosphatase	Acid phosphatase
Control	87.58±1.22g	20.94±1.12g	0.34±0.04g	1.04±0.05g	0.033±0.005g	0.37±0.005f
F	147.7±1.45a	39.02±2.63bc	0.60±0.05bc	2.02±0.15b	0.086±0.005a	0.47±0.005a
A	111.4±1.02e	30.14±1.90e	0.49±0.10d	1.37±0.03ef	0.066±0.005c	0.41±0.005cd
T	118.6±5.32de	27.72±2.09ef	0.47±0.03de	1.40±0.17ef	0.042±0.004f	0.38±0.005e
P	98.48±1.57f	28.44±1.77ef	0.43±0.02f	1.21±0.05fg	0.044±0.005f	0.40±0.005d
FA	134.9±20.9b	30.74±2.65e	0.40±0.02f	1.10±0.04fg	0.066±0.089c	0.42±0.005c
FT	127.2±9.17cd	33.88±3.75de	0.52±0.02cd	1.47±0.28de	0.066±0.054c	0.41±0.004cd
FP	120.3±1.27d	37.74±2.37bc	0.64±0.02a	2.23±0.19a	0.090±0.005a	0.47±0.004a
AT	132.0±1.86bc	26.92±2.02f	0.58±0.01bc	1.72±0.16c	0.064±0.005cd	0.43±0.005bc
AP	130.5±1.67bc	33.30±2.54de	0.51±0.01cd	1.48±0.38de	0.062±0.004de	0.43±0.005bc
TP	121.5±4.98d	36.58±2.52cd	0.50±0.01d	1.39±0.29ef	0.048±0.004f	0.41±0.004cd
FAT	127.5±3.61cd	26.20±2.34f	0.46±0.03e	1.24±0.01f	0.084±0.005a	0.43±0.007bc
FAP	142.0±6.10ab	44.30±2.16a	0.62±0.02ab	2.26±0.10a	0.086±0.005a	0.45±0.005ab
FTP	131.48±1.11bc	39.24±1.88bc	0.54±0.04c	1.76±0.24c	0.056±0.005e	0.43±0.005bc
ATP	123.46±0.94cd	28.08±1.86ef	0.51±0.02cd	1.62±0.06cd	0.054±0.005ef	0.43±0.005bc
FATP	141.12±1.93ab	40.28±1.24b	0.58±0.01bc	2.00±0.17b	0.076±0.055b	0.45±0.005ab
L.S.D (p≤0.05)	8.0535	2.8548	0.0312	0.1569	0.0069	0.0068
ANOVA (F 15,32)	30.167	39.538	53.028	47.854	47.986	120.965

Notes: F: Funneliformis mosseae, A: Acaulospora laevis, T: Trichoderma viride, P: Pseudomonas fluorescens,±: standard deviation, FM: fresh matter.

Values in columns followed by the same letter are not significantly different, p≤0.05, least significant difference test.

Plant phosphatases

As shown in Table 2, with reference to phosphatase activity of bell pepper plants, it was found higher in the roots of inoculated plants as compared to uninoculated control. Acid phosphatase activity was found more active than alkaline phosphatase. F. mosseae when combined with P. fluorescens showed maximum alkaline phosphatase followed by F, FAP, and FAT respectively. On the other hand, F alone influenced maximum acid phosphatase activity followed by FP, FAP, and FATP. Contrary to this, single inoculation of either T. viride or P. fluorescens recorded least enzyme activity, both acid and alkaline.

Growth performance

Soil inoculation with bioinoculants significantly increased the plant height of red bell pepper 120 DAT and maximum increase was observed in combined inoculation of FP, followed by FATP, AT, and F and least in uninoculated control (Table 3). However, in FA treatment the height was less as compared to control but the plant showed better biomass (dry weight) because of side branching. Similarly, increase in shoot fresh weight and dry weight was also found maximum in FP, whereas second best results were observed in single inoculation of F. In Table 3, synergistic effects were observed between AM fungi and P. fluorescens in increasing root parameters. After 120 DAT, maximum increase in root fresh weight, root dry weight, and root length was recorded in consortium of FAP.

Table 3. Effect of bioinoculants on plant growth improvement of bell pepper plants.

Treatment	Change in plant height (cm)	Above ground fresh weight (g)	Above ground dry weight (g)	Root length (cm)	Fresh root weight (g)	Dry root weight (g)
Control	53.18±1.81d	32.39±2.01g	5.14±0.69g	22.10±1.18g	5.23±0.95f	0.90±0.11f
F	84.04±4.53a	66.58±3.84b	13.37±0.53a	33.10±1.68cd	11.47±1.09cd	3.69±0.56ab
A	63.78±4.74bc	45.32±2.82e	8.97±0.53cd	34.84±1.22c	11.34±1.20cd	3.16±0.45bc
T	57.78±2.75c	38.42±2.92ef	8.19±0.23de	32.22±1.17d	6.99±0.79e	2.55±0.86cd
P	57.64±4.80c	51.44±5.86d	10.37±1.09b	31.84±1.85de	8.02±0.68e	2.09±0.74cd
FA	51.52±4.06e	31.75±3.86f	5.72±0.67fg	32.44±1.72d	11.12±1.22cd	2.54±0.36cd
FT	68.28±4.80b	39.63±3.67ef	8.33±0.64de	29.30±1.06f	11.45±0.67cd	2.73±0.60bc
FP	88.76±7.10a	73.77±5.88a	14.2±0.60a	30.00±2.03e	12.94±1.04b	2.96±0.51bc
AT	84.06±6.66a	44.90±3.91e	8.42±0.75de	34.56±1.49c	11.13±1.06cd	3.07±0.51bc
AP	64.44±5.77bc	43.44±3.23ef	9.57±0.57c	26.20±1.17ef	6.90±0.75e	2.14±0.48cd
TP	61.28±4.24bc	40.84±2.67ef	8.44±0.38de	28.32±1.35ef	12.01±0.97bc	2.50±0.42cd
FAT	55.42±4.17cd	34.70±3.62f	6.47±0.41f	29.24±1.57f	5.56±0.45f	1.31±0.27e
FAP	82.40±2.54a	59.70±4.94c	12.0±0.43b	53.18±1.30a	17.64±1.16a	4.30±0.79a
FTP	61.04±3.58bc	34.72±3.65f	5.77±0.23fg	21.82±1.40g	9.65±0.65d	3.53±0.63b
ATP	63.50±3.74bc	40.54±1.97ef	7.64±0.23e	31.38±1.93de	10.18±1.20d	2.51±0.38cd
FATP	85.40±4.14a	54.08±2.68d	7.89±0.51e	47.98±2.26b	17.32±1.21a	3.44±0.60bc
LSD (p≤0.05)	5.743	4.7671	0.7256	1.9757	1.23	0.6963
ANOVA (F 15,32)	39.695	53.444	104.857	134.287	63.364	12.272

Notes: F: Funneliformis mosseae, A: Acaulospora laevis, T: Trichoderma viride, P: Pseudomonas fluorescens,±: standard deviation.

Values in columns followed by the same letter are not significantly different, p≤0.05, least significant difference test.

Fruit yield

Perusal of the data presented in Table 4 revealed that fruit parameters were highly influenced by inoculated microbes. Early flowering was recorded in the inoculated ones as compared to control in which flowering was delayed. Flowers were first seen on 43 DAT in F alone–treated plants, followed by AT and FAP as compared to control in which flowers appeared 85 DAT. Likewise, number of days taken for fruit formation significantly vary among treatment, and fruit formation was first recorded in F-treated plants 56 DAT as compared to control (97 DAT). Healthy fruits appeared in treated plants that were four-lobed, with square blocky shape having thick pericarp. Even though fruits appear in all the treatments, their number and fresh weight varies with each treatment. Table 3 showed that plants that were treated with FAP harbored maximum number of fruits compared to rest of the treatment. Among the five set of replicates used for control, fruits appeared only in three sets 120 DAT. A significant increase in fruit nutrient content (P and N) was noticed in AM-treated plants. Amongst both the AM fungi used, soil inoculation with FP resulted in maximum P uptake while single inoculation of F depicted maximum P and protein content.

Table 4. Effect of amendment of soil with AM fungi, P. fluorescens or T. viride on yield of bell pepper plants.

Treatment	Number of days of flowering	Number of days of fruiting	Number of fruits/plant	Fruit fresh weight/plant (g)	Fruit P (%)	Fruit N (%)	Fruit protein (%)	Fruit color
Control	85.4±2.70g	96.8±2.3f	0.60±0.55d	2.848±2.81h	0.03±0.03g	0.50±0.46h	3.15±2.87g	1
F	43.2±2.77a	56.2±2.68a	1.80±0.45b	45.78±3.50cd	0.17±0.005bc	2.14±0.11a	13.4±0.69a	5
A	66.0±2.73d	74.2±3.19c	1.00±0c	35.16±1.15e	0.12±0e	1.78±0.04c	11.1±o.23bc	5
T	80.6±2.40f	89.4±3.91e	1.00±0c	32.60±0.77e	0.07±0f	1.06±0.04g	6.62±0.27f	5
P	61.6±2.07cd	74.6±3.10c	1.00±0c	21.34±0.94f	0.15±0.005cd	1.47±0.04de	9.23±0.25de	5
FA	80.6±2.40f	90.8±3.70e	1.00±0c	34.81±0.60e	0.13±0.004d	1.34±0.03e	8.40±0.18e	5
FT	77.8±2.28f	88.4±4.51e	1.00±0c	41.36±1.15d	0.16±0.005c	1.24±0.01f	8.00±0.40e	3
FP	83.0±2.00fg	88.4±3.84e	2.00±0b	72.50±1.77b	0.19±0.004a	1.76±0.01c	11.0±0.10bc	5
AT	52.8±1.92b	70.2±3.03c	1.80±0.5b	69.04±1.50bc	0.07±0.004f	1.84±0.01bc	11.6±0.11bc	4
AP	64.4±3.00d	70.0±3.16c	1.00±0c	40.82±1.31d	0.14±0cd	1.01±0.04g	6.34±0.26f	5
TP	59.0±2.00c	65.2±2.59b	1.00±0c	18.08±0.93g	0.14±0.004cd	0.96±0.01g	6.01±0.08f	4
FAT	71.4±2.40e	80.8±2.77d	2.00±0b	52.88±1.77c	0.14±0.005cd	1.73±0.01c	10.8±0.05c	5
FAP	58.6±2.60c	63.4±3.36b	3.00±0a	115.3±4.97a	0.19±0a	1.95±0.01b	12.2±0.05b	5
FTP	71.0±2.91e	82.8±3.70d	1.00±0c	26.58±1.67ef	0.13±0d	1.53±0.01d	9.60±0.07d	5
ATP	69.8±2.86e	80.8±3.70d	2.00±0b	18.36±1.93g	0.13±0.005d	1.51±0.01d	9.47±0.08d	3
FATP	59.6±2.60c	71.2±3.70c	2.00±0b	52.62±2.20c	0.18±0.005ab	1.93±0.01b	12.1±0.05b	5
L.S.D (p≤0.05)	3.1618	40.554	0.2643	2.6779	0.0103	0.1525	0.9603
ANOVA (F 15,32)	133.128	93.026	47.238	800.642	152.533	66.868	65.79

Notes: F: Funneliformis mosseae, A: Acaulospora laevis, T: Trichoderma viride, P: Pseudomonas fluorescens,±: standard deviation.

Values in columns followed by the same letter are not significantly different, p≤0.05, least significant difference test.

Fruit color was measured using a scale of 1–5, where: 1-green: fruit surface is completely green; 2-turning: tannish yellow, red color on over 10% but not more than 20%; 3-pink: red color on over 30% but less than 60%; 4-light red: pinkish red, red color on over 60% but not more than 90%; 5-red: more than 90% fruit surface is red.

All the young fruits were green in color initially, that changed to red color passing through green, turning pink, and light red to red color. At this red color stage fruits were harvested. On 120 DAT, the fruits of all treatments became red in color except for control in which fruits remained green, light red in AT, and pink in ATP.

Plant nutrient uptake

The results of the present investigation reveal positive influence of inoculants on plant nutrient uptake (P and N) as compared to uninoculated control (Table 5). After 120 DAT, plant nutrient content was found maximum in shoots compared to roots. In particular, maximum increment in shoot as well as root P-content was found in dual inoculation of FP followed by single application of F and consortium of FATP respectively. Likewise, shoot N-content was also found highest in FAP compared to control. However, single application of F induced maximum increase in root N-content.

Table 5. Plant nutrient content as affected by amendment with AM fungi, T. viride and P. fluorescens.

Treatment	Shoot P (%)	Root P (%)	Shoot N (%)	Root N (%)
Control	0.12±0.005h	0.010±0f	1.24±0.17i	0.88±0.04g
F	0.22±0.005b	0.030±0bc	2.64±0.17bc	2.36±0.18a
A	0.16±0.005f	0.022±0.004de	2.08±0.11ef	1.81±0.11cd
T	0.13±0.005g	0.012±0.004	1.52±0.18h	1.00±0.09f
P	0.18±0.005d	0.026±0.005cd	2.26±0.13de	1.85±0.10cd
FA	0.18±0.005d	0.020±0e	1.84±0.17g	1.35±0.08ef
FT	0.18±0.005d	0.020±0e	2.36±0.17d	1.45±0.15ef
FP	0.25±0.005a	0.038±0.004a	2.48±0.13cd	1.90±0.09cd
AT	0.17±0.005e	0.020±0e	2.82±0.13b	2.04±0.14bc
AP	0.17±0.005e	0.020±0e	1.98±0.15f	1.42±0.25ef
TP	0.16±0.005f	0.014±0.005f	1.84±0.11g	1.35±0.13ef
FAT	0.17±0.005e	0.014±0.005f	2.50±0.16cd	2.00±0.11bc
FAP	0.21±0.005b	0.032±0.004b	3.06±0.17a	2.15±0.14b
FTP	0.19±0.005c	0.020±0e	1.60±0.19h	1.64±0.19d
ATP	0.13±0.005g	0.010±0f	2.36±0.21d	1.56±0.21de
FATP	0.19±0.005c	0.026±0.005cd	2.54±0.13cd	2.14±0.20b
L.S.D (p≤0.05)	0.0068	0.0045	0.1978	0.1875
ANOVA (F 15,32)	183.4	25.902	50.622	40.763

Notes: F: Funneliformis mosseae, A: Acaulospora laevis, T: Trichoderma viride, P: Pseudomonas fluorescens,±: standard deviation.

Values in columns followed by the same letter are not significantly different, p≤0.05, least significant difference test.

Discussion

Plants inoculated with bioinoculants grew remarkably better as compared to uninoculated control. Among both the AM fungi, F. mosseae was found to be more compatible strain as compared to A. laevis in colonizing host roots when used alone and even when applied along with P. fluorescens and thus exhibit biological specificity between AM fungal strain and host plant (Bever et al. 1996). P. fluorescens is known to influence the growth of the AM hyphae from germinating fungal spore (Barea & Jeffries 1995) and in the present study also such synergistic interaction was observed between AM fungi and P. fluorescens. Moreover, when AM plants are grown on soils with poorly available P, their responsiveness to colonization is higher than when grown on soils with equivalent amounts of readily available P (Smith & Smith 2012). These results are in harmony with those of Kim et al. (2010) that showed the dual inoculation of AM fungi with other bioinoculant increased fresh weight and dry weight of red pepper plants.

The increase in the chlorophyll content of red bell pepper leaves was highly pronounced by the added microbes. These results are in accordance with other workers (Demir 2004; Zuccarini, 2007; Amaya-Carpio et al. 2009). This is due to the enhanced stomatal conductance, photosynthesis, and transpiration in the host plant coupled with the effective synergism of microbial inoculants (Sheng et al. 2008). Previously also it was reported that AM fungi (G. intraradices) increases total chlorophyll and carotenoid content in pepper plants (Çekiç et al. 2012). The increased chlorophyll content may be due to increased uptake of mineral nutrients from the soil especially Mg that increases the photosynthetic activity of the leaves and thus the chlorophyll content.

Several investigations have showed the great potential of different microorganisms for secretion of phosphatases. In the present investigation also enzyme activities (both acidic and alkaline) increased to the greatest extent after application of AM fungi and P. fluorescens. These enzymes produced by the extraradical hyphae of AM fungi has the ability to hydrolyze extracellular phosphate ester bonds and ultimately made P available to the plants (Joner and Johansen 2000) and thus the increase in P content may have been due to the enhanced activities of phosphatases (Feng et al. 2002). According to Tisserant et al. (1993), there is no alkaline phosphatase activity in plant roots, but in the present investigation very low alkaline phosphatase activity was observed in comparison to acidic phosphatase activity, which may be due to AM colonization. The P cycle enzyme activities are actually inversely related to the available P in the soil (Tadano et al. 1993). The experimental soil used in the present study was low in P and with low phosphorus availability P demand increases, resulting in an increase in the phosphatase activity (Tanwar et al. 2012).

Among both the phosphatase enzymes, acid phosphatase activity was found higher than alkaline phosphatase activity in all the inoculated plants. This is due to the slight acidic pH of the experimental soil used in the present study that stimulates the activity of acidic phosphatase, which has a major role in the mineralization of organic P (Rodríguez & Fraga 1999; Tarafdar & Claassen 2005).

It seemed that better AM status enhances activities of phosphatase, as has been reported by others (Koide & Kabir 2000). In the present investigation also we observed increased AM colonization of roots after application of bioinoculants that strongly increases root phosphatase activities in plants.

The results clearly indicate the dependency of plants on bioinoculants for nutrition and growth, and a positive interaction was recorded among them due to the mutual benefits provided by each partner. The increment in plant growth parameters could be due to the establishment of appropriate AM root colonization by potential strains that greatly increases the absorbing surface of plant. AM hyphae colonize the roots; extend deeper in the soil away from depletion zone; and increase uptake of water, P, and other nutrients (Giri and Mukerji 2004), thus making plant healthier and stouter.

Plant growth promoting effect of Pseudomonas could be explained due to their ability to produce indole-3-acetic acid (IAA) that stimulates better root development and hence growth (Patten & Glick 2002). Moreover, the plant growth promoting effect of Pseudomonas is also associated with the solubilization of insoluble P sources (Richardson et al. 2001) and also by regulating plant growth regulator either through their production or through their degradation (Vessey 2003).

The efficacy of bioinoculants to increase fruit yield varied between the treatments, although inoculation always resulted in better fruit yield. Indeed, fruits appeared in the control also, but were fewer in number and also lower in weight. AM fungi might have stimulated the population of P. fluorescens, by directly providing energy rich carbon compounds derived from the host assimilates, transported via fungal hyphae to the rhizosphere, and changes the pH of rhizosphere making conditions more conducive for the bacteria and also by secretion of some stimulatory substances (Johansson et al. 2004). Increased fruit yield by T. viride could be due to the stimulation of nutrient transfer from soil to roots, as Trichoderma can colonize the interior of roots (Kleifeld & Chet 1992), also regulate IAA and ethylene production (Gravel et al. 2007) or through production of growth-promoting compounds such as cytokinin-like molecules, possibly kinetin (Tsavkelova et al. 2006).

Application of P. fluorescens may synergistically interact with AM fungi and produce plant growth–promoting substances, thereby enhancing plant growth and yield (Kaiser et al. 1989) as depicted in our study. It was observed in one another study that inoculation with native AM fungi decreased transplanting stress and resulting in higher and better yield quality of chili pepper plants (Castillo et al. 2009). Increased yield through the application of AM fungi alone and with other bioinoculants has also been reported in tomato (Copetta et al. 2011), sesame (Ziedan et al. 2011), and bell pepper (Tanwar et al. 2011).

Better nutritional status of AM and P. fluorescens-inoculated plants could be due to the secretion of phosphatases by AM fungi and Pseudomonas, which is also considered a common mode of conversion of insoluble forms of P to available forms and thus enhances plant P uptake and growth (Park et al. 2009). Further solubilization of inorganic P source is also done by Pseudomonas by synthesis of organic acids (Illmer & Schinner 1992). AM symbiosis is known to increase the nutrient acquisition of N, P, K, Ca, S, Cu, and Zn from the soil. However, the most prominent nutritional effect is the improved uptake of immobile nutrients, particularly P, Cu, and Zn (Hodge et al. 2010). The AM hyphae increases the absorptive surface area of the plant root systems through the extended mycelia that move several centimeters outwards from the root surface, bridging over the zone of nutrient depletion around the roots, and absorb low-mobile mineral nutrients from the bulk soil like P and N (Johansson et al. 2004; Smith & Smith 2012). Increased nutrient uptake (P and N) could be linked with AM fungal colonization of the host roots. Moreover, in this study also, AM root colonization as well as nutrient uptake was recorded maximum in F-treated plants. Increased nutrient uptake was also observed after the application of T. viride as compared to control. Similar to our results, increased P and N uptake by AM fungi and Trichoderma was also reported by other workers (Yedidia et al. 2001; Harman et al. 2004). However, this uptake increases when T. viride was inoculated along with other bioinoculants. These results are in accordance with those of Latef (2011) who also observed positive effect of AM inoculation on plant biomass, chlorophyll content, and plant nutrient content of Capsicum annuum L. cv. Zhongjiao 105. The increase in the uptake of soil minerals by bioinoculants can increase the production of bell peppers with improved quality and can possibly reduce the input of chemical fertilizers.

Conclusion

The findings of present investigation can help in improving fruit yield of red bell peppers by implementing the use of F. mosseae+P. fluorescens or F. mosseae+A. laevis+P. fluorescens in the production methodology. This is attributed to increased plant growth, plant physiological status and increased nutrients under potted conditions, and significantly reduces the need to use chemical fertilizer, soil less media, and thus can provide an economic advantage for farmers. Since red bell peppers are generally grown under glass house conditions, these bioinoculants can be further tested under field conditions in order to confirm and gain more insight to plant establishment and development under these conditions and after proper trials can be recommended to be adopted by farmers for commercial production.

Acknowledgements

We sincerely thank Kurukshetra University, Kurukshetra for providing financial assistance in the form of university research scholarship to AT for the completion of this work.