Glomus mosseae and Pseudomonas ﬂuorescens Application Sustains Yield and Promote Tolerance to Water Stress in Helianthus annuus L.

: The inoculation of sunﬂower ( Helianthus annuus L.) plants with arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) can signiﬁcantly enhance its growth and yield in a sustainable manner. Drought tolerance is mediated by a combination of direct AMF and PGPR beneﬁts that boost the plant’s natural ability to cope with stress, whereas drought mitigation is mediated by indirect AMF and PGPR beneﬁts and increased water uptake. An experiment was carried out to demonstrate the interactive effects of AMF ( Glomus mosseae ) alone or in association with PGPR ( Pseudomonas ﬂuorescens ) under water-stressed conditions in order to assess their biofertilizer efﬁciency. Accordingly, various morphological and biochemical parameters were studied, and the results suggested that all the co-inoculation treatments displayed beneﬁcial effects. Still, the combination of G. mosseae + P. ﬂuorescens showed the maximum increment in all the parameters considered, i.e., plant height and weight, leaves length and width, number of leaves per plant, speciﬁc leaf weight, relative leaf water content (RLWC), photosynthetic efﬁciency, seed length, width, and area, seed yield per plant, number of seeds per ﬂower, days to 50% ﬂowering, days to maturity, ﬂower and head diameter, harvest index, oil content, fatty acid composition (palmitic acid, oleic acid, stearic acid, and linoleic acid), and total yield. The improvement in different parameters may be attributed to the increased availability of nutrients due to the symbiotic association of AMF and PGPR with plant roots along with enhanced root structures for more water absorption under stressed conditions. Therefore, the results suggested that they offer a promising bio-control strategy for crop protection as biofertilizers combined in one formulation.


Introduction
Sunflower is one of the most valuable sources of edible oils known for its monounsaturated fats. The oil content of about 36-52% and protein content of 28-32% are found in sunflower seeds [1]. It also contains vitamins A, D, E, and K, which renders them beneficial in averting heart diseases [2,3]. Therefore, to enhance the productivity of this important oilseed crop, various biotic or abiotic factors that could affect its growth and development should be considered carefully. Drought (or water deficit) stress is one of these limiting abiotic factors that has received a lot of attention because it is the most likely constraint for agricultural productivity [4]. Drought happens when the plant's water requirements are not fully met, and this occurs when the amount of transpired water exceeds the amount of water taken up by the roots, which is caused by insufficient precipitation, low groundwater levels, or water retention by soil particles [5,6]. Water scarcity causes stomatal closure, which reduces CO 2 influx, lowering photosynthetic activity and carbon partitioning [7], as well as plant productivity and agricultural produce. Further, increasing environmental problems triggered by chemical fertilizers have created the need to use biofertilizers for sustainable crop production [4]. In this context, plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) represent the two most important groups of beneficial rhizospheric micro-organisms which may act as biofertilizers [5]. The microorganisms capable of colonizing the rhizosphere may be categorized based on their way of interaction with plants, and particularly with roots, some act as pathogens while others produce beneficial effects [6,7].
The beneficial effects of PGPR and AMF on sunflower plants have been well-documented through many studies. Mycorrhizal fungi, such as Glomus mosseae, help in increasing water and mineral uptake by roots, subsequently improving the soil structure [8,9]. The other significant benefit of AMF includes protecting the host plant from pathogenic infections caused by various soil microbes such as bacteria, fungi, and nematodes [10,11]. This is because mycorrhizal fungi encourage various defense mechanisms, including competition for photosynthates with pathogenic microbes and the expression of disease-resistance genes [12,13]. They also stimulate the expression of antioxidant enzymes and reduce malondialdehyde (MDA) production, an indicator of plant-membrane damage by lipid peroxidation [3]. Arbuscular mycorrhizal fungi influence the functioning of the root system, affect plant growth at different levels, and help reduce fertilizer input in the agricultural system without any significant yield loss [14,15]. Moreover, numerous studies have suggested that the AMF application in sunflower augmented its growth parameters and seed yield, even under biotic and abiotic stress conditions, via complex interactions with the host plant [16,17].
Plant growth-promoting rhizobacteria influence plant growth either directly by stimulating plant metabolism or by generating phytohormones such as auxins and gibberellins, solubilizing minerals, fixing atmospheric-nitrogen, or indirectly by mitigating detrimental effects of phytopathogenic microbes and improving stress tolerance. They also affect plant growth by increasing the availability of nutrients and reducing incidences of pathogenic infestation [18,19]. Further, inoculation with P. fluorescens may also accelerate seed germination, improve the emergence of seedlings, respond to various external stress factors, and enhance root structures [20,21]. Due to these properties, various inoculants of PGPR that tend to promote plant growth via one of the mechanisms (i.e., suppression of diseases in plants, improved absorption of nutrients, or production of phytohormones) are being commercialized. One such bacterial species is Pseudomonas which can be efficiently used as a bio-control agent for plants. The present study, therefore, aims to investigate the beneficial effects of AMF (G. mosseae) inoculation alone and in combination with PGPR (P. fluorescens) on the biochemical and morphological characteristics of sunflower and their possible use as biofertilizers.

Results
Significant variations were observed among all the treatments (<0.01) for all the characteristics analysed in the present study, except for harvest index and specific leaf weight ( Table 1). The effects of G. mosseae and P. fluorescens inoculation on different morphological and biochemical characteristics of sunflower were investigated and recorded in Tables 2 and 3. Although all the treatments produced significant results, T6 was found to be the most efficient in enhancing the quality, as well as quantity, of the yield.   Means ± SD followed by the same letters are not significantly different at p < 0.05 (Newman-Keuls test).

Discussion
Results of the study as recorded in Table 2 revealed that AMF (G. mosseae) and PGPR (P. fluorescens) inoculation presented significant increments in all the growth parameters of sunflower plants. This may be attributed to the fact that these bioinoculants led to an increase in the surface area of roots, subsequently enhancing their water and mineral absorption efficiency, particularly slow dissolving mineral ions such as phosphorus, and resulting in better plant growth under water stress [22,23]. The reason might be the organic acids produced by plant growth-promoting micro-organisms, chelating the phosphatebound cations, and increasing phosphorus accessibility in the soil [24,25]. The mycorrhiza hyphae extension can penetrate the soil pores which are inaccessible to root hairs subsequently absorbing more water than non-mycorrhizal plants. The improvements in plant growth parameters after AMF and PGPR inoculation were further supported by earlier experiments on sesame [26] and mustard [27,28].
In general, AMF inoculated sunflower plants possess larger leaves than non-inoculated plants as shown in Table 2. The photosynthetic rate associated with the chlorophyll content may be responsible for augmented leaf characteristics (leaf length and width), an increase in the number of leaves, and specific leaf weight [16,29]. Enhanced rate of water uptake, aided by AMF such as G. mosseae through upgraded hydraulic conductivity helps in increasing photosynthetic efficiency and consequently leaf area and leaf conductance [30,31]. These observations confirm the findings of other researchers, which showed an increased number of leaves per plant after AMF and PGPR inoculation in sunflower [32,33]. We observed the positive effects of G. mosseae and P. fluorescens inoculation on seed characteristics as shown in Table 2, which were also in agreement with the analysis of Arif et al. [34] which revealed that the inoculated sunflower plants had a significantly higher number of seeds per flower in addition to improved structural features, i.e., length, width, and area of seed [34]. AMF also interacts with Rhizobacteria, which promotes plant growth. As PSB solubilizes sparingly accessible phosphorous compounds into orthophosphate, which AMF may absorb and transport to the host plant, AMF and phosphate solubilizing bacteria (PSB) could interact synergistically and improve plant health and productivity. Further, the study also indicated a significant increase in the yield of sunflower seeds. This may be explained by the fact that mycorrhizae symbiosis develops the photosynthetic source for plants through increased leaf area so that the crops might lead to a greater yield of seeds [35,36].
Furthermore, we also observed the augmented flower characteristics (flower diameter and head diameter) of sunflower after application of G. mosseae and P. fluorescens. However, the G. mosseae and P. fluorescens inoculations did not produce many variations in the number of days to 50% flowering and number of days to maturity ( Table 2). The large size of flowers in AMF treated sunflower plants (which was again due to enhanced photosynthetic rate and increased photosynthate accumulation) subsequently leads to an increase in plant weight, [29,37] also observed in this investigation. The findings of other researchers also suggested a larger head diameter in AMF inoculated sunflower plants than in non-inoculated plants [33,38]. The increase in flower head diameter and the corresponding increase in plant weight, along with the increase in plant height can be ascribed to the improved uptake of some nutrients such as boron through root colonization with AMF. It should be noted that sunflower is considered sensitive to a lower boron supply, and deficiency may hinder its vegetative growth [39]. According to different studies, plant height, inter-nodal length, and flower head diameter increased significantly in sunflower plants with sufficient boron nutrition as compared to the control [40,41]. However, further investigations are required to confirm the significance of boron nutrition in other plant growth parameters. The increase in total yield, as per our observations, might also have resulted from improved root structure in inoculated sunflower plants leading to more water and nutrient absorption. The increase in the total yield of sunflower observed after AMF and PGPR application has been supported by numerous other studies on various crops such as switchgrass (Panicum virgatum) [42] and maize (Zea mays) [43,44].
Arbuscular mycorrhizal fungi pre-treated sunflower plants displayed more water absorption over other solutions of sugar, salt, kinetin, and salicylic acid [45]. Mechanisms significant for drought resistance in plants include reduced loss of water via transpiration, maintenance of membrane stability, and regulated osmotic adjustment. Application of G. mosseae enhances the root hydraulic conductivity at a low water potential of soil, which eventually influences the rate of transpiration, the resistance of leaves, and crop yield [46]. The increase in RLWC after G. mosseae inoculation also suggested more osmotic adjustment in the roots of inoculated plants at a low-water potential. Arbuscular mycorrhizal fungi are known to stimulate plant growth and produce plant hormones such as auxin, cytokinin, and gibberellin [47,48]. Furthermore, PGPR can significantly affect plant growth through increased nitrogen uptake, phytohormones synthesis, and mineral dissolution [49]. Inoculation with both AMF and PGPR thereby increased spore abundance, root colonization rate, and phosphorus content in tissues of sunflower at later stages of experiment [47,50,51]. Nevertheless, AMF species differ in the extent of benefits conferred by them on the development of host plants [36].
The higher colonization rates of G. mosseae, irrespective of water content in the soil, suggested greater adaptability of this species to native soils even under drought conditions. [52]. The present study also reported an increase in oil content under the influence of both G. mosseae and P. fluorescens. Any factor which is known to augment the biomass will improve the grain yield and eventually the oil yield owing to a higher rate of photosynthesis and increased photosynthates generation [53]. Majeed et al. [54] reported a substantial increase in the oil content of sunflower with the help of PGPR-based fertilizers [54]. Moreover, the fatty acid composition (palmitic acid, stearic acid, oleic acid, linoleic acid) of seed oil was also found to be enhanced significantly, as shown in Table 3. Owing to the beneficial effects of these bioinoculants on sunflower plants, it may be concluded that G. mosseae and P. fluorescens provide a promising strategy to upgrade their structural and biochemical characteristics with low chemical input and result in a better crop yield.

Planting Material and Experimental Details
The experiment used a randomized complete block design with five replicates of each treatment to compare the findings using the popular hybrid PSH-996 (Punjab Agricultural University, Ludhiana, India). Polyhouse used in this study was settled in, Jharkhand, India (24 • 22 18 N, 86 • 19 27 E). There were 10 pots of every treatment. The soil was obtained from the experimental site and mixed in a 3:1 ratio with sand (soil:sand). Further, this mixture was sieved through a 2 mm sieve and autoclaved for two days at 121 • C for two h. Sunflower seeds were surface sterilized and grown in earthen pots in a polyhouse setting. Each pot was administered 10% of G. mosseae inoculum, either alone or in combination with P. fluorescens. The effect of these bio inoculants on various growth parameters of sunflower was determined under limited water conditions. The performance under reduced irrigations was determined by withholding irrigation during anthesis and soft dough crop growth stages [55]. Under normal conditions, six irrigations were applied during the crop season as suggested by Punjab Agricultural University, Ludhiana for a successful sunflower production under Northern Indian plains. Whereas in the treatments screened for water stress, only two irrigation schedules were performed during the crop season to generate a water stress environment as described in our previous work [55]. Hoagland's nutrient solution without P (100 mL/pot) was applied after every 15th day of transplantation [56]. A package of practices was followed for various agronomical practices as defined elsewhere [57].

Plant Harvest and Analysis
Plants were harvested after 120 days of inoculation (DOI), and the effect of bioinoculants on various growth parameters was reported.

Morphological Characteristics
Plant height (cm) was recorded after 120 DOI by a 1 m scale from ground level to the head attachment of the plant (after attaining physiological maturity), and plant weight (kg) was measured using the weighing balance. Leaf length (cm) and leaf width (cm) were determined using a centimetre scale, and the number of leaves (per plant) was calculated as the average number of leaves at the stage of maturity. Specific leaf weight (g) (SLW) was measured as the total weight of the leaves divided by the number of plant leaves. The head diameter (cm) and flower diameter (cm) were recorded as the distance between two ends of the head and flower, respectively, at the time of maturity. The seed length (mm), seed width (mm), and seed area (mm 2 ) were measured after harvesting the crop, and the seed yield per plant (g) was determined as the average weight of the harvested seeds. The number of seeds per flower was determined by calculating the average value of the total number of seeds. Days to 50% flowering were counted from the sowing date until the opening of approximately 50% of the flower buds into flowers. Similarly, days to maturity were recorded from the sowing date until complete maturity (when the heads' backside turned brown). The harvest index (HI) was predicted at maturity using the formula HI = 100 × seed yield/total biomass (seed yield + vegetative mass).

Biochemical Characteristics and Root Colonization (%)
The relative leaf water content (RLWC) was estimated using 100 mg fresh weight of leaf discs, kept in distilled water till saturation. The leaf discs were taken out after 6 h, then water was blotted off from the surface of the discs without any pressure. The leaf discs were then weighed carefully to obtain a saturated weight. Finally, the dry weight was estimated after drying the leaf discs for 72 h at 70 • C. RLWC was then calculated using the formula, RLWC = 100 × ((fresh weight − dry weight)/(saturated weight − dry weight)). The photosynthetic efficiency of leaves was measured using a soil plant analysis development (SPAD) meter as SPAD reading (SPAD 502 Plus Chlorophyll Meter, Minolta, Japan). The oil content (%) of sunflower seeds was determined with the help of a nuclear magnetic resonance (NMR) analyzer (Newport Analyzer MK III A, Newport Instruments Ltd., Milton Keynes, England). The standardization of NMR was performed with 4 g seeds of known oil content. The seed samples were cleaned and dried for 3 h at 11 • C in an oven and a representative sample of 2 g was then employed for oil content estimation. The oil content in each seed was calculated using the formula: CONSTANT = NMR reading of calibration standard/(weight of seed) × (oil content of the standard, by extraction) and % OIL = NMR reading of seed/(weight of dry sample) × (constant). The fatty acid composition (palmitic acid, stearic acid, oleic acid, linoleic acid) of sunflower oil was further determined using near-infrared (NIR) spectroscopy. The sunflower seeds were husked manually and their NIR spectra were scanned between 1100 to 2500 nm according to methods proposed by Sato et al. [58]. In order to assess the amount of root colonisation (percentage), the Phillips and Hayman staining technique [59] was used first, followed by the 'Giovannetti and Mosse' [60] method, both of which were carried out using a Lab Digital Trinocular Compound LED Microscope (Omax 40X-2500X, Kent, WA, USA). In order to calculate the proportion of AMF infected root segments, the following formula was used: 100/(number of root segments colonised/total number of root segments).

Data Analysis
Statgraphics Centurion XVI software was used for data analysis testing (StatPoint Technologies, Warrenton, VA, USA). A Newman-Keuls multiple-range test was used to statistically examine the findings, which were represented as mean values ± standard deviation. The variance analysis and the Newman-Keuls multiple-range test were used to compute and statistically analyse the data acquired. In all cases, unless otherwise indicated, statistical significance was denoted by the value of p < 0.05.

Conclusions
Drought is a widespread adverse limiting factor that affects plant growth, physiology, and metabolism in different ways. Drought stress has an impact on the morphological, physiological, biochemical, and metabolic pathways of plants, resulting in a decrease in plant productivity. AMF and PGPR are vital in enhancing plant response to biotic and abiotic challenges, as well as reducing the consequences of these stresses on plants. Their role in enhancing plant growth and output, disease resistance, biotic and abiotic tolerance, and reducing the use of harmful pesticides and industrial fertilizers is an environmentally acceptable approach for reducing the use of pesticides and industrial fertilizers. Inoculation with mycorrhizal fungi and growth-promoting rhizobacteria can be considered efficient in augmenting growth parameters and total crop yield. Arbuscular mycorrhizal fungi promote plant growth and crop yield by supplying the nutrients essential for the plants by developing an extra-radical hyphae network that increases the soil volume used by plant roots. Further, AMF root colonization helps in the absorption of nutrients and water from the soil, biocontrol of pathogens, production of plant hormones, and settlement of plantlets in unfavorable conditions.
Our experiment on sunflowers demonstrated distinct improvements in the quality and quantity attributes of almost all the plant parts including leaves, flowers, and seeds. All the morphological and biochemical parameters produced favorable results under integrated AMF and PGPR treatment, reflecting their efficiency to be used as biofertilizers. Further, it may be observed that bio-inoculation could be of much significance in water-stressed soils. The exogenic application of G. mosseae alone or with P. fluorescens alleviates the adverse effects of water stress on the sunflower yield and physiology. Moreover, inoculation of the studied micro-organisms G. mosseae and P. fluorescens are economically viable as well as sustainable, thus, they can be applied in fields for better growth and yield. Thus, we conclude that AMF and PGPR can possibly increase sunflower productivity while mitigating environmental degradation simultaneously.