Response of Capsicum annuum L. Seedlings Raised in Pro Trays to Inoculation with AM Fungus Glomus bagyarajii and K Solubilizing Bacterium Frateuria aurantia

Abstract

Raising vegetable seedlings in pro trays is becoming an innovative approach to producing quality seedlings in horticulture. The present investigation was conducted to evaluate the effect of arbuscular mycorrhizal fungus (AMF) Glomus bagyarajii and potassium (K) solubilizing bacterium Frateuria aurantia singly and together in enhancing the growth of capsicum seedlings raised in pro trays under polyhouse conditions. Different growth parameters such as shoot and root length, total seedling length, stem diameter, dry weight of seedlings, biovolume index, plant strength, vigour index, macro and micronutrient uptake, mycorrhizal root colonization, and the population of F. aurantia in the root zone soil were monitored. Significantly higher shoot length, root length, stem diameter, and biovolume index were recorded in the treatments inoculated with G. bagyarajii alone, followed by F. aurantia alone. Most of the plant growth parameters were significantly less in the dual inoculated treatment with G. bagyarajii + F. aurantia compared to single inoculation with either of them. This brings out the negative influence of the two inoculants on each other leading to a reduced effect on plant growth.

1. Introduction

Sustainable agriculture aims to maintain soil fertility for a long time and achieve optimized yield using low input [1]. It recommends reduced chemical fertilizer application and increased organic inputs for long-term crop production, which benefits the environment and reduces the cost of cultivation [2]. It also increases biodiversity by providing a healthy environment for the organisms to live [3]. Soil harbours a large population of microorganisms. The highest concentration of microbial population is around the roots, i.e., the rhizosphere region [4]. This is due particularly to the presence of sugars, amino acids, organic acids, etc., in the root exudates [5,6]. Microorganisms such as arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) colonize the rhizosphere to form a beneficial association with the root system of plants. This association aids plant growth by increasing its ability to take nutrients from the soil. Biofertilizers developed from microorganisms such as PGPR and AMF are less expensive and environmentally friendly compared to chemical fertilizers and pesticides and thus promoting sustainable agriculture [1,7,8,9].

AMF form a symbiotic association with nearly 80% of plants, and inoculation with AMF improving plant growth under phosphorus (P) deficient conditions is well documented [1]. They facilitate the uptake of diffusion-limited nutrients and protect the plants against biotic and abiotic stresses in exchange for photosynthates of the plant [10,11]. It also has synergistic interactions with other beneficial soil microorganisms, thereby enhancing plant growth [1]. PGPR are the rhizosphere bacteria that can enhance plant growth by a wide variety of mechanisms such as biological nitrogen fixation, phytohormone production, P, and potassium (K) solubilization, etc. [12].

Potassium solubilizing bacteria (KSB) viz. F. aurantia are PGPR which are effective in releasing potassium from potassium-bearing minerals through the production of organic acids such as tartaric acid, citric acid, succinic acid, α-ketogluconic acid, and oxalic acid, and thus solubilizing the insoluble potassium to soluble forms of potassium, which can be easily and readily taken up by the plants [13]. Usually, 90–98% of the total potassium in soil occurs in the form of mineral potassium, and most of this is unavailable for plant uptake [11]. K is an essential element required for plant growth. It is required to activate numerous enzymes involved in various processes such as sugar degradation, energy metabolism, and starch synthesis [14,15].

Capsicum (Capsicum annuum L.), also known as bell pepper or chilli pepper, belongs to the family Solanaceae. It is a popular vegetable crop which is native to the tropical and subtropical regions of America, and is presently cultivated all over the world. Capsicum is known for its high nutritional content. It is high in vitamins C, E, and A, a good range of minerals, phytochemicals, and dietary fibre, which play an important role in decreasing human micronutrient deficiencies [16,17]. The phytochemicals present in capsicum also have many industrial applications [18]. India contributes to one-fourth of the world’s production of capsicum, with an average annual production of 518,000 metric tons from an area of 24,000 ha, with a productivity of 1266 kg per ha [19]. Cultivation of capsicum is highly encouraged in peri-urban regions because of easy access to urban markets.

There are numerous methods for the application of beneficial microbes for improving plant growth [20]. The pro tray nursery is a recent technology widely gaining popularity for quality seedling production. Such seedlings have an independent area for each seedling; hence, improved seed germination, better root development, easy handling, cheaper transportation, and better establishment of the crop when transplanted in the main field [21,22]. The objective of the current work was to evaluate the effect of the AMF G. bagyarajii and the KSB F. aurantia inoculated singly and together on the growth of capsicum seedlings raised in pro trays under polyhouse conditions.

2. Materials and Methods

The polyhouse experiment was conducted at the Centre for Natural Biological Resources and Community Development (CNBRCD), Bangalore, India. The capsicum seeds of Arka Mohini, a F₁ hybrid, commonly used by farmers in India, were obtained from the Indian Institute of Horticultural Research (IIHR), Bangalore, and used in the present study.

2.1. Inoculum Preparation

Pure culture of F. aurantia maintained at CNBRCD was used in the experiment. The culture was inoculated in a Glucose Yeast extract Calcium Carbonate (GYCC) broth and was incubated for 24 h. The culture suspension was centrifuged at 4 °C for 10 min at 10,000 rpm and the centrifuged pellet was mixed in an appropriate amount of phosphate buffer. This culture buffer suspension was used as inoculum to inoculate seedlings in pro trays. The CFU of the KSB F. aurantia inoculum was determined by serial dilution and the spread plate method technique [23]. G. bagyarajii used in the study was maintained at CNBRCD culture collection in the polyhouse using Chloris gayana (Rhodes grass) as the host, using vermiculite, perlite, and soilrite as the substrate, in the ratio 3:1:1 (v/v/v). Seventy-five days after sowing, the shoots of Rhodes grass was harvested and the roots were cut into 1 cm length, mixed with the grown substrate and air-dried. This air-dried mixture was used as inoculum to inoculate in pro trays. The number of infective propagules of the inoculum was estimated through the most probable number (MPN) method [24].

2.2. Pro Tray Inoculation and Growth Condition

The capsicum seedlings were raised in one hundred cells of two pro trays (each with 50 cells) for each treatment holding 20 g substrate per cell. Each cell was filled with 18.5 g of the vermiculite, soilrite and perlite mixture, and 1.5 g of compost. The experiment had four treatments viz. T1—Uninoculated control; T2—Treated with KSB F. aurantia; T3—Treated with AMF G. bagyarajii, and T4—Treated with F. aurantia + G. bagyarajii. The experimental design used for the study was a randomized complete block design (RCBD). A depression was made in each cell and 0.8 g of the G. bagyarajii inoculum containing 7000 IP/g and/or 2 mL of F. aurantia containing 9 × 10⁵ CFU/mL were added depending on the treatment. Uninoculated pro trays with no inoculation received autoclaved GYCC broth at the rate of 2 mL/cell, and washings of AMF inoculum suspension passed through a 45 µm sieve which contained associated microorganisms but not AMF propagules at the rate of 2 mL/cell. Two seeds were sown to each cell of the pro trays and thinned to one after germination. Ruakura nutrient solution (5 mL) with P [25] was applied to each cell on the tenth day after sowing, and thereafter 5 mL of the same nutrient solution without P was applied once in 7 days to all the cells. The plants were maintained in a polyhouse and watered regularly to maintain the optimum moisture status in the soil.

2.3. Parameters Evaluated

The seedlings were harvested 55 days after sowing (DAS). Various plant parameters such as shoot, root, seedling length, and stem diameter of the seedlings were measured. The biovolume index (BI) was calculated using the formula given by Hatchell et al. [26], and the seedling vigour index and plant strength were calculated using the standard formula [27,28].

$$Bio volume index=plant height \left(cm\right)\times Stem girth \left(mm\right)$$

$$Seedling vigour=Germination percentage\times seedling length \left(cm\right)$$

$$Plant stength=\frac{Dry weight of plant \left(g\right)}{Height of plant \left(cm\right)}$$

The dry weight of the seedlings was determined after drying in an oven at 60 °C to a constant weight. The dried seedlings were powdered, and the nitrogen (N) concentrations of the shoot and root were estimated following the micro-Kjeldahl method [29]. The phosphorus concentration was determined by the vanadomolybdate phosphoric yellow colour method [30]. The potassium concentration was estimated by the flame photometer method [31]. The micronutrient contents were determined using an atomic absorption spectrophotometer with a hollow cathode lamp set to standard wavelengths [32]. Roots were cut into 1 cm size, the root bits were stained using trypan blue as described by Philips and Hayman [33], and the per cent mycorrhizal root colonization was estimated by adopting the gridline intersect method [34]. The rhizosphere population of F. aurantia was enumerated by the standard dilution plating technique [35] using GYCC agar.

The results were analyzed using ANOVA with the help of the software Web Agri Stat Package 2.0 https://ccari.icar.gov.in/wasp2.0/index.php (accessed on 22 October 2022) and the means were compared by Duncan’s multiple range test at 5% level.

3. Results and Discussion

There was a significant increase in shoot and root length, stem diameter, and BI of capsicum seedlings treated with microbial inoculants compared to uninoculated seedlings (Figure 1). Shoot length was the highest in G. bagyarajii treated seedlings but statistically did not differ from F. aurantia treated seedlings. Seedlings inoculated with F. aurantia + G. bagyarajii had significantly lesser shoot and root length compared to seedlings inoculated singly with either of them. Uninoculated seedlings had the least shoot and root length. The BI followed a similar trend. Stem diameter was significantly higher in G. bagyarajii alone treatment followed by F. aurantia alone treatment. The least stem diameter was observed in seedlings inoculated with F. aurantia + G. bagyarajii and uninoculated seedlings both being statistically on par with each other (

Table 1. Influence of the microbial inoculants on shoot length, stem diameter, root length, and biovolume index of capsicum seedlings grown in pro trays 55 days after sowing.

Treatments	Shoot Length (cm)	Stem Diameter (mm)	Root Length (cm)	Biovolume Index
Uninoculated control	17.24 ± 1.22 c	2.64 ± 0.19 c	10.81 ± 0.76 c	45.51 ± 3.22 c
Inoculated with Frateuria aurantia (Fa)	19.8 ± 1.40 ab	2.82 ± 0.20 b	13.65 ± 0.97 ab	55.84 ± 3.95 a
Inoculated with Glomus bagyarajii (Gb)	20.46 ± 1.45 a	3.01 ± 0.21 a	14.38 ± 1.02 a	61.58 ± 4.35 ab
Inoculated with Fa + Gb	19.35 ± 1.37 b	2.64 ± 0.19 c	12.75 ± 0.90 b	51.08 ± 3.61 b
SEd	0.47	0.05	0.67	0.94
LSD (0.05)	0.93	0.11	1.36	1.86

Means in the column with same alphabets are not significantly different at p ≤ 0.05.

).

AMF enhancing plant growth is well known and documented [1,36,37]. The improved growth by AMF is mainly attributed to the uptake of diffusion-limited nutrients from soil. The other beneficial effects are their role in the biological control of root pathogens, phytohormone production, greater ability to withstand abiotic stress, and synergistic interaction with PGPR. Increased shoot length, root length, stem diameter, and BI in AMF-inoculated seedlings raised in pro trays has been reported earlier in other plants [36,37,38].

The seedling vigour index was significantly the highest in G. bagyarajii inoculated treatment, followed by seedlings inoculated with F. aurantia alone. The dry weight of seedlings was higher in G. bagyarajii inoculated treatment, followed by seedlings inoculated with F. aurantia alone and with F. aurantia + G. bagyarajii both not differing significantly (

Table 2. Influence of the microbial inoculants on seed vigour, plant strength, fresh weight, and dry weight of capsicum seedlings grown in pro trays 55 days after sowing.

Treatments	Seeding Vigour Index	Plant Strength/Seedling	Fresh Weight/Seedling (g)	Dry Weight/Seedling (g)
Uninoculated control	2167 ± 153.2 d	0.02	2.81 ± 0.20 b	0.46 ± 0.03 c
Inoculated with Frateuria aurantia (Fa)	2585 ± 182.8 b	0.02	3.21 ± 0.23 ab	0.52 ± 0.04 b
Inoculated with Glomus bagyarajii (Gb)	2692 ± 190.3 a	0.02	3.68 ± 0.24 a	0.55 ± 0.04 a
Inoculated with Fa + Gb	2481 ± 175.4 c	0.02	3.29 ± 0.21 a	0.50 ± 0.04 b
SEd	0.046	NS	0.26	0.003
LSD (0.05)	8.51		0.53	0.007

Means in the column with same alphabets are not significantly different at p ≤ 0.05; NS-Not significant.

). Uninoculated seedlings had the lowest dry weight. Inoculation with AMF improving plant dry weight has been reported in many crop plants [37,38]. G. bagyarajii enhancing plant growth has also been reported [39,40]. The various mechanisms by which AMF help in increasing plant growth and dry weight have been explained in the earlier paragraph. K being one of the major nutrients required for plant growth is present in an unavailable form in soils. KSB are effective in releasing these K bound to minerals in the soil through the production of various acids solubilizing the insoluble K to soluble forms which are taken up by the plants [41,42]. Inoculation with KSB improving the dry weight of several other plants has also been reported earlier [42,43]. In the present study, the differences in plant strength because of inoculation were not statistically significant (Table 2).

Regarding N concentration of seedlings, the maximum concentration was encountered in G. bagyarajii alone treatment, followed by F. aurantia alone treatment, followed by F. aurantia + G. bagyarajii treatment, with all three differing significantly. The least N concentration was recorded in uninoculated seedlings. The P concentration was highest in G. bagyarajii inoculated seedlings, followed by F. aurantia alone, and F. aurantia + G. bagyarajii inoculated seedlings, both being statistically on par, and the least P concentration was observed in uninoculated seedlings. P is a diffusion-limited nutrient. So, its uptake depends on the movement of these ions to the root surface and then the absorbing capacity of the root [44]. Increased uptake in G. bagyarajii inoculated seedlings is in conformity with earlier reports made on other plants [36,37]. K concentration was significantly higher in F. aurantia treatment, followed by G. bagyarajii alone, and F. aurantia + G. bagyarajii and uninoculated treatment, with all four treatments differing significantly from each other. The differences in the Ca and Mg concentrations in the different treatments were not statistically significant (

Table 3. Influence of microbial inoculants on uptake of macronutrients (%) by capsicum seedlings grown in pro trays 55 days after sowing.

Treatments	Nitrogen	Phosphorus	Potassium	Calcium	Magnesium
Uninoculated control	3.01 ± 0.21 d	0.66 ± 0.05 c	4.22 ± 0.30 d	0.78	0.33
Inoculated with Frateuria aurantia (Fa)	3.47 ± 0.25 b	0.72 ± 0.05 b	4.57 ± 0.32 a	0.83	0.37
Inoculated with Glomus bagyarajii (Gb)	3.82 ± 0.27 a	0.83 ± 0.06 a	4.45 ± 0.31 b	0.88	0.34
Inoculated with Fa + Gb	3.33 ± 0.24 c	0.72 ± 0.05 b	4.42 ± 0.31 c	0.81	0.40
SEd	0.007	0.009	0.009	NS	NS
LSD (0.05)	0.018	0.02	0.02

Means in the column with same alphabets are not significantly different at p ≤ 0.05. NS-Not significant.

).

The concentration of the micronutrients Zn, Mg, B, Mo, and Fe were highest in G. bagyarajii inoculated seedlings, followed by F. aurantia treatment, followed by F. aurantia + G. bagyarajii treatment, followed by uninoculated treatment, with all the treatments differing significantly from each other. The concentration of Cu was the highest in G. bagyarajii treatment, followed by F. aurantia treatment, with both not differing statistically. Seedlings dually inoculated with F. aurantia + G. bagyarajii had significantly less micronutrient uptake compared to seedlings singly inoculated. Higher uptake of micronutrients like Zn, B, Mo, and Fe in capsicum seedlings inoculated with AM fungi upholds the observations made earlier on other plants [45]. KSB improving K and micronutrient uptake has also been reported [41,42,43]. Dual inoculation of AMF + PGPR, such as N-fixers and P-solubilizers enhancing the uptake of micronutrients has been reported in many plants [38,44,45]. But in the present study, dual inoculation with F. aurantia + G. bagyarajii resulted in a reduced uptake of Zn, B, Mn, Fe, and Cu (

Table 4. Influence of microbial inoculants on uptake of micronutrients (ppm) by capsicum seedlings grown in pro trays 55 days after sowing.

Treatments	Zinc	Copper	Manganese	Boron	Molybdenum	Iron
Uninoculated control	51.78 ± 3.7 d	16.84 ± 1.19 c	47.47 ± 3.36 c	36.69 ± 2.6 d	67.45 ± 4.8 d	2622 ± 185.4 d
Inoculated with Frateuria aurantia (Fa)	54.68 ± 3.9 b	18.56 ± 1.31 a	53.49 ± 3.37 b	41.89 ± 3.0 b	73.55 ± 5.2 b	2922 ± 206.6 b
Inoculated with Glomus bagyarajii (Gb)	56.97 ± 4.0 a	18.84 ± 1.33 a	56.68 ± 4.01 a	46.40 ± 3.3 a	86.27 ± 6.1 a	3468 ± 245.2 a
Inoculated with Fa + Gb	53.23 ± 3.8 c	17.24 ± 1.22 b	53.76 ± 3.80 b	38.86 ± 2.8 c	69.66 ± 5.0 c	2717 ± 192.1 c
SEd	0.17	0.12	0.14	0.20	0.20	13.23
LSD (0.05)	0.4	0.29	0.33	0.4	0.48	30.53

Means in the column with same alphabets are not significantly different at p ≤ 0.05.

). Though it is strange, it agrees with the observations made on tomatoes with dual inoculation with the AMF Funneliformis mosseae and the KSB Bacillus mucilaginosus [43].

The per cent mycorrhizal root colonization was significantly higher in G. bagyarajii alone and F. aurantia + G. bagyarajii treatments, with both being statistically on par, followed by F. aurantia alone treatment (

Table 5. Influence of the microbial inoculants on the percent mycorrhizal colonization of Glomus bagyarajii and microbial load of Frateuria aurantia in rhizosphere of capsicum seedlings grown in pro trays 55 days after sowing.

Treatments	Percent Mycorrhizal Colonization (%)	Bacterial CFU (×105 per mL)
Uninoculated control	20 ± 1.41 c	0 ± 0.35 c
Inoculated with Frateuria aurantia (Fa)	30 ± 2.12 b	16 ± 1.33 a
Inoculated with Glomus bagyarajii (Gb)	96 ± 6.79 a	0 ± 0.35 c
Inoculated with Fa + Gb	94 ± 6.65 a	5 ± 0.35 b
SEd	3.5	0.3
LSD (0.05)	7.4	0.6

Means in the column with same alphabets are not significantly different at p ≤ 0.05.

). Uninoculated seedlings had the least mycorrhizal root colonization. Significantly higher mycorrhizal root colonization in G. bagyarajii alone and F. aurantia + G. bagyarajii treatments compared to uninoculated plants indicate the better proliferating ability of G. bagyarajii with capsicum as the host [45]. The highest CFU of F. aurantia was recorded in F. aurantia alone treatment followed by F. aurantia + G. bagyarajii treatment, with both differing significantly. F. aurantia colonies could not be encountered in uninoculated and G. bagyarajii alone inoculated treatments. Higher CFU of F. aurantia in the root zone of capsicum inoculated with this KSB brings out the ability of the bacterium to establish in the rhizosphere.

Significantly lower plant growth, dry weight, seedling vigour, and macro and micro-nutrients uptake in dual inoculated treatment revealed the negative interaction between the two inoculants. Earlier, Walker et al. [46] reported that the co-inoculated treatments involving Glomus + Azospirillum + Pseudomonas did not show positive effects on maize seedlings compared to their single inoculation. The result of the present study brings out that the AMF G. bagyarajii and KSB F. aurantia are not compatible. Another recent study also reported incompatibility between the AMF F. mosseae and the KSB B. mucilaginosus [40]. This suggests warranting more investigations between different AMF and KSB to uphold such negative interaction.

4. Conclusions

The synergistic interaction between AMF and PGPR is well documented. The result of the present study brings out that single inoculation with the AMF G. bagyarajii or the KSB F. aurantia promoted better plant growth compared to dual inoculation with both organisms. This brings out the negative effect of the two inoculants on each other, leading to a reduced effect on plant growth. Negative interaction between AMF and PGPR is rare and unusual. Detailed studies are required to understand the mechanisms involved in such negative interaction.