Native Rhizospheric Microbes Mediated Management of Biotic Stress and Growth Promotion of Tomato

Abstract

The incidence of biotic perturbation in plants has been amplified due to increased resistance and the resurgence of pathogens. To mitigate stress and promote food production, agrochemicals are being used boundlessly and they have augmented the problem of disease re-occurrence and agroecosystem degradation. With the perception of urgency to reduce biotic stress sustainably, the present study was undertaken. Four native rhizospheric microbes: Trichoderma lixii, T. brevicompactum and two strains of Bacillus subtilis, were evaluated for their antagonistic potential toward soil-borne and foliar pathogens of tomato under pot conditions. The data obtained revealed T. lixii as the most effective isolate, which substantially reduced the disease severity and promoted plant growth. In two consecutive pot experiments, T. lixii was observed to reduce the fusarium wilt and early blight severity by 32% and 31%; and 30% and 25%, respectively, compared to the untreated control. Moreover, T. lixii was reported to colonize the plant roots, which was evident from the result obtained for biofilm formation and spores colonization on root cells. TvR1 also improved the photosynthetic content of both infected and non-infected plants. The conclusion drawn from the result suggested that the native microbial rhizospheric isolate T. lixii was effective in ameliorating the biotic stress, which might be due to root colonizing ability, and therefore, it could be designed into a bioinoculant for green agriculture.

1. Introduction

Biotic stress is one of the major constraints facing the agriculture sector. The increased incidence of pathogenic infections in plants has reduced crop yields and lowered their nutritional content. These outcomes have aggravated the problem of food scarcity and malnutrition worldwide. To ameliorate the biotic stress, chemical pesticides are constantly being used in ample quantities, which have eventually abridged plant growth, their photosynthetic efficiency and yield, induced ecosystem degradation, and increased pathogen resistance [1,2]. Moreover, the resurgence of pathogenic diseases due to climate change has escalated the incidence of biotic stress [3].

Tomato (Lycopersicon esculentum Mill.) is the second most cultivated crop, which is widely grown due to its rich nutritional and antioxidant content [4,5,6]. Tomato plants have been documented to be affected severely by the biotic stresses triggered by bacteria, fungi, viruses and nematodes [7,8]. Fusarium oxysporum f. sp. lycopersici (Fol) and Alternaria solani are two devastating fungal pathogens that affect tomato and cause fusarium wilt and early blight, respectively [4,8]. Fol is a soil-borne pathogen that affects the plant’s vascular system [9], whereas A. solani is a foliar pathogen that causes distress in the above-ground parts [7,10]. Fol causes the browning of the vascular tissues, resulting in wilting, stunted plant growth, and plant death in condition of severe infection [9]. A. solani infection produces necrotic lesions on the leaves in the form of concentric rings, which result in defoliation and lower productivity due to poor fruit quality [6,10]. The infestation of A. solani has been reported to induce a fruit yield loss of 35–78% [7].

The management of fusarium wilt and early blight is based on the use of resistant cultivars and/or toxic chemical fungicides [5,11,12,13]. The extensive application of chemical fungicides can alter the health and functioning of the ecosystem by developing pathogen resistance, promoting the recurrence of secondary phytopathogens, inducing environmental pollution and increasing contamination in the food chain due to toxic residues that pose a risk to human health and biodiversity [5,11,12,13,14,15]. To manage biotic stress sustainably, the adoption of alternative approaches, such as using agriculturally important microorganisms (AIMs) in fields, is needed [16]. AIMs are beneficial microorganisms that generally dwell in the rhizosphere and facilitate the mitigation of abiotic/biotic stress in plants via mechanisms such as antibiosis, mycoparasitism, induced systemic resistance (ISR), the solubilization of micronutrients, and others [17,18,19,20]. These microorganisms induce disease-suppressing and plant-growth-promoting mechanisms -after successfully colonizing the plant roots [17,18,19,20,21,22,23]. Effective root colonization aids microbes in inducing ISR efficiently and, thus, help them to render pathogens, including those that infect plants at spatially different locations [17,22,23,24]. Trichoderma, Pseudomonas, Bacillus, and arbuscular mycorrhizal fungi (AMF) are some microbial isolates that have the potential to abridge plant stress [12,19,25,26,27,28,29,30]. For instance, the application of compost containing Bacillus subtilis QST713 and Trichoderma sp. TW2 reduced the abundance of Fol and lowered disease severity [31]. The inoculation of plants with AIMs improves plant growth and their biochemical properties as well as changes microbial communities in the rhizosphere, which indirectly alleviates plant stress, particularly by alleviating the stress-induced production of reactive oxygen species (ROS), improving photosynthetic activity, and improving nutrient uptake [2,32,33]. For instance, the inoculation of alfalfa grown in copper-contaminated soil with plant-growth-promoting rhizobacteria (PGPR) Paenibacillus mucilaginosus and rhizobium Sinorhizobium meliloti, improved the nutritional content and plant growth, and reduced Cu-induced ROS generation as well as oxidative damage by enhancing the activity of antioxidants. The inoculation of PGPR along with rhizobium was found to improve soil biochemical properties, such as soil microbial biomass, nitrogen content, and others [34]. Similarly, the treatment of ginseng with Penicillium citrinum reduced the root rot disease of plants by inhibiting the growth of Fusarium oxysporum and significantly improved the community structure of the rhizosphere [35].

Several microbial isolates have been determined to have potential as effective biocontrol agents; however, in the field, their application is limited. Several factors reduce the action potential of these microorganisms, such as low consistency, narrow activity spectrum, inability to survive in localized environments, unable to compete with the existing microbiome, and poor shelf life [36,37,38]. To address these problems, the present study was conducted. The current work aims to evaluate the ability of native rhizospheric microbes to (1) survive and manage a broad range of pathogens, particularly those that have different sites of infection in the local environment; (2) promote plant-growth promotion; (3) show biocontrol consistency in the long run through two consecutive pot experiments; and (4) colonize roots effectively.

2. Materials and Methods

2.1. Microbial Cultures

Fungal pathogens Fol and A. solani were isolated from the diseased tomato plants collected from agriculture fields in Lucknow, Uttar Pradesh, India. The collected plants were thoroughly washed with sterilized distilled water (SDW) and cut into 1 cm long sections. The sections were surface sterilized with a 0.2% solution of sodium hypochlorite for 3 min, rinsed with SDW, and dried using tissue paper. The plant sections were maintained on potato dextrose agar (PDA) plates and incubated at 28 ± 2 °C. The colonies were identified on the basis of the morphological characteristics observed under the microscope [4,39] and a pathogenicity test. The pathogenicity tests for the Fol and A. solani isolates were performed by using the cup assay [40] and leaf detached [41] methods, respectively. The identified cultures of the pathogens were purified and stored on PDA slants at 4 °C for future use.

Four native microbial strains, TvR1, TbS2, BS6, and CS13 were isolated from the root surface of the tomato plants and the rhizospheric soil of tomato, bean, and chilli plants, respectively, which were collected from the agriculture field in Lucknow, Uttar Pradesh, India [42]. The isolated strains were identified at the molecular level as Trichoderma lixii TvR1 (accession number MF780730), T. brevicompactum TbS2 (accession number MF780729), Bacillus subtilis BS6 (accession number MF780728), and B. subtilis CS13 (accession number MF678835) and evaluated for biocontrol potential under in vitro conditions in a previous study [42]. In the present study, the isolated strains were assessed for their biocontrol and plant-growth-promoting activity under in vivo conditions.

2.2. Plant Growth Promoting Activities

The quantitative estimation of Indole-3-Acetic Acid (IAA) production by using biocontrol agents was performed using the method described by Brick et al. [43]. The bacterial and fungal isolates were grown on a minimal media containing 0.2 g/l L-tryptophan for 48 h and 7 days, respectively, and then centrifuged at 10,000 rpm for 10 min at 4 °C. After centrifugation, 2 mL of the supernatant was taken to which two drops of orthophosphoric acid and 2 mL of Salkowski reagent was added, and incubated for 30 min at 28 °C. The pink color that developed was estimated quantitatively by measuring the absorbance in a spectrophotometer at 530 nm. The quantity of IAA was calculated in µg/mL by using the standard curve.

Phosphate and zinc solubilization was screened on Pikovskaya agar media, as described by Gaur [44], and on media containing zinc oxide, as described by Saravanan et al. [45], respectively. For phosphate solubilization, the microbial isolates were incubated at 28 ± 2 °C for 4 days in Pikovskaya agar media. The clear halo zone that developed around the microbial colony indicated the solubilization of the phosphate. The phosphate solubilization index (PSI) was calculated according to the ratio of the total diameter to colony diameter (colony diameter + clear halo/colony diameter) [46]. Zinc solubilization was estimated by using the spot-inoculation of the microbial isolates on the respective media. The plates were incubated at 28 ± 2 °C for 48 h and then observed for the development of a clear zone around the colony, and the diameter was calculated.

2.3. Exopolysaccharides and Biofilm Study

Exopolysaccharide (EPS) production was assayed by inoculating the bacterial and fungal isolates for 48–72 h in a nutrient broth modified with tryptone soy broth. After the incubation, the broth was centrifuged at 10,000 rpm for 10 min, and the supernatant collected was mixed with an equal amount of chilled acetone and kept in the refrigerator overnight. The formation of precipitate indicated the presence of EPS. The ability of the microbial strains to form a biofilm was assessed by the following method described by Yadav and Sundari [47].

2.4. Seed Germination Assay

The effect of the microbial isolates on seed germination was assessed under in vitro conditions. The tomato seeds (Lycopersicon esculentum var. Pusa ruby) were purchased from the local market in Lucknow, Uttar Pradesh, India, and were surface sterilized by using 1% mercuric chloride (HgCl₂) and washed thrice with sterile distilled water. Few disinfected seeds were grown on agar plates and checked for the growth of microbes, if any, to ensure the proper sterilization. The seeds were individually coated with a conidial solution of T. lixii TvR1 and T. brevicompactum TbS2 @ 1 × 10⁶ spores/mL; and a cell suspension of B. subtilis BS6 and CS13 @ 1 × 10⁹ cfu with 1% carboxymethyl cellulose (CMC) as an adhesive for one hour. For control, the seeds were soaked in sterilized distilled water (SDW). The seeds were dried overnight at room temperature in laminar flow. Thirty seeds from each treatment were assessed, and the experiment was performed in three replicates. The disinfected and treated seeds were placed in Petri plates with a 100 mm diameter and lined with Whatman filter paper no. 1 and were moistened with SDW and kept for germination at 24 °C ± 2 under a 12/12 h day/night light cycle.

The germination was checked every 24 h for twenty days, and at the end of the experiment, the shoot and root lengths of all of the germinated seedlings were measured. The germination index and vigor index were calculated using the Formulas (1) and (2), as described by Thakkar and Saraf [48].

$$Germination percentage=\frac{Number of seeds germinated}{Total number of seeds}\times 100$$

(1)

$$Vigor index \left(VI\right)=Germination percentage\times mean of seedling length \left(\mathrm{cm}\right)$$

(2)

2.5. Pot Experiment: In Vivo Study

The pot experiment was conducted twice across two consecutive years, from December to March, under open field conditions. The first pot experiment was undertaken to assess the biocontrol potential of all four of the isolated microbial strains, and the second consecutive pot experiment was performed to evaluate the consistency of the results produced by the effective microbial isolate. Both of the pot experiments were performed in a similar manner; however, the first experiment was undertaken for 60 days after transplantation (DAT), and the second experiment was conducted for 90 DAT to study the impact of the effective microbial isolate on the fruit yield and quality.

The tomato seeds were disinfected with 1% HgCl₂ and then treated with microbial isolates, as described in the section describing the seed germination assay. The treated seeds were sown in a germination tray and transplanted in 15 cm diameter (8 kg capacity) pots after 28 days of sowing (DAS). Sandy loam soil was used in the tray and pot, which was sterilized at 121 °C and 15 psi for one hour for three consecutive days. To ensure the proper sterilization of the soil, the serial dilution method was used, and the soil was cultured on nutrient agar (NA) and PDA plates. The pH, bulk density, water-holding capacity, electrical conductivity, organic carbon, and available N, P, and K of the soil were 7.24, 1.14%, 39.9%, 0.729 dS/m, 1.2%, and 154.2 mg/kg, 11.33 mg/kg, and 132.81 mg/kg, respectively. In each pot, three seedlings were transplanted, and for each treatment, four replicates were used. In total, 12 plants per treatment were maintained and watered regularly. The mass multiplication of pathogen Fol was undertaken by following the method described by Sundaramoorthy and Balabasker [49] with some modifications. Fol was mass multiplied on sterilized wheat porridge for two weeks and then inoculated in soil @ 5 g/kg one week prior to transplantation for the establishment of the pathogen in the soil. In the case of A. solani, a spore suspension of 1 × 10⁵ spores/mL was prepared as per the method used by Attia et al. [4], and seven DAT 20 mL of the spore suspension was sprayed on the adaxial or abaxial surface of the leaves. The plants were covered with poly bags for 48 h to maintain the moisture and the development of disease incidence [50]. The experimental setup was a completely randomized design in replicates of four [12].

2.6. Scanning Electron Microscope (SEM) Analysis

To assess the root-colonization potential of the microbial isolate, the root section of the plants harvested 90 days after treatment (DAT) were examined using a Scanning Electron Microscope (SEM). The roots were washed thrice thoroughly with distilled water and prepared for SEM analysis, as described by Mycock and Berjek [51] with some modification. The tomato roots were cut into small pieces using a sharp blade and immediately fixed in 2.5% glutaraldehyde in a 0.1 M phosphate buffer of pH 7.0. The root sections were incubated at 4 °C for 6 h and then washed with buffer thrice. Each washing was performed for 15 min at 4 °C for the removal of the unreactive fixatives. Then, the sections were dehydrated for 20 min in each graded series of ethanol (30, 50, 70, 90, 95, and 100%). The sections were dried at critical point drying and then mounted on an aluminum stub with carbon tape. The samples were analyzed under SEM (model: Joel Quanta 250) at 2000×.

2.7. Percentage Disease Index (PDI)

The percentage of disease index (PDI) for Fusarium wilt and Early blight was calculated after 90 days of treatment (60 days after treatment in the case of the first pot experiment) as per the Formulas (3) and (4) described by Akkopru and Demir [52] and Wheeler [53], respectively, using the disease severity score given by Bora et al. [54] for Fusarium wilt and Horsfall and Barratt [55] with some modification, as described by Sahu et al. [56] for Early blight.

$$PDI=\frac{\Sigma \left(Disease score\times Number of plants rated\right) }{Total number of plants\times 4}\times 100$$

(3)

where 4 = highest disease score for the incidence of fusarium wilt

$$PDI=\frac{\Sigma \left(Disease score\times Number of leaves rated\right) }{Total number of leaves\times 5}\times 100$$

(4)

where 5 = highest disease score for the incidence of Early blight.

2.8. Photosynthetic Pigments

Photosynthetic pigments were estimated in the plants grown during the second experiment after 90 days of transplantation. Then, 0.5 gm of fresh leaves was homogenized in 10 mL of 80% chilled acetone in the dark and centrifuged at 5000 rpm at 10 °C for 15 min. The supernatant was taken, and the optical density was measured at wavelengths of 663 nm, 645 nm, and 480 nm with a spectrophotometer. For the estimation of the chlorophyll and carotenoid content, the formulas described by Maclachlan and Zalik [57] and Duxbury and Yentsch [58] were adopted, respectively. The obtained result was expressed as mg/g fresh weight.

2.9. Statistical Analysis

The data were statistically tested to identify significant treatment by using one-way Analysis of Variance (ANOVA) followed by a DUNCAN test at a significance level of 5% (p ≤ 0.05) using SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA) [59]. The mean and the standard error (S.E.) were calculated using Microsoft Excel 2007.

3. Results

3.1. Plant Growth Promoting, EPS Production and Biofilm Activity

The results of the quantitative estimation of Indole-3-acetic acid production and solubilization index of mineral (phosphate and zinc) for the microbial isolates are presented in

Table 1. Quantitative estimation of IAA and solubilization index value for inorganic minerals (phosphate and zinc) and ability to produce EPS and biofilm.

Treatments	Phosphate Solubilization Index (PSI Value)	IAA (μg/mL)	Zinc Solubilization Index (ZSI Value)	EPS Production	Biofilm Formation
Control #	-	-	-	-	-
BS6	-	-	-	-	-
CS13	-	-	-	-	-
TvR1	-	1.82 ± 0.01 b	1.62 ± 0.02 b	+	+
TbS2	-	1.28 ± 0.03 a	1.44 ± 0.01 a	+	+

^# = treated only with distilled water; Values are mean of triplicate; ± Standard error; values followed by different letters are significantly different according to DUNCAN test at 5% significance level.

. IAA production and Zn solubilization were displayed by the fungal isolates TvR1 and TbS2. TvR1 was reported to produce 1.82 ± 0.01 μg/mL IAA, which was significantly higher than the values reported for TbS2 (1.28 ± 0.03 μg/mL). Likewise, the trend was observed for the Zn solubilization index (ZSI). TvR1 showed 1.62 ± 0.02 ZSI, which was significantly more than the ZSI value 1.44 ± 0.01 reported for TbS2. None of the biocontrol isolates displayed phosphate solubilization activity. The ability to produce EPS and biofilm was confirmed by the formation of precipitate and biofilm on the walls of the glass test tubes, respectively (Supplementary Figure S1). Trichoderma lixii TvR1 and T. brevicompactum TbS2, both displayed positive results for EPS and biofilm production (Table 1).

3.2. Seed Germination

The seed germination assay revealed an increase in the germination percentage and seedling length in all of the biocontrol isolates treated seeds except T. brevicompactum TbS2. The data are presented in

Table 2. Effect of microbial antagonists on seed germination and vigor index.

Treatments	Mean Seedling Length (cm)	Germination Percentage (%)	Seedling Vigor Index
Control #	6.66 ± 0.30 a	66.67	444.02 ± 16.08 a
BS6	7.60 ± 0.27 b	70	532.33 ± 18.85 b
CS13	9.42 ± 0.31 c	76.67	722.36 ± 23.80 c
TvR1	11.10 ± 0.26 d	90	999 ± 22.96 d
TbS2	6.49 ± 0.25 a	66.67	432.69 ± 16.37 a

^# = treated only with distilled water; Values are mean of the number of seeds germinated; ± = Standard error; values followed by different letters are significantly different according to DUNCAN test at 5% significance level.

. The best result for seed germination and seedling vigor index was exhibited by TvR1. The seeds treated with TvR1 showed a 90% germination rate and 999 ± 22.98 seedling vigor index, which was 35% and 125%, higher than the respective parameters reported for the non-treated control.

3.3. In Vivo Study-Pot Experiment

The biocontrol and plant-growth-promoting activities of the microbial isolates were screened using a pot experiment under in vivo conditions. The results of plant-growth promotion obtained in the first pot experiment after 60 DAT are outlined in

Table 3. First pot experiment: plant growth 60 days after transplantation.

Treatments	Plant Height (cm)	Shoot fw (g)	Root fw (g)	Shoot dw (g)	Root dw (g)	Percentage Disease Index	Disease Reduction (%)
Control	52.6 ± 2.6 de	29.8 ± 0.8 b	2.2 ± 0.1 ab	6.8 ± 0.3 ab	0.6 ± 0.04 bcd	0	0
BS6	57.2 ± 2.3 efg	32.4 ± 1.1 bc	2.7 ± 0.3 bcd	7.9 ± 0.2 d	0.8 ± 0.01 de	-	-
CS13	63.7 ± 2.2 hi	37.7 ± 1.6 d	3.3 ± 0.3 efg	8.0 ± 0.2 d	0.9 ± 0.03 ef	-	-
TvR1	77.5 ± 1.8 k	43.9 ± 1.6 e	4.0 ± 0.2 g	8.9 ± 0.2 e	1.1 ± 0.12 g	-	-
TbS2	51.3 ± 1.2 cde	29.2 ± 1.1 b	2.0 ± 0.2 a	6.6 ± 0.1 ab	0.7 ± 0.03 cde	-	-
Fol	40.4 ± 0.9 a	22.2 ± 1.0 a	1.8 ± 0.1 a	6.2 ± 0.2 a	0.4 ± 0.02 a	52.78	-
Fol + BS6	54.4 ± 1.4 ef	31.6 ± 0.7 b	2.9 ± 0.2 cde	7.1 ± 0.2 bc	0.7 ± 0.05 cde	47.22	10.53
Fol + CS13	60.4 ± 1.3 fgh	35.8 ± 1.9 cd	3.1 ± 0.2 def	7.6 ± 0.2 cd	0.7 ± 0.03 cde	44.44	15.80
Fol + TvR1	70.3 ± 1.7 j	45.7 ± 1.1 e	3.7 ± 0.1 fg	8.8 ± 0.1 e	1.0 ± 0.06 fg	36.11	31.58
Fol + TbS2	45.6 ± 1.2 abc	21.4 ± 1.2 a	2.0 ± 0.2 a	6.5 ± 0.1 ab	0.6 ± 0.04 bc	42.00	20.42
A. solani	45 ± 1.5 ab	22.8 ± 1.2 a	2.2 ± 0.1 ab	6.4 ± 0.2 ab	0.5 ± 0.08 ab	43.70	-
A. solani + BS6	55.7 ± 2.6 efg	30.3 ± 1.0 b	2.8 ± 0.1 cde	7.1 ± 0.3 bc	0.6 ± 0.04 bc	37.78	13.55
A. solani + CS13	61.6 ± 1.7 gh	35.8 ± 1.2 cd	3.1 ± 0.2 def	7.8 ± 0.2 cd	0.7 ± 0.03 cde	36.88	15.61
A. solani + TvR1	67.9 ± 2.3 ij	44.3 ± 1.6 e	3.8 ± 0.2 g	8.7 ± 0.3 e	1.0 ± 0.08 g	30.37	30.50
A. solani + TbS2	47.3 ± 2.7 bcd	20.6 ± 0.6 a	2.4 ± 0.1 abc	6.4 ± 0.3 ab	0.6 ± 0.04 bc	31.80	13.90

Values are mean of quadruplicate; ± = Standard error; values followed by different letters are significantly different according to DUNCAN test at 5% significance level; fw = Fresh weight; dw = Dry weight.

. All of the biocontrol microbial isolates effectively improved the growth parameters (plant height, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight), except TbS2, which showed growth on par with the non-infected control. The plants treated with TvR1 displayed the highest plant-growth-promoting activity, which was significantly higher than the values reported for plants challenged with pathogens alone and the control. The heights of the non-infected, Fol-infected, and A. solani-infected plants inoculated with TvR1 were documented with an increase of 47.30, 74.01, and 50.84%, respectively, as compared to their counterpart control. The TvR1 treatment increased the fresh and dry shoot weight of the non-infected, Fol-infected, and A. solani-challenged plants by 47.32 and 30.88%; 105.86 and 41.94%; and 94.30 and 35.94%, respectively, compared to their respective control. The fresh and dry root weights of the TvR1-treated plants in comparison to the non-infected, Fol-, and A. solani-challenged control plants increased by 81.82 and 45.46%; 105.56 and 150%; and 72.73 and 100%, respectively.

Biocontrol activity was exhibited by all of the microbial biocontrol isolates under pot conditions; however, the fungal isolate of T. lixii TvR1 significantly enhanced plant growth and reduced disease severity. The percentage reduction in fusarium wilt and early blight by TvR1 were recorded as 31.58 and 30.50%, respectively. The isolate of BS6 was the least effective in controlling the fungal pathogens and resulted in only a 10.53 and 13.55% reduction in fusarium wilt and early blight, respectively. The fungal isolate of TbS2 was not found to render beneficial effects on plant growth but reduced disease severity more than that displayed by the bacterial isolates of BS6 and CS13. Reduced fusarium wilt due to TbS2 was reported to be 1.94- and 1.29-fold higher than the disease-reduction potential displayed by BS6 and CS13, respectively.

On the basis of the results obtained, the consecutive pot experiment was performed to assess the long-term activity consistency of significant isolate T. lixii TvR1. The second pot study was conducted for 90 DAT (Supplementary Figure S2). After the completion of the experiment, the plants were assessed for increased growth parameters and disease reduction. The data generated are presented in

Table 4. Optimized pot experiment: plant growth 90 days after transplantation.

Treatments	Shoot Length (cm)	Root Length (cm)	Shoot Fresh Weight (g)	Shoot Dry Weight (g)	Root Fresh Weight (g)	Root Dry Weight (g)	No. of Leaves	No. of Fruits	Percentage Disease Index	Disease Reduction (%)
Control	69.8 ± 2.4 c	27.2 ± 0.7 c	50.8 ± 0.5 c	11.0 ± 0.6 bc	5.8 ± 0.2 b	1.2 ± 0.06 b	17 ± 1.5 bc	5.33 ± 0.7 bcd	0	0
TvR1	82.4 ± 1.7 g	36.3 ± 0.9 ef	57.3 ± 0.6 fg	12.7 ± 0.6 cd	7.7 ± 0.2 def	2.0 ± 0.12 de	23.3 ± 0.9 ef	6 ± 0 bcd	-	-
Fol	50.7 ± 1.0 a	15.7 ± 0.8 a	39.9± 1.0 a	8.3 ± 0.5 a	4.6 ± 0.2 a	0.9 ± 0.06 a	14.3 ± 0.9 ab	2.7 ± 0.3 a	50	-
Fol + TvR1	77.6 ± 1.2 ef	31.4 ± 0.7 d	52.8± 1.4 cde	11.7 ± 1.0 bcd	7.2 ± 0.2 c	1.7 ± 0.06 c	21.3 ± 1.2 de	5.7 ± 0.3 bcd	35	30
A. solani	62.5 ± 1.2 b	20.7 ± 0.9 b	45.4 ± 1.2 b	9.6 ± 0.8 ab	5.6 ± 0.2 ab	1.1 ± 0.07 ab	13 ± 1.0 a	2.3 ± 0.7 a	35	-
A. solani + TvR1	72.0 ± 0.8 cd	31.2 ± 0.9 d	54.4± 0.9 def	11.4 ± 0.7 bcd	7.6 ± 0.2 c	1.7 ± 0.04 c	19.7 ± 0.7 cd	5 ± 0.6 bc	26.20	25.14

Values are mean of quadruplicate; ± = Standard error; values followed by different letters are significantly different according to DUNCAN test at 5% significance level.

. The treatment of the pathogen-challenged and non-challenged plants with TvR1 showed improved root and shoot length, fresh and dry weight, and increased leaves and fruit yield. The maximum shoot (82.4 ± 1.7 cm) and root length (36.3 ± 0.9 cm) was observed in the non-infected TvR1-inoculated plants. The percentage reduction in fusarium wilt and early blight was recorded as 30 and 25.14%, respectively, showing the consistent performance of TvR1 under in vivo conditions.

To confirm the root-colonizing activity of TvR1, the root section of the treated plant was assessed using a scanning electron microscope (SEM) after 90 DAT. The SEM images clearly showed the presence of conidia of T. lixii TvR1 on the root sections of the treated tomato plants (Figure 1), confirming the ability of TvR1 to colonize root surfaces successfully.

3.4. Effect on Photosynthetic Pigment

Chlorophyll estimation is an important parameter that indicates the photosynthetic and metabolic activity of plants [60,61]. The effect of the microbial isolates on photosynthetic pigments, i.e., the chlorophyll and carotenoid content in plant leaves were determined after 90 DAT (

Table 5. Photosynthetic pigments.

Treatments	Chlorophyll a (mg/g fwt)	Chlorophyll b (mg/g fwt)	Total Chlorophyll (mg/g fwt)	Carotenoid (mg/g fwt)
Control	0.13 ± 0.002 b	0.09 ± 0.006 a	0.22 ± 0.008 b	0.24 ± 0.014 cd
TvR1	0.33 ± 0.012 f	0.33 ± 0.010 e	0.65 ± 0.006 g	0.26 ± 0.004 de
Fol	0.07 ± 0.001 a	0.09 ± 0.003 a	0.17 ± 0.003 a	0.18 ± 0.005 a
Fol + TvR1	0.28 ± 0.008 e	0.29 ± 0.005 d	0.57 ± 0.013 e	0.32 ± 0.004 g
A. solani	0.1 ± 0.001 a	0.11 ± 0.004 a	0.2 ± 0.005 b	0.21 ± 0.005 b
A. solani + TvR1	0.25 ± 0.011 d	0.21 ± 0.004 c	0.46 ± 0.011 d	0.21 ± 0.003 b

Values are mean of quadruplicate; ± = Standard error; values followed by different letters are significantly different according to DUNCAN test at 5% significance level.

). The maximum total chlorophyll (0.65 ± 0.006 mg/g fwt) was recorded in the non-infected plants treated with TvR1. The highest value of carotenoid (0.32 ± 0.004 mg/g fwt) was documented in the leaves of Fol-infected plants treated with TvR1. The photosynthetic pigments in infected as well as non-infected plants were significantly improved after the inoculation of T. lixii TvR1. The carotenoid content of the plants infected with A. solani, remained significantly unaltered upon inoculation with TvR1.

4. Discussion

Several antagonistic microbes have been reported to have properties that reduce pathogenic diseases in plants. However, the field results of the biocontrol agents have demonstrated inconsistency due to poor competence against the native microbial communities and interaction with the host plants [38,62,63]. The compatibility between the biocontrol agents, host plant, and the natural rhizospheric microbiome is crucial forachieving the effective management of plant diseases and growth stimulation, as native isolates are well adapted to the localized environmental conditions [64]. Moreover, the biocontrol species isolated from soil or plants affected by pathogenic infestation show better biocontrol activity [64,65]. Therefore, in the present work, native rhizospheric microbes were assessed for biocontrol and plant-growth-promoting attributes. The isolated strains were screened for their potential to produce phytohormones and solubilize insoluble minerals from growth media, which is a prerequisite step to collect baseline data for selecting strains with plant-growth-promoting attributes in a short time span [66]. Phosphorus and zinc are essential growth nutrients, and 90% of them are present in the soil in unavailable forms [67]. The plant-growth-promoting microbes improve the growth and productivity of plants by facilitating the uptake of minerals via enhanced solubilization; synthesizing growth hormones such as Indole-3-acetic acid (IAA) that stimulate cell elongation and division, thereby altering the root and stem architecture [68] and by influencing ACC deaminase activity [69,70]. In the present study, TvR1 and TbS2 were reported to have IAA synthesizing and Zn solubilization activity, confirming their potential role in facilitating nutrient accessibility to plants and improving growth parameters. Mwashasha et al. [71] and Riddech et al. [72] reported increased plant growth and yield after the inoculation of plants with microbial isolates that possess the ability to produce IAA, supporting the findings of the present study. Another work undertaken by Yaish et al. [73] showed improved root elongation under saline conditions with reduced ethylene concentration and enhanced nutrients uptake in plants treated with bacterial strains synthesizing IAA and 1-Aminocyclopropane 1-carboxylic acid (ACC) deaminase and possessing the ability to solubilize zinc and potassium. Analogously, mung bean cuttings soaked in a suspension of a wild-type strain of Pseudomonas putida GR12-2 containing a high level of IAA stimulated the formation of several very small, adventitious roots, whereas the IAA-deficient mutant resulted in the formation of fewer roots, suggesting the role of IAA in the development of the plant root system [69].

The seed germination assay and pot experiment conducted to study the growth-promoting activity of the rhizospheric microbial isolates revealed an increase in seed germination, vigor index, and plant biometric parameters (plant height, fresh and dry weight of shoot and root) by all of the microbial isolates except TbS2. The two species of Trichoderma TvR1 and TbS2 used in the present study displayed contradictory effects on plant growth, highlighting species-specific activity [74]. Some species of Trichoderma have been cited in the literature to show neutral or negative stimulating effects on plant growth [75]. Although T. brevicompactum TbS2 was observed to produce IAA under in vitro conditions, it had no positive effect on the seed germination recorded; this may be due to the production of phytotoxic compounds, such as Trichodermin, which play an influential role in biocontrol [76]. Hajieghrari [77] also found similar results, where the inoculation of T. hamatum and T. harzianum produced no effect on the chlorophyll content and root length of maize seedlings compared to a non-inoculated control while a reduction in seedling fresh shoot and root weight by strains was documented. Similarly, Tijerino et al. [78] reported reduced plant size, root length, and the number of lateral roots in tomato seedlings due to inoculation with T. brevicompactum compared to the control.

Apart from plant growth, the microbial isolates were evaluated for their biocontrol activity against two pathogens that have spatially different sites of infections, i.e., the roots and aerial parts of the plant, under in vivo conditions. Induced systemic resistance (ISR) in plants mediated by microorganisms facilitates the management of local and distally located pathogens [79]. Several studies have reported on the management of pathogenic fungi by using rhizospheric microbial isolates, such as Trichoderma spp. and PGPR, via multifarious mechanisms. Reduced Fusarium ear rot and Gibberella ear rot disease of maize caused by fungal pathogens Fusarium verticillioides and F. graminearum by 37.1% and 30.7%, respectively, upon treatment with Trichoderma harzianum INAT11 was documented by Ferrigo et al. [80]. The reduction in disease incidence was attributed to the up-regulation of defense-related gene expression associated with ISR and systemic acquired resistance (SAR) due to Trichoderma sp. Similarly, Attia and co-workers [4] found an increase in the growth and yield of tomato, with a 13% reduction in early blight disease severity when using PGPR. Antagonistic PGPRs were found to disrupt the cell walls of pathogens and inhibit conidial development. The inhibition of multiple pathogens by Bacillus cereus KTMA4 was reported by Karthika et al. [81], who documented a 54 and 66% reduction in early blight and fusarium wilt, respectively, with an enhanced seed germination percentage, vigor index, and plant growth in tomato. The ability to produce biofilms, biosurfactants, and cell lytic enzymes (cellulase, xylanase, protease, lipase, and amylase) with growth-promoting factors, such as ammonia, siderophores, IAA, and others were reported to actively participate in disease control and plant-growth promotion. In the present study, T. lixii TvR1 was observed to produce biofilms and IAA. Moreover, in our previous study [42], under in vitro conditions, TvR1 was reported to synthesize cell lytic enzymes and other growth-promoting factors, such as ammonia and siderophores, which could probably be involved in the biocontrol of fungal pathogens and the growth promotion of tomato plants.

The ability of microbial isolates to produce EPS and biofilms facilitates them in adherence to different surfaces, including the plant roots, aggregation, and root colonization and mediate protection against stresses and plant-growth promotion [17,82,83,84]. In the study, TvR1 was observed using a biofilm and EPS production assay, and the SEM analysis of the colonization of the treated plant roots indicated the probable involvement of root-colonizing mechanisms in effective biocontrol and plant-growth-promoting activity. Eslahi et al. [85] discovered a significant stimulatory effect of recombinant T. harzianum strain T13 on bean plant growth in comparison to a wild strain. The plant-growth-stimulating activity was due to the effective root colonization that triggered plant metabolism by up-regulating the expression of genes responsible for the growth and the enhanced production of auxin and siderophores. Similarly, Song et al. [86], reported on the suppression of foliar pathogens (Pseudomonas syringae pv. tomato DC 3000 and P. carotovorum subsp. carotovorum SCC1) and the improved growth of Arabidopsis thaliana when using Archaeon Nitrosocosmicus oleophilus MY3, which efficiently colonized the root surface and elicited ISR against spatially separated pathogens. Chowdappa et al. [87] reported on the suppression of pathogens directly through mycoparasitism, antibiosis, and competition for space and nutrition and/or indirectly by ISR in case of distally located pathogens on the effective root colonization and proliferation of Trichoderma species. These findings correlated with the result of our study and highlight the probable role of root colonization by TvR1 in the control of pathogens, particularly A. solani, infecting plants at distally located sites.

Chlorophyll estimation is an important parameter that indicates the photosynthetic and metabolic activity of plants [60,61]. In our study, TvR1 was reported to improve the photosynthetic pigment in both the infected and non-infected plants, which aligned with the results of Tanwar et al. [88], who observed an increased rate of photosynthesis and chlorophyll content in broccoli upon inoculation with T. viride. The modification of soil with T. lixii TvR1 mass multiplied on sugarcane bagasse increased the rate of photosynthesis in spinach [89]. The increase in the chlorophyll content of plants may be due to the enhanced uptake of nutrients from the soil and the increased rate of photosynthesis [90]. Trichoderma species are reported to affect chemical communication on efficient root colonization, altering gene expression and rendering various benefits, such as increased nitrogen-use efficiency, resistance to biotic and abiotic stress, and enhanced photosynthetic activity [91,92].

5. Conclusions

The study concludes that the native rhizospheric isolates were effective against both of the pathogens and stimulated the growth of tomato plants under a localized environment. Among the studied isolates, Trichoderma lixii TvR1 displayed the most effective results in terms of disease reduction and plant-growth promotion. T. lixii TvR1 managed two pathogens having different sites of infection. It displayed the ability to produce IAA, solubilize zinc, and form exopolysaccharides and biofilms that could have assisted fungus in the colonization of the root surface effectively and facilitated a significant reduction in disease severity with improved plant biochemical parameters and growth. The result suggested that the multiple mechanisms demonstrated by T. lixii TvR1 were instrumental in triggering the effective results.

As climatic situations are changing continuously, there is a constant need for searching and developing a better biological formulation to manage plant diseases and improve plant growth. The microbial bio-agents recruit various direct and indirect mechanisms to cure plant diseases and promote plant growth. Deciphering these mechanisms under different environmental conditions could aid in understanding the outcome of the interaction of microbial agents with plants and their microbiome. Thus, exploring such microbial agents that could improve ecological competence and biocontrol prospectus is imperative to harness biological potential effectively and perform sustainable agricultural activities. Further, it has been recognized that the application of a consortium consisting of multiple bio-compatible microbial isolates shows the augmented impact on plant and soil biochemical properties. Hence, future research should focus on improving the biocontrol and bio-stimulating prospects of T. lixii TvR1 by developing consortia with compatible strains.