Evaluation potential of PGPR to protect tomato against Fusarium wilt and promote plant growth

Growth of PGPR and FOL

B. aryabhattai SRB02 was isolated previously from the rhizosphere of a soybean field in the Chungcheong buk-do region of South Korea (Park et al., 2017b). Bacteria were cultured on LB agar or in broth (AppliChem, Darmstadt, Germany) media at 28 °C for 24 h. F. oxysporum f. sp. lycopersici strain KACC 40032 was obtained from the Korean Agricultural Culture Collection (KACC,  http://genebank.rda.go.kr/) and grown on potato dextrose agar plates at 28 °C for 7 d. The antifungal activity of B. aryabhattai SRB02 against FOL was evaluated following the protocol of Shahzad et al. (2017a).

Briefly, a 0.5 cm² disc of active fungal mycelia of FOL was placed at the center of a 90 mm disposable plastic Petri dish (SPL, Korea) containing LB agar (Becton, Dickinson and Company, France). The overnight bacterial culture of B. aryabhatttai SRB02 was aseptically streaked around the fungal disc at equal distances in a square pattern. For the untreated control, a fungal disc was placed on LB agar, as mentioned earlier, but instead of B. aryabhatttai SRB02, only sterile water was streaked. For comparison, the effects of fungal growth inhibition of organic acids against the pathogen were also evaluated. All of the plates were incubated at 28 °C for 7 d. After the incubation period, the inhibition zone was measured and the percent inhibition was calculated according to the following formula.

${\displaystyle \text{Inhibition}\text{\%}=\frac{\left(\text{diameter of fungus on control plate}-\text{diameter of fungus on SRB02 co-cultured plate}\right)\times 100}{\text{diameter of fungus on control plate}}.}$

Screening of tomato varieties for resistance to FOL

In the current study, tomato seeds of four Korean cultivars (IT 252842-13 (Cultivar-1), IT 252869-14 (Cultivar-2), IT 260627-16 (Cultivar-3), IT 259462-15 (Cultivar-4) ) were selected for their response to the pathogen. Seeds were sterilized with 2.5% sodium hypochlorite for 10 min and kept on wet paper towels inside Petri plates in an incubator at 25 °C for 5 d. Horticultural soil, distilled water, and pots were autoclaved at 121 °C for 20 min. Uniformly germinated seeds were transferred to separate trays filled with sterilized horticultural soil (Soil and Fertilizer Technology, Korea). After one week, uniformly grown seedlings were transplanted to big pots with the dimensions (LxWxH)-3. 5 × 3 × 3 inches and volume 85–90 g. Plants were allowed to acclimatize for a few days, and the experimental treatments were set up in triplicates, with each replicate containing at least six plants. The fungal spore suspension of FOL strain KACC 40032 was prepared according to the protocol described by Lichtenzveig et al. (2006). Control plants were treated with distilled water, and plants were allowed to grow for 5 d. Plants to be treated with the pathogen were inoculated by applying a spore suspension (10⁶ conidia/mL) to the exposed roots of tomato plants. The roots were then covered with soil. Plants were allowed to grow at relatively high humidity of 80 ± 2%. After 14 d of growth under the conditions mentioned above, the inoculated plants were assessed based on symptomatology (severity of plant wilting) and growth.

In planta biocontrol assessment

After the screening test, two cultivars (resistant and susceptible, one each) were selected based on disease symptoms and growth under biotic stress. Seeds of the selected cultivars were surface-sterilized, germinated, and grown before being transplanted to pots as mentioned previously. The plants were allowed to acclimatize for a few days, and the experimental treatments were set up in triplicate, with each replicate containing at least six plants. SRB02 was applied to plants by soil drenching with 10 mL SRB02 broth culture (4 × 10⁸ cfu/mL) in the root zone. The fungal spore suspension FOL strain KACC 40032 was prepared as mentioned previously. Control plants were treated with distilled water, and plants were allowed to grow for 5 d. Plants to be treated with the pathogen were inoculated by applying spore suspension (10⁶ conidia/mL) to the exposed roots of tomato plants. The roots were then covered with soil. The plants were allowed to grow at relatively high humidity of 80 ± 2% because to further exploit the pathogenic impact of fungus. Data were recorded on growth parameters such as plant height (PH), root length (RL), fresh weight (FW), dry weight (DW), and chlorophyll content (Chl. Cont.) to determine the response of plants to infection in the presence or absence of SRB02. For fresh plant biomasses, the plants were uprooted, carefully washed, and frozen in liquid nitrogen, and then transferred to storage at −80 °C until further analysis.

Extraction and quantification of amino acid content

The plant amino acids were extracted according to the protocol described by Khan et al. (2017), with some modifications. Briefly, the freeze-dried whole plant samples were ground to homogenate, and 100 mg powdered samples were hydrolyzed under a vacuum in 6N HCl at 110 °C followed by 80 °C for 24 h. The dried residue was suspended in 0.02N HCl and filtered through a 0.45 µm filter. The amino acids were then quantified using an automatic amino acid analyzer (Hitachi, Japan; L-8900). The experiments were conducted in triplicate, and each replicate was comprised of six plants. The amino acid concentration was determined using relevant standards. This standard known as amino acid standard mixture solution (type H) used for the automatic amino acid investigation was procured through Wako Pure Chemical Industries Ltd (Japan), and used for endogenous amino acids assessment.

Jasmonic acid quantification

For the quantification of endogenous jasmonic acid (JA) content, the optimized protocol described by McCloud & Baldwin (1997) was used. Briefly, homogenized powder (0.3 g) from the immediately freeze-dried whole plant samples was suspended in extraction buffer (70:30 v/v acetone and 50 mm citric acid), and 25 ng JA internal standard ([9, 10-2H²]-9, 10-dihydro-JA) was also added to the suspension. The extract suspension was kept overnight at room temperature for evaporation of highly volatile organic solvents and to retain the less-volatile fatty acids. The subsequent aqueous phase was filtered and then extracted with 30 mL diethyl ether three times. The collective extracts were subsequently loaded onto a solid-phase extraction cartridge (500 mg of sorbent, aminopropyl). In addition, 7.0 mL of trichloromethane and 2-propanol (2:1 v/v) were used to wash the loaded cartridges. Then, the exogenous JA and relevant standard were eluted with one mL of diethyl ether and acetic acid (98:2 v/v). Following evaporation, the samples were esterified and analyzed by GCMS (6890N network GC system) and a 5973 network mass selective detector (Agilent Technologies, Palo Alto, CA, USA) in the relevant ion mode. The relevant ion mode was selected for JA determination. The ion fragment was examined at m/z = 83 AMU, corresponding to the base peaks of JA and [9, 10-2H²]-9, 10-dihydro-JA. The endogenous JA values were determined from the peak areas for relevant standards.

Salicylic acid (SA) quantification

The SA of SRB02-treated tomato plants was extracted and quantified according to the protocol described by  Enyedi et al. (1992), Seskar, Shulaev & Raskin (1998). Immediately freeze-dried whole plant tissues were homogenized, and 0.2 g of homogenate powder was used for the extraction using 90% and 100% methanol. The pellets were dried and re-suspended in 2.5 mL 5% trichloroacetic acid (TCA) and further partitioned with ethyl acetate, cyclopentane, and isopropanol (ratio of 100:99:1, v/v). The upper organic layer containing free SA was used for air-drying with nitrogen gas. The dry SA was again suspended in one mL 70% methanol and subjected to high-performance liquid chromatography (HPLC) using a Shimadzu device outfitted with a fluorescence indicator (Shimadzu RF-10AxL) with excitation at 305 nm and emission at 365 nm filled with a C18 reverse-phase HPLC column (HP Hypersil ODS, particle size 5 µm, pore size 120 Å, Waters). The flow rate was maintained at 1.0 mL/min.

Statistical analysis

All experiments were replicated three times, and each replicate was comprised of six plants. Data were statistically evaluated with Duncan multiple range tests and t-tests where appropriate, using SAS version 9.2 software (Cary, NC, USA).

In vitro antifungal assay

The in vitro antifungal activity of PGPR B. aryabhattai SRB02 was assessed against pathogenic Fusarium oxysporum in dual culture. The results revealed that the PGPR B. aryabhattai SRB02 significantly inhibited the growth of pathogenic F. oxysporum, as shown in Fig. S1.

Response of tomato cultivars under pathogenic infection by F. oxysporum

To determine the response of four tomato cultivars, the plants were challenged with a spore suspension of the pathogen. The pathogen was applied to the exposed roots of tomato plants and incubated under higher relative humidity to create a conducive environment for successful infection. After 14 d of inoculation, the cultivars revealed a differential level of tolerance to the pathogen (Fig. 1). The plant tolerance level was determined based on the symptomatology (severity of plant wilting). In the susceptible plants, clear symptoms of wilting were evident. Susceptible plants were also observed with retarded growth as compared to the tolerant plants. Based on the plant growth attributes and resistance level, as shown in Fig. 1, the most tolerant and susceptible tomato cultivars were selected for further experiments.

Plant growth-promoting and ameliorative effects of B. aryabhattai SRB02 against FOL

Based on screening, the plant-growth-promoting and biocontrol efficiency of PGPR B. aryabhattai SRB02 against a virulent strain of F. oxysporum was investigated in both the selected tolerant and susceptible varieties (Fig. 2). B. aryabhattai SRB02 significantly promoted plant growth, and interestingly reduced the disease in both tolerant and susceptible tomato cultivars (Fig. 2).

The growth-related traits of the disease-tolerant plants were significantly improved when applied with SRB02 alone. The plant height (PH) was improved by 37.4%, while RL was improved by 26.8% as compared to the water-treated control plants. Other traits including seedling FW, seedling DW, and chlorophyll content were also improved by 15.3%, 23.3%, and 5.8%, respectively. A similar trend was also observed when the tolerant plants were treated with the pathogen and SRB02 together, compared to the pathogen-treated plants. The PH, RL, seedling FW, DW, and chlorophyll content were improved by 124%, 6.4%, 15.8%, 42.3%, and 39.7%, respectively, compared to the plants inoculated with the pathogen alone. SRB02 also improved the growth attributes of the disease-susceptible plants with or without co-treatment by the pathogen. The PH of the disease-susceptible plants was improved by 14.1% with the application of SRB02 in comparison with the water-treated control plants; however, the increase in PH was significantly greater (105.7%) in plants treated with SRB02 and F. oxysporum combined as compared to the pathogen-treated plants. Likewise, other traits were also improved in plants treated with PGPR alone as compared to the water-treated plants and also in the PGPR and pathogen co-treated susceptible plants in comparison with the plants treated with the pathogen alone. The RL, seedling FW, seedling DW, and chlorophyll content were improved by 9% and 44.5%, 10.4% and 32.6%, 3%, and 24.6%, and 4% and 61.3% in plants treated with SRB02 alone and in plants co-treated with PGPR and the pathogen, respectively (Fig. 2, Table 1).

B. aryabhattai regulates defense against F. oxysporum by modulating defense-related hormones in tomato

Measurement of basal and induced levels of the plant defense-related hormones SA and JA following inoculation with FOL in the absence or presence of SRB02 revealed strict regulation of plant defense responses in SRB02-treated plants due to the regulation of the synthesis of both of these hormones (Figs. 3 and 4). Interestingly, these results were observed in both the resistant and susceptible cultivars, indicating the high utility of SRB02 for field use even in susceptible crops. More specifically, SRB02-treated infected plants (tolerant and susceptible) produced significantly lower JA (11.10% and 10.30%, respectively) compared to control plants (Fig. 3). Even the SRB02-treated plants in the absence of FOL accumulated lower JA (6.92% and 17.91%).

Furthermore, SRB02 treatment with F. oxysporum-inoculated plants of the tolerant cultivar accumulated 48.48% more SA compared to plants not treated with the PGPR. More interestingly, the response of the F. oxysporum-inoculated plants of the susceptible cultivar was more robust in the presence of SRB02, as these plants produced 74.60% more SA as compared to plants not treated with PGPR (Fig. 4). However, no significant differences in SA accumulation were observed in SRB02-treated plants of either tolerant and susceptible cultivars in the absence of F. oxysporum.

B. aryabhattai SRB02 regulates amino acids in plants with or without biotic stress

The current study showed that B. aryabhattai SRB02 regulates amino acids in both disease-tolerant and susceptible tomato plants in the presence or absence of F. oxysporum (Table 2). Under pathogenic infection by F. oxysporum, B. aryabhattai SRB02 inoculation significantly enhanced aspartic acid (115.57% and 147.48%), threonine (123.18% and 118.56%), serine (123.13% and 158.91%), glutamic acid (4.86% and 157.89%), glycine (131.82% and 143.58%), alanine (99.61% and 109.67%), valine (98.13% and 74.62%), methionine (239.06% and 172.93%), isoleucine (42.60% and 97.82%), leucine (103.21% and 58.03%), tyrosine (138.45% and 65.45%), phenylalanine (39.86% and 34.16%), lysine (113.15% and 98.03%), histidine (98.42% and 111.74%), arginine (108.69% and 157.18%), and proline (90.09% and 115.25%) in disease-susceptible and tolerant tomato plants, respectively (Table 2). Only cysteine was decreased by 9.65% and 21.82% in B. aryabhattai SRB02 applied to susceptible and tolerant plants, respectively (Table 2).

Likewise, in the absence of the pathogen, B. aryabhattai SRB02 significantly enhanced aspartic acid (3.35% and 24.98%), threonine (32.99% and 118.56%), glutamic acid (4.86% and 157.89%), glycine (30.78% and 143.58%), alanine (29.70% and 4.65%), cysteine (44.22% and 60.20%), valine (21.91% and 31.81%), methionine (132.35% and 31.17%), isoleucine (97.76% and 29.48%), leucine (36.52% and 32.40%), phenylalanine (114.92% and 77.20%), histidine (22.81% and 41.48%), and arginine (35.98% and 23.95%) in the susceptible and tolerant plants, respectively (Table 2). However, B. aryabhattai SRB02 showed an increase in serine-intolerant (9.73%) plants and a decrease of 13.22% in susceptible plants. Tyrosine was increased by 45.03% only in the tolerant plants when applied with PGPR, while it was decreased by 51.52% in susceptible plants. Similarly, lysine was increased (35.12%) in the PGPR-applied tolerant plants, while no significant difference was observed in the susceptible plants. In contrast, proline was increased by 35.77% only in susceptible plants, while no significant difference was recorded in intolerant plants when challenged with PGPR (Table 2).