Leaves and roots of healthy plants (Cinnamon basil, Ricinus communis, Epipremunum aureum, Citrus jambhiri, Hibiscus rosasinensis were collected from “Botanical Garden” of Guru Nanak Dev University, Amritsar, Punjab, India (31°37′45″N and 74°49′36″N). Plant samples were wrapped aseptically in plastic bags and taken to the laboratory where they were processed within 4 hours.
Different test phytopathogenic fungi viz. Alternaria brassicicola (MTCC2102), Alternaria solani (MTCC2101), Colletotrichum acutatum (MTCC1037), Fusarium oxysporum (MTCC284), Cladosporium herbarum (MTCC351), were procured from Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. Whereas, Fusarium solani (NFCCI 91) was obtained from the National Fungal Culture Collection of India NFCCI, Pune. Fusarium moniliforme and Alternaria alternata were isolated in the lab and all of the fungal cultures were maintained at 4ºC on Potato dextrose agar (PDA).
Isolation of endophytic strains from plant samples
The collected plant samples were washed under running tap water for 1-2 minutes to remove soil particles. The samples were then surface sterilized by 70% ethanol for 10 min followed by treatment with 1% sodium hypochlorite solution for 15 min (21). Plant samples were then repeatedly washed with sterilized water, air-dried in a laminar airflow hood, and cut into small pieces using a sterile razor blade. The small pieces of sterile samples were placed on SCNA medium (starch casein nitrate agar). To inhibit the growth of fungi and other non-Streptomyces bacteria, cycloheximide (50g/ml) and nalidixic acid (50g/ml) were added to the medium. The plates were then incubated at 28°C for 7–21 days. Isolated actinobacteria colonies were subcultured and purified on SCNA plates. Isolate spores were reserved as a stock in 20% glycerol at -20 °C for potential use.
Screening and selection of the isolate
Primary screening determines the ability of the microorganisms to produce an antifungal metabolite without providing a significant idea about the production potential of the organism. Modified Kirby Bauer antibiotic susceptibility test was used for primary screening (22). The actinobacterial isolates were cultured for seven days on the SCNA medium at 28 ºC. Six mm agar discs from well-grown culture of actinobacterial isolates were placed on PDA plates which were already seeded with the test phytopathogenic fungi (100 µl of 106spore ml-1). The plates were then kept at 4ºC for 30 min for the diffusion and then incubated at 28ºC. The zone of inhibition was determined after 48 h.
Following primary screening, the active isolates were subjected to secondary screening. Three discs of actinobacterial culture with a diameter of 6 mm were transferred to 250 ml Erlenmeyer flasks containing 50 ml of starch casein nitrate broth and incubated for 10 days at 28 ºC in a rotary shaker at 180 rpm. Mycelia were extracted by centrifuging at 10,000 g for 20 minutes at 4 ºC and the supernatant was used for bioassay. The PDA plates inoculated with test fungi (100 µl of 106spore ml-1) were punctured with sterile cork borer to create wells (6 mm in diameter) and 200µl of culture supernatant was transferred to each well under aseptic conditions. The plates were incubated at 28 ºC for three days and observed for antifungal activity of the isolates as clear zones of inhibition (in mm) around well. Out of 9 isolates from secondary screening, isolate SP5 was selected for further studies based on its broad-spectrum antifungal activity.
Morphological, physiological, and biochemical characterization of Streptomyces sp. SP5
Cultural characteristics were determined as per methods described by Shirling and Gottlieb (23) in the International Streptomyces Project (ISP). Bright light microscopy and electron microscopy were used to examine the strain morphological characteristic. Physiological and biochemical characterization such as growth at different temperatures (20-50°C), pH (5.0–12.0), salt concentration (0–20% w/v), and capacity to generate different hydrolytic enzymes were carried out according standard protocols (24). According to Shirling and Gottlieb (1966), sugars assimilation as carbon source was investigated. The major diagnostic features of Streptomyces, a sugar pattern in whole-cell hydrolysate and an isomer of diaminopimelic acid (DAP) in the cell wall, were determined using Lechevalier and Lechevalier (1980).
The genomic DNA of the isolate was extracted using Marmur (26) method for 16S rRNA gene sequencing. The 16S rRNA gene sequence was amplified by polymerase chain reaction (PCR) using primers 27f (5′-AGAGTTTGATCC TGGCTCAG-3′) and 1492r (5′-AGAAAGGAGGTGATC CAGGC-3′). Amplification was done for 40 cycles in an PCR machine (Eppendorf-gradient). Each cycle included a denaturation stage of 1 minute at 94 0C, an annealing step of 1 minute at 50 0C, and an extension step of 2 minutes at 72 0C and a final extension of 10 minutes at 72 0C. The QIA quick gel extraction kit (Qiagen, Germany) was used to purify the obtained PCR product. The Institute of Microbial Technology (IMTECH), Chandigarh, India, sequenced the 16S rRNA gene of Streptomyces sp. SP5. For the detection of phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarities, the EzTaxon server (http://www.ezbiocloud.net) was used (27). The Clustal W program was used to align the nearly complete sequence (1400 bp). Phylogenetic trees based on bootstrap values (1000 replications with MEGA6 software) were constructed using neighbor-joining and maximum-parsimony methods (28-29).
Time course experiment related to antifungal metabolites production and growth
For antifungal profiling determination, seed culture was prepared, three discs (6mm diameter) of 7 days old Streptomyces sp. SP5 culture were inoculated into Erlenmeyer flasks (250 ml) containing 50 ml of starch casein nitrate broth and incubated at 28°C at 180 rpm for a week. Inoculating the production medium (SCN broth) with seed culture (2%) and incubating at 28 °C for 10 days with agitation at 180 rpm was used for fermentation. After every 24 hours, the flasks were harvested, and the culture broth was centrifuged at 10,000g for 10 minutes to remove the biomass. The biomass was dried for 2 days at 60 °C and measured (mg). The activity against test fungal phytopathogens was determined using the remaining cell-free culture supernatant. The test fungi (100 µl of 106spore ml-1) were seeded on PDA, which were punctured with sterile cork borer to create 6 mm wells. The plates were held in the refrigerator for 1 hour after adding 200 µl of culture supernatant to allow active metabolites to diffuse. After diffusion, the plates were incubated at 28 °C for 4-5 days. The antifungal activity of the isolate was measured in millimetres as zones of inhibition around wells.
Recovery of antifungal metabolites
For the production of antifungal active metabolites, fermentation was carried out by inoculating the strain (at a concentration of 2.5% from seed culture) in a production medium and incubating for five days at 28°C under shaking (180 rpm). Fermentation was terminated when the maximum antifungal activity was observed (5th day) and culture broth was centrifuged at 10,000g at 4 ºC for 10 min to separate the mycelium.
The active metabolites from culture supernatant were recovered by solvent extraction. Different organic solvents (hexane, diethyl ether, chloroform, ethyl acetate, butanol) having a wide range of polarity were screened to work out the best extractant. The supernatant of Streptomyces sp. SP5 was extracted twice in the ratio of 1:1 (supernatant: solvent). Rotavapor (BUCHI Rotavapor R-210) was used to concentrate the obtained organic phase to dryness. The obtained crude extracts were redissolved in methanol (1ml) and tested for antifungal activity against test strains. Acetone precipitation was also employed to extract out the antifungal metabolites from the culture supernatant. For acetone precipitation, two volumes of 100% chilled acetone were added to the culture supernatant and kept for 1 hour. at -20oC. The precipitates were recovered by centrifugation at 10,000 rpm for 10 minutes at 4oC, and were redissolved in distilled water. The antifungal activity of precipitates was determined against different test phytopathogenic fungi using the agar well diffusion method.
Stability of active metabolites in the culture supernatant
The supernatant was heated at various temperatures (37 0C, 50 0C, 70 0C, 100 0C and autoclaving at 121 0C) for 1hr, and was also exposed to a lower temperature (-20 0C) for one hour to determine thermostability. The effect of pH on activity was determined by adjusting the pH from 2 to 14, followed by incubation for one h at 28 0C. Photostability was tested by exposing the supernatant separately to UV (200 – 600nm) and sunlight (1hr). The antifungal activity of all the treated samples was then tested using a well diffusion assay.
Plant growth promoting potential
Indole acetic acid (IAA) production
For Indole acetic acid (IAA) production 50 ml of yeast malt broth (YMB; g/lt: yeast extract 4.0, malt extract 10.0, dextrose 4.0) containing 0.2% L-tryptophan was inoculated with three plugs (6mm) of seven-day-old Streptomyces sp. SP5 culture and then incubated at 28ºC for 7 days under shaking (180rpm). After incubation, the broth was centrifuged at 10,000g for 15 minutes to remove mycelia. After centrifugation 2ml Salkowski reagent, as modified by Gordon et al (1950) was added into tubes containing 1ml of cell-free supernatant and incubated for 25 min at room temperature. The developed pink color intensity measured at 530nm, which indicates indole acetic acid production. The concentration of IAA produced was estimated against the standard curve of IAA using a concentration range of 10-100 μg/ml.
Extraction, purification, and identification of Indole acetic acid
The culture supernatant was acidified to pH 2.5 with HCl and extracted twice with ethyl acetate using solvent-solvent extraction. The separated organic phase was concentrated using a rotary evaporator and redissolved in ethyl acetate (1ml). For IAA analysis, the ethyl acetate extract was separated using thin-layer chromatography (TLC) with propanol: water (8:2, v/v) as solvent system, and the chromatogram was observed after spraying the plates with Salkowski regent. IAA was partially purified from the crude solvent extract by using silica gel column chromatography and fractions were collected with the solvent system of ethyl acetate and hexane (20:80 v/v). Each fraction was checked for the presence of IAA by TLC using Salkowski regent. The indole containing fraction was further quantified by high-performance liquid chromatography (HPLC) using C-18 reversed-phase column eluting with methanol: 1 % acetic acid in water (40:60 v/v as mobile phase) at 1 ml min-1 flow rate. The pure IAA compound was spiked into the column as standard and from partially purified IAA extract 20 µl aliquot was analyzed. The identical retention time of the respective standard was used to assess the presence of IAA in the specimens.
Optimization of IAA production
Various factors such as incubation period, pH, temperature, and tryptophan concentration were studied using one variable at a time approach or classical approach to optimise IAA production. The effect of incubation time on IAA synthesis was investigated by growing the culture in yeast malt broth for 10 days and IAA production was checked after every 24 h. The optimal for production was determined by changing the initial pH of the medium from 2-10 using 1N HCl and 1N NaOH. Similarly, the effect temperature of incubation on IAA production was investigated by incubating Streptomyces sp. SP5 into the same medium at 20, 25, 28, 30, 35, and 40 ºC for six days. The effect of L-tryptophan concentration was calculated by adding 0, 1, 2, 3, 4, and 5 mg/ml of L-tryptophan to medium (pH 7.0) and incubating for six days. The bacterial growth rate was measured, culture broth was centrifuged at 10,000g for 10 min and the biomass was dried at 60 °C for 2 days and weighed (mg). All experimentations were performed in triplicates and average values were observed.
Streptomyces sp. SP5 In vivo biocontrol of Alternaria solani and its effect on tomato plant growth promotion
Efficacy of Streptomyces sp. SP5 against early blight disease was tested by conducting three independent pot trials at Guru Nanak Dev University (Amritsar) using soil drenching method. The aim was to see whether Streptomyces sp. SP5 culture cells, culture supernatant, solvent extract as well as acetone precipitates could regulate Alternaria solani (the causal fungal phytopathogen of early blight in tomato) and promote various plant growth traits. Tomato seeds susceptible to Alternaria solani (Solanum lycopersicum Mill. “Pusa Ruby”) were sown in sterile soil for 3 weeks at 28±2.0 °C and after true leaf stage, a single plant was transplanted into a pot of 8cm diameter containing 100g sterile soil for each treatment. The plants were subjected to a variety of treatments by Streptomyces sp. SP5 antagonists viz. Streptomyces sp. SP5 cells, supernatant, solvent extract and Acetone precipitates, with and without pathogen (Table.1). Three replications were maintained for each treatment and the pots were kept under natural conditions (temperature 22±2.0°C, photoperiod L16: D8). Plants were watered and disease symptoms, flowering, and fruiting stages were observed daily. After disease appearance in the control plants, all treated plants were uprooted carefully, and plants were washed with running tap water to remove the adhered soil, shoot & root length, and fresh & dry weights of tomato seedlings were recorded. Data obtained from in vivo were subjected to statistical analysis. Values were represented as their mean ± SE of three independent experiments and to compare the mean difference, one-way analysis of variance (ANOVA) with Tukey’s post hoc test was carried out using SPSS statistical analysis software (Version 20.0, IBM SPSS)