2.1 Sample Collection and Isolation of Endophytes
Roots (Healthy and Nematode infected) of 90 days aged Corchorus capsularis and C. olitorius were collected from Bangladesh Jute Research Institute, Manikganj Regional Station (23°52′ N, 90°1.4′E), Bangladesh.
2.2 Isolation and Preservation of Jute Endophytes
The roots were thoroughly washed to remove soil and organic matter, and surface sterilization was performed with a 60-second wash with 70% ethanol followed by a 20-minute wash in 0.1% Mercuric chloride and a 30-second wash with 70% ethanol. Then the residual Mercuric chloride was removed by a ten times wash with autoclaved distilled followed by a final rinse with sterile reverse osmosis-treated (RO) water (Sameer et al. 2016). Then the root (Healthy jute) and root galls (Nematode Infected plant) were carefully collected and soaked for 4 hours at room temperature in a pectinase solution and gently mashed with a pestle. The resultant slurry was transferred to a 250 mL glass flask with 50 mL distilled 0.2 M PBS buffer (pH 7.0) and then shaken for 10 minutes to disperse the root materials. The homogenized solution was filtered through a series of stacked sieves: 250-μm, a 75-μm, and a 25-μm pore sieve to remove the host plant and nematode material (Jenora et al. 2006). Then the 1000 μl (1 ml) of the filtrate was serially diluted up to 10-5 and spread onto Nutrient Agar (Peptone 5g/L, Yeast extract 2g/L, Meat extract 1g/L, NaCl 5g/L, Agar 15g/L; pH 7.2±0.2) and incubated both aerobic and anaerobic condition for 48 hours at 37°C. A triplicate set of plates were used for all the treatments throughout the study. Bacterial isolates were preserved in 30% glycerol at -80°C.
2.3 Phosphate and lignin hydrolysis test
The phosphatase and lignase activity were tested on Pikovskaya’s agar (PA) containing Tricalcium phosphate as an insoluble phosphate source (Pikovskaya et al. 1948) and lignin agar respectively. Pikovskaya’s agar plates were prepared as per following composition (g/l): C6H12O6 10.0g, CaHPO4 5.0g, (NH4)2 SO4 0.5g, NaCl 0.2g, MgSO4.7H2O 0.1g, KCl 0.2g, yeast extract 0.5g, MnSO4.H2O 0.002g, FeSO4.7H2O 0.002g containing Bromo phenol blue 0.24g, pH 7.2±0.2. Freshly grown cultures of bacteria were spot inoculated on PA and lignin agar plates with the help of a sterile loop. Inoculated plates were incubated at 37°C for 72h. The formation of a halo zone around the colonies is indicative of positive enzyme activity. Solubilizing index (SI) of the isolates was determined by using the formula as follows:
SI = (Halo zone + Colony diameter)/Colony diameter.
2.4 Extra-cellular enzyme assay
For the screening of extra-cellular enzyme producing the isolates, the procedure described by Dingle et. al. (Dingle et al. 1953) was followed where bacteria were cultured in 100 ml Nutrient Broth (Peptone 5g/L, Yeast extract 2g/L, Meat extract 1g/L, NaCl 5g/L; pH7.2±0.2) and incubated at 37°C in shaking incubator with 200 rpm for 24 hours. After incubation, a suitable volume of bacterial cultures was centrifuged at 22,000x g rpm for 20 min and filtered the supernatant through a Millipore filter (0.22 μm). The filtrate was subjected to the screening of Pectinase, Xylanase, and Cellulase enzyme (Dingle et al. 1953). The 6 mm diameter wells were prepared in agar plates containing pectin, xylan, and CMC substrate with sterile cork borer to inoculate 50 μl of filtrate. After 24 hours of incubation at 37°C, the plates were flooded with iodine solution and the presence of a clear zone around the well indicates the enzymatic activity of the isolates. The average diameter (mm) of clear zones of 3 replications for each isolate was recorded.
2.5 Identification of bacteria by sequencing the 16S rRNA gene
Comparison of the bacterial 16S rRNA gene sequence allows differentiation among organisms at the genus level across all major phyla of bacteria along with classifying strains at the species and subspecies level (Jill et al. 2004; Woese et al. 1985; Woese et al. 2000). The bacterial communities were analyzed by genomic DNA isolation, PCR amplification, and sequencing of the 16S rDNA fragments.
DNA Isolation
Total genomic DNA was isolated according to the “Bacterial genomic DNA isolation using CTAB” protocol described by JGI (William et al. 2004) briefly bacterial isolates were grown in Nutrient broth at 37°C for 16 hours at 180 rpm shaking. Then the culture was centrifuged at 10,000 rpm for 5 minutes and the pellet was re-suspended in TE (10mM Tris; 1 mM EDTA, pH 8.0) to adjust OD600 ≅ 1.0 concentration. Then added lysozyme at 100 mg/ml concentration and incubated at 37°C for 30 minutes. Then 10% SDS was added and mixed thoroughly followed by the addition of Proteinase K (10mg/ml) and incubation for 1 hour at 56°C. Finally Incubated at 65°C for 10 min after the addition of 5 M NaCl and CTAB/NaCl (heated to 65°C) and mixing. Afterward phenol: chloroform: isoamyl alcohol (25:24:1) was added and mixed well and centrifuged at max speed for 10 min at room temperature and the aqueous phase was transferred to a clean microcentrifuge tube followed by another wash with chloroform: isoamyl alcohol (24:1) and finally 0.6 volume isopropanol (-20°C) was added to the collected aqueous phase and incubated at -20°C for overnight. After centrifuged at max speed for 15 min at 4°C the pellet was washed with ice-cold 70% ethanol (directly from -20°C freezers) and spun at max speed for 5 min and the pellet was air dried at room temperature and was re-suspended in ~170 μl of DNase-free water and RNAse treated (with a final concentration of 0.5 U/μl) and incubated at 37°C for 1 hr. The purity and concentration of DNA were estimated by visual examination on ethidium bromide-stained agarose gel as well as using the Thermo Scientific NanoDrop 2000.
Amplification of 16S rDNA and PCR product purification
PCR amplification of 16s rDNA gene was carried out in GeneAmp PCR System 9700 (Applied Biosystems, Life Technologies, USA) by using forward primer 533F (5'-GAGTTTGATCCTGGCTCAG-3') and the reverse primer 1492RI (5'-ACCTTGTTACGACTT-3') (Idriss et al. 2002). The final volume of PCR mixture was 50 μl containing 5μl of 50ng/μl genomic DNA, 0.25μl of Platinum Taq DNA Polymerase (Invitrogen, Life Technologies, USA), 5μl of 10x PCR buffer with 2μl of 20mM MgSO4, 1μl of 10mM dNTP (Invitrogen, Life Technologies, USA), 2μl Dimethyl sulfoxide (DMSO), 2μl of 10μM each primer and 30.75μl of Nano pure distilled water. The cycling parameter consisted of 35 cycles: denaturation at 94°C, 30s; primer annealing at 55°C, 45s; extension at 72°C, 1min. Before the amplification cycle, DNA was denatured for 5 min at 94°C and after amplification, an extension step for 7 min at 72°C was performed. All the amplified PCR products were eluted from agarose gel using Qiagen QuickSpin PCR purification columns (Qiagen, Catalog No. 28706).
Sequencing, assembly, and BLAST
The amplified and purified PCR fragments were sequenced in ABI 3730XL DNA Analyzer (Applied Biosystems, Life Technologies, USA) with the 533F (5'-TTA CCGCGGCTGCTGGCAC-3'), 1492RI (5'-ACCTTGTTACGACTT-3') primer. The sequencing reaction was performed by using Big Dye V3.1 sequencing reagents (Applied Biosystems, Life Technologies, USA) following the manufacturer’s protocol. The two sequences for each sample were assembled using CAP3 (Buensanteai et al. 2008) and the contig of each isolate was compared to public databases available in NCBI and species level identification was determined as a 16S rDNA sequence similarity of >99% with that of the prototype strain sequences in the GenBank.
Phylogenetic Analysis
Phylogenetic analysis was conducted using a multiple sequence alignment program Clustal omega. Then Newick file of the tree was given to MEGAX (Kumar et al. 2018) to construct a phylogenetic tree by the neighbor-joining method program where significant levels of interior branch points obtained were determined by bootstrap analysis (1000 data re-sampling).
Effects of Endophytes on Seed Germination
The selected endophytic bacterial strain was grown in Nutrient Broth for 16 h (OD600=1) at 34°C with 180 rpm shaking followed by the collection of bacterial cells by centrifugation (13,000 rpm for 10 min at 4°C). Cells were re-suspended with sterile 0.05 mM PBS (pH 7.0) to prepare a final concentration of 108 CFU/ml for the uniform population of bacteria for seedlings inoculation (Taghavi et al. 2009). 1 gm of healthy seeds were treated with endophytic bacteria (1x108 CFU/ml) for 2h with 180 rpm shaking, placed on moist germination paper, and incubated at room temperature. Seeds treated with sterile distilled water served as control. Vigor index (VI = Mean Root Length + Mean Shoot Length x % Germination) was calculated at the end of 7 days (Baki et al. 1973). Three replicates of 10 seeds were used per treatment.
Effect of Endophytic Bacteria on Jute Growth
Seeds treated with endophytic bacterial isolates as well as sterile distilled water were sown to clay pots containing a sterilized mixture of Coconut husk: Soil: Manure (1:1:1) in a greenhouse (30°C, 70% humidity). Two ml of the endophytic bacteria cells were added around the roots of 5-day-old aseptic seedlings of C. olitorius, C. capsularis, and Robi-1, and 2 ml of phosphate buffer of heat-killed endophytic bacteria cells were added as a negative control. Vegetative growth parameters such as Shoot length, Shoot diameter, root length, fresh weight, number of leaves, and leaf area were recorded after 30 days of growth. The fresh weights of the plants were determined by weighing the individual plants immediately after harvesting.
Primer design, RNA extraction, and expression profile
The primers of GA-20, GA-3, and GA-2 oxidase were designed using a web-based tool from IDT (http://sg.idtdna.com) and GenScript (www.genscript.com) followed by verification with Multiple Primer Analyzer (https://www.thermofisher.com). Primer specificity was evaluated by 1% agarose gel electrophoresis and melting curve analysis during qRT-PCR.
One gram of sample tissues was disrupted into a fine powder with mortar-pestle, and an in-house modified CTAB (Hossain et al. 2019) protocol was used to extract total RNA. Genomic DNA contamination was removed by amplification grade DNase I (Sigma Aldrich, Germany). NanoDrop 2000 spectrophotometer (NanoDrop, Thermo Scientific) determined the concentration of RNA, while the integrity was evaluated by 1% agarose gel electrophoresis. Complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) and random primers from 1 µg of total RNA according to the manufacturer’s protocol. Then the samples were treated with RNaseH (Thermo Fisher Scientific, USA) to remove residual RNA. The quantitative RT-PCR was performed with a Quantstudio Real-time PCR system (Applied Biosystems, USA) using 96 well-plate with a final volume of 20 μL containing 5 ng cDNA, 10 ul of 2X PowerUpTMSYBRTM Green Master Mix (Applied Biosystems, USA) and 300 nM of each primer. The qRT-PCR cycling for all genes was carried out according to the following conditions: an initial activation step of 2 min at 50°C, then a denaturation step for 10 min at 95°C followed by 40 cycles of 95°C for 15 s, the primer specific annealing temperatures (Supplementary table 1) for 20 s, and 72°C for 20 s. To check the specificity of the primers, a melting curve was generated at end of qRT-PCR cycles by a constant increment of temperature from 60°C to 95°C. The qRT-PCR reactions were performed with three biological replicates and three technical replicates for each biological replicate along with no template control. The expression level of the selected Gibberellin genes was analyzed with the comparative CT method (2-∆∆Ct) [Donoso et al. 2010] and two housekeeping genes such as HK-9 [Pedrosa et al. 2011] were used as internal control genes for normalization of qRT-PCR analysis.