2.1 Experimental site:
The experiment was carried out at the experimental station of Department of Plant Pathology, (31º29’42.2664”N, 74º17’49.1316”E, 217 m altitude) Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan, from March 2019 to April 2021. The local climate is semi-arid (Köppen climate classification BSh) with an average temperature of 40◦C and 350 mm annual rainfall and rainy season July-September.
2.2 Plant material and soil substrate:
Capsicum annum L. seeds (Yolo Wonder) were purchased from local seed market (Ghula Mandi, Lahore) and surface sterilised with 70% ethanol followed by 50% NaOHCl solution (100 mL of NaOHCl + 100 mL of distilled + 50 µL tween-20 detergent) and thrice washing with distilled deionized water. These seeds were sown in clay pots ¾ filled with sterilized 20% leaf compost soil (The soil texture was a sandy loam (82.88% sand, 13.04% silt, and 4.08% clay) with a pH of 7.88 and an electrical conductivity (EC) of 1.55 dS/m (measured using a pH meter and an EC meter); organic matter content (OM) of 0.54%; containing 3% total N and 1.5% total C; having a C/N ratio of 0.5; and containing 12, 68, and 100 mg·kg−1 of Ca, P, and K, respectively). Fully developed plants at 4-5 leaf stage were transplanted, into clay pots of bigger size (Volume: 2 L, 15.5 cm height × 14 cm width) (Joshi et al., 2019; Dhaliwal et al., 2017) with the same soil composition up to 1-2 inches depth with 2-3 plants per pot. Lighter irrigations were applied on day-to-day basis to keep water level as desired. Then established young plants in pots were transferred to open area where seedlings were exposed to light so that they can carry their photosynthetic activity (Al-Herbi et al., 2020).
2.3 Xanthomonas campestris pv. vesicatoria culture acquisition:
Pure culture of Xanthomonas campestris pv. vesicatoria (FCBP-DNA B0003) was acquired from First Fungal culture Bank of Pakistan (FCBP), Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan. The inoculum was prepared by re-culturing in LB broth (MERCK, USA) based on Lennox formulation and incubating on shaker at 120 rpm for 36 h at 28 ± 2 °C. Bacterial culture was then centrifuged at 5,000 rpm for 10 min at 4 °C. The suspension was diluted through serial dilution process to obtain bacterial concentration of 108 at 600 nm wavelength having optical density of 0.3 in spectrophotometer (Ibrahim and Al-Saleh, 2012).
2.4 Biochar and nano particles production:
TLUD portable kiln method (McLaughlin, 2010) on farm biochar production was used, with minor adjustments, to prepare biochar from Rice Straws collected from field area of University of the Punjab, new campus, Lahore, at pyrolysis temperature of 500 °C to be used in this experiment. Fly ash was procured from textile industry as left over after burning of corn cobs and coal as fuel. Physico-chemical properties of rice straw biochar and fly ash used for further nanoparticles production are summarized in table 1 and 2.
Nanocomposite of rice starw biochar (RSBNPs) and fly ash (FNPs) were isolated from their bulk materials following protocols of Yeu et al., 2019; Guo et al., 2020, by grinding bulk-biochar into a commercial blender to produce fine biochar powder. Fly ash obtained was already packed in sealed polythene bags. Fly ash (30 g) and fine biochar powder were mixed in 800 mL of sterile water, separately. Both the solutions were shaken vigorously and autoclaved to physically and thermally disperse the bulk forms of biochar fine powder and fly ash. After dispersion of bulk material, prepared solutions were passed through 500 µm filter membrane to remove large particles. Filtrates were centrifuged twice at 3500 rpm for 25 minutes to isolate the nanoparticles in supernatant based on density gradient. XRD analysis of Biochar and fly ash nanoparticles was done by following Du et al., 2020, from the Department of Physics, Lahore College for Women University, Lahore, Pakistan.
Biochar and fly ash nanoparticles were applied through drenching (Imada et al., 2016) to chilli plants by applying 50 mL of solution, containing nanoparticles (RSBNPs and FNPs), in the root zone by injecting with the help of disposable syringe (Telemart: 10cc, Bd).
Table 1: Physico-chemical characterization of rice straw biochar.
Parameter measured
|
Value
|
pH
|
9.3
|
Basic gps (meq/g)
|
7.8
|
Acidic gps (meq/g)
|
1.8
|
Ash%
|
50
|
Density (g/cm3)
|
0.28
|
Surface area (S BET ) (m2/g)
|
100
|
C wt%
|
53
|
H wt%
|
3.0
|
N wt%
|
1.5
|
O
|
42.4
|
C/N
|
25
|
H/C
|
0.08
|
O/C
|
0.79
|
Alkaline elements (ppm (ug/g) by dry weight)
|
K
|
14,000
|
Mg
|
3500
|
Na
|
2190
|
Ca
|
7354
|
Other Essential Elements
|
Fe
|
5754
|
P
|
2765
|
Heavy Toxic Elements
|
Zn
|
0.01
|
Mn
|
575
|
Al
|
4231
|
Cu
|
4
|
Table 2: Physico-chemical characterization of fly ash.
Parameter measured
|
Value
|
Ash %
|
46
|
pH
|
9.75
|
EC dSm-1
|
2.43
|
C%
|
39.3
|
N (g Kg-1)
|
6.71
|
P (g Kg-1)
|
2.97
|
K (g Kg-1)
|
0.31
|
Ca (g Kg-1)
|
2.51
|
Mg (g Kg-1)
|
1.37
|
S (g Kg-1)
|
7.52
|
2.5 Chili pepper plant inoculation and disease assessment:
The plants were grown for 7-8 days before the inoculation of the pathogen (X. campestris pv. vesicatoria). Leaves of chilli plant were injured by needle prick method of bacterial inoculation (Gao et al., 2021). In this method 8-10 clean needles were tightly held by rubber band at equal heights. These needles were used to damage the leaves. Slight gentle injuries were done to leaves to provide entry sites to bacteria. Then bacterial suspension was sprayed with the help of atomizer. Inoculated plants were covered again with polythene bags and water was sprinkled on inner bag surface to maintain high relative humidity.
The whole research trail was comprised of two groups. Each group was further divided into three treatments, having five replicates. Group I: Inoculated set: (1. Fly-ash nanoparticles + Xnth, 2. Rice Straw Biochar Nanoparticles + Xnth 3. Only Soil + Xnth). Group II: Un-inoculated set/Control group: (1. Fly-ash nanoparticles, 2. Rice Straw Biochar Nanoparticles, 3. Only Soil).
After application of nanoparticles and inoculation of pathogen, agronomic data were recorded as shoot and root length, weight (Sharma, 2018). Disease incidence was calculated by following formula
Disease severity was calculated by formulating a disease grading scale in which severity was rated from 0 to 4 grades with zero indicating minimal or no disease symptoms to grade four showing 76% or above leaf area infected (Chiang et al., 2017).
2.6 In vitro X. campestris pv. vesicatoria and other isolated potential bacterial and fungal pathogens growth inhibition assay:
Antimicrobial activity of RSBNPs and FNPs was investigated against X. campestris pv. vesicatoria used in this experiment. Agar well diffusion method (Irshad et al., 2018) was employed for the estimation of antimicrobial potential of RSBNPs and FNPs.
Antimicrobial activity of RSBNPs and fly FNPs was also investigated against microflora isolated from soil used in this experiment. Fungi and bacteria were isolated through serial dilution method on Malt Extract Agar (MEA) and Luria Bertani Agar (LBA), respectively. Agar well diffusion method (Irshad et al., 2018) was employed for estimation of antimicrobial potential of RSBNPs and FNPs against isolated fungal and bacterial isolates including Escherichia coli, Erwinia spp, Pseudomonas syringae, Xanthomonas campestris pv. citri, X. campestris pv. vesicatoria, Fusarium solani, F. oxysporum, Alternaria alternata and Alternaria solani,.
2.7 Statistical analysis:
The experimental data were analysed by ‘Statistix version 8.1’ analytical software by analysis of variance (ANOVA), while the means were differentiated by Tuckey’s HSD test at P = 0.05. Additionally, the percentage data were transformed for disease incidence, severity and in vitro bacterial growth inhibition before analysis.