Incorporation of Engineered Nanoparticles of Biochar and Fly Ash Against Bacterial Leaf Spot of Pepper

Abstract

In agriculture, the search for higher net profit is the main challenge in the economy of the producers and nano biochar attracts increasing interest in recent years due to its unique environmental behaviour and increasing the productivity of plants by inducing resistance against phyto-pathogens. The effect of rice straw biochar and fly ash nanoparticles (RSBNPs and FNPs, respectively) in combination with compost soil on bacterial leaf spot of pepper caused by Xanthomonas campestris pv. vesicatoria was investigated both in vitro and in vivo. The application of nanoparticles as soil amendment significantly improved the chili pepper plant growth. However, RSBNPs were more effective in enhancing the above and belowground plant biomass production. Moreover, both RSBNPs and FNPs, significantly reduced (30.5 and 22.5 %, respectively), while RSBNPs had shown in vitro growth inhibition of X. campestris pv. vesicatoria by more than 50%. The X-ray diffractometry of RSBNPs and FNPs highlighted the unique composition of nano forms which possibly contributed in enhancing the plant defence against invading X. campestris pv. vesicatoria.

On the basis of our findings, it is suggested that biochar and fly ash nanoparticles can be used for reclaiming the problem soil and enhance the crop productivity depending upon the nature of soil and the pathosystem under investigation.

1 Introduction

Capsicum or bell pepper or sweet pepper (Capsicum annum L.) is a crop of solanaceae family and genus ‘capsicum’. Basically characterized by their green color and bell shape. These medium-sized fruit pods have wonderful colors (green, red, orange and yellow) thick and brittle skin with a glossy outer cover, and a fleshy texture. It is highly appreciated crop being good source of vitamin A, C, E, thiamine, beta carotene, folic acid and vitamin B6 and great therapeutic values (El-Ghorab, et al., 2013). Pepper is a globally produced except Antarctica and economically very important and valuable crop throughout the world. Production of capsicum was 11 million metric tons in 1990 and became more than 30 million metric tons globally in two decades and further increases are still expected (Parisi et al., 2020). In Pakistan, the area under pepper has been 62,742 hectares in 2018-19 with a total production of 145,856 tonnes and comes at 5th position worldwide. Bacterial leaf spot (BLS) caused by Xanthomonas campestris pv. vesicatoria result in severe damage to sweet pepper. The bacterium attacks leaves, fruits, and stems causing blemishes on these plant parts. It is a gram-negative, rod-shaped bacterium that can survive in seeds and plant debris from one season to another (Frank et al. 2005; Osdaghi et al., 2016; Ramzan et al., 2021). Pathogen can devastate a pepper crop by early defoliation of infected leaves and disfiguring fruit. In severe cases, complete crop failure has occurred due to this disease. Marketable yield is reduced both by defoliation and damaged fruits (Stall et al., 2009; Parisi et al., 2020). For the management of BLS different techniques have been under application such as chemical control (Fayette et al., 2012), cultural methods (Roberts et al., 2004, Sevic et al., 2019), bio control strategies (Le et al., 2020), and use of resistant plant genome (Potnis et al., 2015).

In the recent years, organic amendment, including crop residues, compost, organic waste and biochar application has become auspicious strategy for the control of soil-borne diseases because of its strengths as, cost-effectiveness, resource utilization and environmental protection (Scotti et al. 2015; Bonanomi et al. 2018, Zahra et al., 2021). Biochar (BC) or black gold is noval organic soil amendment with some special physical and chemical properties has been increasingly discussed in agriculture as a strategy for the sequestration of recalcitrant carbon into soils to increase soil fertility, (Zahar et al., 2021) improve plant growth and suppression of soil borne diseases (Wang et al., 2019; Alharbi et al., 2020; Rasool et al., 2021). Fly ash has been defined as the fine particulate by-product released into the atmosphere together with gases as a result of combustion processes (Rose, 2018). The dynamic physic chemical properties (low bulk density (1.01–1.43 g cm − 3), hydraulic conductivity and specific gravity (1.6–3.1 g cm − 3), while the moisture retention ranging from 6.1% at 15 bar to 13.4% at 1/3 bar and being rich in P, K, Ca, Mg and S and micronutrients like Fe, Mn, Zn, Cu, Co, Ba, Mo, Cd and Ni) (Basu et al., 2009; Sharma and Sharma, 2017) of fly ash make it a potential source in agricultural applications, as improving biological and physic-chemical properties of soil (Sahu et al., 2017; Singh and Sukkul, 2019) as competent as the compost and Biochar are.

Incorporation of engineered nanoparticles has gained an undeniable importance in our daily life from electronics to medicine and agriculture. In agriculture, for instance, nano-pesticides, nano-fertilizers and nano-sensors are in direct applications to agricultural soils to get an enhanced crop productivity and reduce cost (Servin et al., 2015), or control plant pathogens (Sevin et al., 2017; Ashraf et al., 2021). Characterized nanoparticles of Fly ash (Liang et al., 2020; Uda et al., 2021) and Biochar (Das et al., 2021) have been extensively used in agriculture sector not only to reduce the hazards of deposited chemical pesticides and fertilizers but also to control infectious pathogen and to improve crop yields (Narayanasamy, 2019; Ramezani et al., 2019; Salama et al., 2021).

In this study we explored the synthesis of nanoparticles form rice straw biochar (RSBNPs) and fly ash (FNPs) and secondly to access the prepared nanoparticles potential against bacterial leaf spot of pepper caused by Xanthomonas campestris pv. vesicatoria.

2 Materials And Methods

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.

Table 2: Physico-chemical characterization of fly ash.

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.

4 Results

5 Discussion

The rise in unprecedented climatic changes like temperature and changing weather patterns, had worsened the situation over the past few decades. While annual crop losses due to insect pests and diseases is estimated to range from 20 to 40% of total agricultural produce worldwide further escalating the hostility to existing food insecurity (Savary et al., 2012). Discovery of innovative technological advancements in agriculture sector is mandatory, to supersede an otherwise deteriorating global food scenario, in a sustainable manner. The recent innovations in scientific research, particularly, the advent of molecular nanotechnology have provided a ray of hope against all the odds through its effective role in drug delivery, target specificity, diagnostics, anti-microbial activity in pharmacology and medicine industry (Prasad et al., 2017). Nanotechnology have marked its foot prints in the field of agricultural research byits utility in establishing disease and pest diagnostic systems, phytohormonal delivery systems, nano-barcoding, enhancing germination of seeds, providing nano-vector for successful transfer of genes, establishing efficient and targeted slow releasing chemical pesticides (Khandelwal et al., 2016).

Rangaraj et al., (2014) has reported that silica NPs as effective agents for building resistance against Fusarium oxysporum and Aspergillus niger in maize. Nanotechnology is being widely used in plant pathological studies (Abd-Elsalam & Prasad 2018). There exist thorough studies on effect of biochar in controlling plant diseases (Akhter et al., 2016). But major portion of studies on biochar involves macro fraction of biochar and material behaves differently when used in nano (10^− 9) size in contrast to their bulk/macro forms. The present study was designed to fulfil the research needs on nano fractions of biochar and their role in controlling plant disease. Yue et al. (2019) attributed the increase in plant growth in response to biochar NPs in negating the effect of allelopathic materials in soil (Yue et al., 2019). In accordance with our results, Xu et al. (2017) demonstrated that nano biochar possessed unique set of physical and chemical traits other than in their bulk forms which enhanced root lengths. High, surface reactive tendencies and capacities to disperse allow them to attach and interact easily to root surfaces that is quite beneficial for protection of roots by physical means against heavy metal adversities.

Moreover, the nano biochar due to its smaller particle size have high mobility in soils and helps to transport water (Chen et al., 2017; Lahiani et al., 2016). Bashir et al. (2020) used composts and ZnO-nanoparticles to evaluate their effect on growth parameters like dry weight of roots, shoots, husk, and kernels, plant height and spike length of the plant concerned. Results obtained suggested strong effect of used nanoparticles and compost material on the growth promotion. Furthermore, increased photosynthetic activity owing to nanoparticles inoculation, which reduce the effects of osmotic and oxidative stress, is well documented as the process increase the plant biomass (Haghighi & Pessarakli 2013; Siddiqui et al., 2014).

The decrease in disease incidence and severity can be attributed to, up regulation of innate immune response of plants against pathogens, due to induction of nanoparticles (Sathiyabama and Manikandan, 2016). Chandra et al. (2015) reported sufficient enhancement in plant’s response though activation of innate immunity by induction of chitosan nanoparticles which, in return, increased the activity of defense related enzymes. Enhancement in total phenolic compounds, anti-oxidative enzymes and genes involved in defense mechanism was also reported due to treatment of carbon base chitosan nanoparticles. Studies indicate that induction of carbon-based nanoparticles stimulate the production of enzymes related to defense mechanisms, like Peroxidase (PO), Phenyl Alanine amonia Lyase (PAL), Poly Phenol Oxidase (PPO), and plant defense regulating molecules such as beta 1–3 glucanase, nitric oxide (NO) and etc. Nitric oxide is involved in many physiological processes (Baudouin, 2011) including regulation of defense process in plants (Chandra et al., 2014; Acharya et al., 2011).

Disease incidence and severity percentages of bacterial pathogen were decreased that can be due to direct destructive effects of nanoparticles on the bacterial membrane as nano forms of materials are electro-statically active and interact with lipo-polysaccarhide structure of bacterial membrane. As, XRD data revealed novel characteristics of BNPs including azimuthal and parallel orientation of aromaticity, partly carbonized lamellae (Huang et al., 2009). The hump around 42–46^o due to C(100) proves a large amount of carbon present in sample and due to this carbon presence, a crystalline orientation is appeared in simple in the form of peaks (Huang et al., 2016; Chen et al., 2016; Mohan et al., 2018), While in case of fly ash NPs, the XRD data is the evidence of presence of different phases of Al and fly ash particles (Sobiecka 2013).

Secondly, nanoparticles constituting, mostly, heavy metals bind with DNA/RNA molecules of bacteria by passing through cell membrane and hinder transcription- translation process thus inhibiting bacterial proliferation (Xing et al., 2015). Nanoparticles trigger the production of salicylic acid (SA), a phytohormone that activates SAR mechanism in plants (Bari & Jones, 2009). Carbon material based nanoparticles triggered systemic acquired resistance mechanism that provides resistance to infection to remote plant tissue from the site of its production (García-sánchez et al., 2015).

Fly ash is known previously (Eswaran and Manivannan, 2007), to limit the papaya leaf curl disease spread along with the regulation of vector population (Bemisia tabaci). However, there are also risks associated with fly ash use including leaching of heavy metals or changes in microbial composition of the soil. So, caution should be practiced while using fly ash for agricultural purposes (Singh et al., 2010).

Owing to increasing food demand, rapidly changing climate, high pathogens adaptability to climatic changes and hazardous effects of least efficient chemical control measures, the need of natural, effective, climate friendly way of disease control is inevitable.

The NPs induced changes were significant regarding chilies growth and bacterial leaf spot suppression. However, the plant response to NPs was dependent on the source or material used for their production. RSBNPs could provide a better alternative to unchecked bulk use of pesticides. There is need to check the possible hazards like dose, toxicological issues and eco-acceptability of these nano forms. Further exploration NPs utility obtained from fly ash and biochar would certainly help in managing plant diseases and addressing environmental concerns associated with toxic pesticides.

6 Conclusion

In agriculture, the search for higher net profit is the main challenge in the economy of the producers and nano biochar attracts increasing interest in recent years due to its unique environmental behaviour and increasing the productivity of plants by inducing resistance against phyto-pathogens. The effect of rice straw biochar and fly ash nanoparticles (RSBNPs and FNPs, respectively) in combination with compost soil on bacterial leaf spot of pepper caused by Xanthomonas campestris pv. vesicatoria was investigated both in vitro and in vivo. The application of nanoparticles as soil amendment significantly improved the chili pepper plant growth. However, RSBNPs were more effective in enhancing the above and belowground plant biomass production. Moreover, both RSBNPs and FNPs, significantly reduced (30.5 and 22.5 %, respectively), while RSBNPs had shown in vitro growth inhibition of X. campestris pv. vesicatoria by more than 50%. The X-ray diffractometry of RSBNPs and FNPs highlighted the unique composition of nano forms which possibly contributed in enhancing the plant defence against invading X. campestris pv. vesicatoria.

On the basis of our findings, it is suggested that biochar and fly ash nanoparticles can be used for reclaiming the problem soil and enhance the crop productivity depending upon the nature of soil and the pathosystem under investigation.

Declarations

Availability of data and materials:

Not Applicable

Author Contributions: Authors contribute their part in performing experiments and drafting of the manuscript: M.D.A, W.A, M.A, performing additional experiments and characterizations: F.M and Z.A, U.A, A.A, I.A, and A.A plaining and supervision. All authors have read the manuscript and agreed for publishing.

Ethics approval and consent participate: 

Not Applicable

Consent for Publications:

All authors have read the manuscript and agreed for publishing.

Funding: This research received no external funding.

Acknowledgements:

The authors are thankful to the University of the Punjab Lahore for providing the resources to conduct the study.

Competing Interest: The authors declare no conflict of interest.

References

1. Abd-Elsalam KA, Prasad R (2018). Nanobiotechnology applications in plant protection. Springer. Akhter A, Hage-Ahmed K, Soja G, Steinkellner. S. (2016).
2. Potential of Fusarium wilt-inducing chlamydospores, in vitro behaviour in root exudates and physiology of tomato in biochar and compost amended soil. Plant Soil. 406:425–440.
3. Al-harbi AR, Obadi A, Al-OmranA M, Abdel-Razzaq H. Sweet peppers yield and quality as affected by biochar and compost as soil amendments under partial root irrigation. Journal of the Saudi Society of Agricultural Sciences. 2020;19(7):452–60.
4. Ashraf A, Anjum T, Riaz S, Ahmed IS, Irudayaraj J, Javed S, Qaiser U, Naseem S. (2021). Inhibition mechanism of green-synthesized copper oxide nanoparticles from Cassia fistula towards Fusarium oxysporum by boosting growth and defense response in tomatoes. Environmental Scince: Nano 6: https://doi.org/10.1039/d0en01281e.
5. Bari R, Jones JDG. Role of plant hormones in plant defence responses. Plant Mol Biol. 2009;69(4):473–88.
6. Basu M. Pande M. Bhadoria PBS, Mahapatra SC. Potential fly-ash utilization in agriculture: A global review. Prog Nat Sci. 2009;19(10):1173–86.
7. Bonanomi G, Lorito M, Vinale F, Woo SL. Organic amendments, beneficial microbes, and soil microbiota: toward a unified framework for disease suppression. Annual Review of Phytopathology. 2018;56:1–20.
8. Chen T, Liu R, Scott NR. Characterization of energy carriers obtained from the pyrolysis of white ash, switchgrass and corn stover—Biochar, syngas and bio-oil. Fuel Process Technol. 2016;142:124–34.
9. Chiang KS, Liu HI, Tsai JW, Bock CH. A discussion on disease severity index values. Part II: using the disease severity index for null hypothesis testing. Ann Appl Biol. 2017;171(3):490–505. https://doi.org/10.1111/aab.12396.
10. Das Sk, Ghosh GK, Avasthe R. Applications of biomass derived biochar in modern science and technology. Environmental Technology Innovation. 2021;21:101306. https://doi.org/10.1016/j.eti.2020.101306.
11. Dhaliwal MS, Sharma SP, Jindal SK, Dhaliwal LK, Gaikwad AK. Growth and yield of bell pepper as influenced by growing environment, mulch, and planting date. Journal of Crop Improvement. 2017;31(6):830–46. https://doi.org/10.1080/15427528.2017.1391146.
12. Eswaran A, Manivannan K. Effect of foliar application of lignite fly ash on the management of papaya leaf curl disease. Acta Hort (ISHS). 2007;740:271–5. http://www.actahort.org/books/740/740_33.htm.
13. El-Ghorab AH, Javed Q, Anjum FM, Hamed SF, Shaaban HA. Pakistani bell pepper (Capsicum annum L.): chemical compositions and its antioxidant activity. Int J Food Prop. 2013;16(1):18–32.
14. Fayette J, Roberts PD, Pernezny KL, Jones JB. The role of cymoxanil and famoxadone in the management of bacterial spot on tomato and pepper and bacterial leaf spot on lettuce. Crop Protection. 2012;31:107–12. http://dx.doi.org/10.1016/j.cropro.2011.09.006.
15. Frank T. Insights into Genome Plasticity and Pathogenicity of the Plant Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria Revealed by the Complete Genome Sequence. J Bacteriol. 2005;187(21):7254–66.
16. Frenkel O, Jaiswal AK, Elad Y, Lew B, Kammann C, Graber ER. The effect of biochar on plant diseases: what should we learn while designing biochar substrates? Journal of Environmental Engineering Landscape Management. 2017;25(02):105–13. https://doi.org/10.3846/16486897.2017.1307202.
17. Gao S, Wang F, Niran J, Li N, Yin Y, Yu C, Jiao C, Yao M. Transcriptome analysis reveals defense-related genes and pathways against Xanthomonas campestris pv. vesicatoria in pepper (Capsicum annuum L.). Plos One. 2021. https://doi.org/10.1371/journal.pone.0240279.
18. García-sánchez S, Bernales I, Cristobal S. (2015). Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair development through salicylic acid signalling Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair. ??? https://doi.org/10.1186/s12864-015-1530-4.
19. Huang Y, Yin X, Wu C, Wang C, Xie J, Zhou Z, … & Li H. Effects of metal catalysts on CO2 gasification reactivity of biomass char. Biotechnology advances. 2009;27(5):568–72.
20. Ibrahim Y, Al-Selah M. First Report of Bacterial Spot Caused by Xanthomonas campestris pv. vesicatoria on Sweet Pepper (Capsicum annuum L.) in Saudi Arabia. Plant Disease. 2012;96(11):1690. doi:10.1094/PDIS-04-12-0354-PDN.
21. Imada K, Sakai S, Kajihara H, Tanaka S, Ito S. Magnesium oxide nanoparticles induce systemic resistance in tomato against bacterial wilt disease. Plant Pathol. 2016;65(4):551–60. https://doi.org/10.1111/ppa.12443.
22. Inam A. Use of flyash in turnip (Brassica rapa L.) cultivation. Pollution Research. 2007;26(1):39–42.
23. Irshad S, Salamat A, Anjum AA, Sana S, Saleem RS, Naheed A. Green tea leaves mediated ZnO nanoparticles and its antimicrobial activity. Cogent Chemistry. 2018;4:1. https://doi.org/10.1080/23312009.2018.1469207.
24. Joshi NC, Ratner K, Eidelman O, Bednarczyk D, Zur N, Many Y, Shahak Y, Aviv-Sharon E, Achiam M, Gilad Z, Charuvi D. Effects of daytime intra-canopy LED illumination on photosynthesis and productivity of bell pepper grown in protected cultivation. Scientia Horticulturae. 2019;250:81–8. https://doi.org/10.1016/j.scienta.2019.02.039.
25. Khandelwal N, Barbole RS, Banerjee SS, Chate GP, Biradar AV, Khandare JJ, Giri AP. 2016. Budding trends in integrated pest management using advanced micro-and nano-materials: Challenges and perspectives. Journal of environmental management, 184, pp.157–169.
26. Kumar V, Mathur M, Kharia Sharma Preeti. Flyash management: vision for the new millennium. In: Proceedings of 2nd international conference on flyash disposal and utilization, vol. I, FAM & CBIP, New Delhi; 2–4 February 2000. p. (i)1–9.
27. Le KD, Kim J, Yu NH, Kim B, Lee CW, Kim J. 2020. Biological Control of Tomato Bacterial Wilt, Kimchi Cabbage Soft Rot, and Red Pepper Bacterial Leaf Spot Using Paenibacillus elgii JCK-5075. Frontiers in Plant Sciences. https://doi.org/10.3389/fpls.2020.00775.
28. Lee SB, Lee YB, Lee CH, Hong CO, Kim PJ, Yu C. Characteristics of boron accumulation by fly ash application in paddy soil. Biores Technol. 2008;99(13):5928–32. https://doi.org/10.1016/j.biortech.2007.11.022.
29. Liang G, Li Y, Yang C, Zi C, Zhang Y, Hu X, Zhao W. Production of biosilica nanoparticles from biomass power plant fly ash. Waste Management. 2020;105:8–17. https://doi.org/10.1016/j.wasman.2020.01.033.
30. Mohan D, Abhishek K, Sarswat A, Patel M, Singh P, Pittman CU. Biochar production and applications in soil fertility and carbon sequestration–a sustainable solution to crop-residue burning in India. RSC Adv. 2018;8(1):508–20.
31. Narayanasamy P. Potential and Futuristics of Fly Ash Nanoparticle Technology in Pest Control in Agriculture and Synthesis of Chemical and Herbal Insecticides Formulations. In: Ghosh S, Kumar V, editors. Circular Economy and Fly Ash Management. Singapore: Springer; 2020. https://doi.org/10.1007/978-981-15-0014-5_7.
32. Osdaghi E, Taghavi SM, Hamzehzarghani H, Lamichhane JR. Occurrence and characterization of the bacterial spot pathogen Xanthomonas euvesicatoria on pepper. Iranian Journal of Phytopathology. 2016;164:722–34.
33. Parisi M, Alioto D, Tripodi P. Overview of Biotic Stresses in Pepper (Capsicum spp.): Sources of Genetic Resistance, Molecular Breeding and Genomics. International Journal of Molecular Sciences. 2020;21:2587. doi:10.3390/ijms21072587.
34. Prasad R, Bhattacharyya A, Nguyen QD. Nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives. Front Microbiol. 2017;8:1014.
35. Potnis N, Timilsina S, Strayer A, Shantharaj D, Barak DJ, Paret ML, Vallad GE, Jones JB. Bacterial spot of tomato and pepper: diverse Xanthomonas species with a wide variety of virulence factors posing a worldwide challenge. Molecular Plant Pathology. 2015;16(9):907–20. https://doi.org/10.1111/mpp.12244.
36. Ramezani M, Ramezani F, Gerami M. Nanoparticles in Pest Incidences and Plant Disease Control. In: Panpatte D, Jhala Y, editors. Nanotechnology for Agriculture: Crop Production & Protection. Singapore: Springer; 2019. https://doi.org/10.1007/978-981-32-9374-8_12.
37. Ramzan M, Sana S, Javaid N, Shah AA, Ejaz S, Malik WN, Yasin NA, Alamri S, Siddiqui MH, Dutta R, Fahad S, Tahir N, Mubeen S, Ali MA, El Sabagh A, Danish S. 2021 Mitigation of bacterial spot disease induced biotic stress in Capsicum annuum L. cultivars via antioxidant enzymes and isoforms. Sci Rep. 2021;11:9445. https://doi.org/10.1038/s41598-021-88797-1.
38. Rangaraj S, Gopalu K, Muthusamy P, Rathinam Y, Venkatachalam R, Narayanasamy K. Augmented biocontrol action of silica nanoparticles and Pseudomonas fluorescens bioformulant in maize (Zea mays L.). RSC Advances. 2014;4(17):8461–5.
39. Rasool M, Akhtar A, Soja G, Haider MS. 2021. Role of biochar, compost and plant growth promoting rhizobacteria in the management of tomato early blight disease. Scientific Reports 11: 6092 https://dx.doi.org/10.1038%2Fs41598-021-85633-4.
40. Roberts PD, Adkins S, Pernezny K. Jones JB. Diseases of Pepper and their Management. Vol. II: Diseases of Fruits and Vegetables; 2004. pp. 333–87.
41. Sahu G, Bag AG, Chatterjee N, Mukherjee AK. Potential use of flyash in agriculture: A way to improve soil health. Journal of Pharmacgnosy Phytochemistry. 2017;6(6):873–80.
42. Salama DM, Abdel-Aziz ME, El-Naggar ME, Shaaban EA, Abd -El-Wahed MS. Synthesis of an eco-friendly nanocomposite fertilizer for common bean based on carbon nanoparticles from agricultural waste biochar. Pedosphere. 2021;31(6):923–33. https://doi.org/10.1016/S1002-0160(21)60024-3.
43. Savary S, Ficke A, Aubertot J, Hollier C. (2012). Crop losses due to diseases and their implications for global food production losses and food security. (Smil 2000). https://doi.org/10.1007/s12571-012-0200-5.
44. Scotti R, Bonanomi G, Scelza R, Zoina A, Rao MA. Organic amendments as sustainable tool to recovery fertility in intensive agricultural systems. Journal of Soil Science Plant Nutrition. 2015;15:333–52.
45. Servin AD, Torre-roche RDL, Castillo-Michel H, Pagano L, Hawthorne J, Musante C, Pignatello J, Uchimiya M, White JC. Exposure of agricultural crops to nanoparticle CeO₂ in biochar-amended soil. Plant Physiology Biochemistry. 2017;110:147–15. https://doi.org/10.1016/j.plaphy.2016.06.003.
46. Singh L, Sukul P. Effect of organic manure, organic fertilizers, and fly ash on physical and electrochemical properties of soil under maize cultivation. Plant Archives. 2019;19(2):2797–800.
47. Singh RP, Gupta AK, Ibrahim MH, Mittal AK. Coal fly ash utilization in agriculture: its potential benefits and risks. Reviews in Environmental Science Bio/Technology. 2010;9(4):345–58.
48. Sevic M, Gasic K, Ignjatov M, Mijatovic M, Prokic A, Obradovic A. Integration of biological and conventional treatments in control of pepper bacterial spot. Crop Protection. 2019;119:46–51.
49. Sharma DK. Bio-efficacy of fungal and bacterial antagonists against pv. Xanthomonas axonopodis vesicatoria Capsicum (Doidge) Dye in chilli (spp.) grown in Rajasthan. Asian Journal of Pharmacy Pharmacology. 2018;4(2):207–13. https://doi.org/10.31024/ajpp.2018.4.2.18.
50. Sharma IP, Sharma AK. Short Communication Use of Fly-ash (Industrial Residue) for Improving Alkaline Soil Status. Int J Soil Sci. 2017;12(1):39–42. 10.3923/ijss.2017.39.42.. ( :. DOI.
51. Sobiecka E. Investigating the chemical stabilization of hazardous waste material (fly ash) encapsulated in Portland cement. Int J Environ Sci Technol. 2013;10(6):pp.1219–24.
52. Stall RE, Jones JB, and Minsavage G. V. Durability of Resistance in Tomato and Pepper to Xanthomonads Causing Bacterial Spot. Annual Review of Phytopathology. 2009;47:265–84.
53. Uda MNA, Gopinath SCB, Hashim U, Halim NH, Parmin NA, Uda MNA, Anbu P. 2021. Production and characterization of silica nanoparticles from fly ash: conversion of agro-waste into resource. Preparative Biochemistry Biotechnology 51(1): https://doi.org/10.1080/10826068.2020.1793174.
54. Wang G, Govinden R, Chenia HY, Ma Y, Guo D, Ren G. 2019. Suppression of Phytophthora blight of pepper by biochar amendment is associated with improved soil bacterial properties. Biology and Fertility of Soils 55, 813–824 (2019). https://doi.org/10.1007/s00374-019-01391-6.
55. Xing K, Zhu X, Peng X, Qin S. Chitosan antimicrobial and eliciting properties for pest control in agriculture: a review. Agron Sustain Dev. 2015;35(2):569–88.
56. Yue L, Lian F, Han Y, Bao Q, Wang Z, Xing B. The effect of biochar nanoparticles on rice plant growth and the uptake of heavy metals: Implications for agronomic benefits and potential risk. Science of The Total Environment. 2019;656:9 – 1.
57. Zahra MB, Aftab ZH, Haider MS. Water productivity, yield and agronomic attributes of maize crop in response to varied irrigation levels and biochar-compost application. Journal of the Science of Food Agriculture. 2021;101(11):4591–604. 10.1002/jsfa.11102.. ). DOI.
58. Zahra MB, Aftab ZH, Akhtar A, Haider. M.S. (2021). Cumulative effect of biochar and compost on nutritional profile of soil and maize productivity. Journal of Plant Nutrition. 44:11, 1664–1676 https://doi.org/10.1080/01904167.2021.1871743.