Assessment of a nanobiofungicide with antimicrobial potential against mycopathogenic Fusarium species

1 Introduction

Nanoformulation of fungicides can be environmentally advantageous with respect to traditional ways of application (e.g. spray). With the more controlled delivery of the active ingredients, the evaporation and leaching of substances to the environment is reduced (de Oliveira, 2021). They also help to reduce the application dosage level (Nuruzzaman et al., 2016) and be designed improve the solubility and stability of biopesticide (Damalas & Koutroubas, 2018). Biotechnological approaches to control phytopathogens and enhanced plant health are in use in agriculture; they aim to be environment friendly without neglecting crops’ needs (Alina et al., 2015). In contrast, synthetic fungicides can have adverse effects on the ecosystem and public health due to their toxicity and uncontrolled released to the surrounding environment of the crop where they are applied (Mullin et al., 2010). Particular idiosyncrasies have arisen in innovative nano-agrochemicals due to the viable applications of nanotechnology in a myriad of agriculture sectors. Furthermore, the fungicides exposure is of various types for instance direct occupational, direct non-occupational and indirect exposure. Occupational exposure is the most unsafe one, as interrelated to broad range illnesses for instance airway obstruction and lungs diseases. The possible genetic damage initiated by occupational pesticide exposure is much more than smoking and alcohol consumption (Nascimento et al., 2022). Numerous epidemiological and molecular researches have established a close relationship between persistent pesticides exposure and increased diseases threat for instance endocrine disruptors, neurodegenerative and reproductive disorders, respiratory problems and birth deficiencies (Gea et al., 2022). Besides, teratogenic, carcinogenic and mutagenic natures of fungicides are assumed to be a causative source of cancer growth in humans (Gea et al., 2022).

The Fusarium genus contains one of the utmost complex and adaptive species the Eumycota. Fusarium oxysporum (Fo) species complex includes plant, animals and human pathogens and a diverse of non-pathogens (Gordon, 2017). Members of Fusarium species are ubiquitous soil borne pathogens of a wide range of horticultural and food crops which cause destructive vascular wilts, rots and damping off diseases (Bodah, 2017; Xia et al., 2021). Some Fusarium species are capable of producing mycotoxins, which are the most important natural contaminants of food and agricultural commodities (Khan et al., 2013; Kumar et al., 2016) and suspected to be implicated in numerous diseases among living beings (Barajas-Ramirez et al., 2021; Zou et al., 2022). Fusarium oxysporium (Fo) is an important soil inhabiting fungus known for its phylogenetic diversity, some of its strain are saprophytic or non-pathogenic. Nevertheless, pathogenic strains cause destructive vascular wilt disease and often limit the production of economically important agricultural commodities (Shahzad et al., 2017).

A major concern in agriculture is the phytopathological diseases caused by several pathogens for instance citrus black spot, speck rot, Fusariosis, rice blast and dessert/beer bananas by guignardia citricarpa, phacidiopycnis washingtonensis, Fusarium guttiforme, magnaporthe oryzae and Fusarium oxysporium f. sp., respectively that resulting in billions of dollars lost due to the spoilage and damage of crops (Carnielli-Queiroz et al., 2019; Faganello et al., 2017; Ndayihanzamaso et al., 2020; Sikdar et al., 2014). To protect crops from the damaging effects of phytopathogens, it has been estimated that more than 600 million dollars are spent on fungicides each year (González-Fernández et al., 2010). About 25 % of food crops are damaged by mycotoxins only (Patel et al., 2014). Soils are involved in suppressing plant diseases and microorganisms and are proposed to be involved in suppressiveness. Formulations with enzymes from bacterial showing anti-phytopathogenic activities could be used as an alternative source for biological control. A very promising candidate for biocontrol are microbial Chitinases (Zhang et al., 2001).

With reference to Chitinases hydrolyse, are the second most abundant polysaccharides into its residues. They have been classified into Glycosyl Hydrolase (GH) family 18 and 19. The GH family-18 Chitinases have been found extensively in bacteria and its existence in rectangle area (IDGIDDYE) in the amino acid sequence that specifies its association to the glycosyl hydrolase family-18. In addition, Chitinases can be endochitinases, which cleave the chitin polymers at internal sites to generate monomers of N-acetyl-D-glucosamine, and exochitinases hydrolyze the chitin into chitotriose, chitobiose and N-acetyl-D-glucosamine (Essghaier et al., 2021).

The potential use of Chitinolytic (Chitinase) enzymes makes them an attractive candidate for a wide range of biotechnological approaches, such as recycling nitrogen and carbon through chitin hydrolyzation. The Chitinase enzyme is responsible for the degradation of chitin, which is found to be produced by wide range of organisms. Though chitin is a major constituent of fungal cell walls and invertebrate exoskeleton. Bacterial Chitinases can be generated at low cost for industrial purpose. Because bacterial Chitinase is thermostable, hydrophobic in nature, with less occurrence of thermolabile residues (Dutta et al., 2021). The use of nano-materials as novel approaches has been widely studied to enhance crop yields because they offer advantages (Servin et al., 2015). They offer increase surface area with subsequent affinity for the active substance as well as greater coverage of the surface area of the plant. In relation to pros and cons of this study, nanobiofungicide can be prepared in a simple cost effective way as biohybrid nanocide materials, a new environmentally friendly nanobiofungicide against numerous fungal pathogenic organisms. Hybrids are prepared by means of the bonding between harmless antimicrobial agents; comprising Chitinases (microbial), usually known as safe (GRAS) constituents that improve the synergistic activity. The current work will describe the preparation of a new hybrid functional nan- antimicrobial with inorganic particles, biopolymers, and to test it’s effect against the most devastating fungus: Fusarium species.

2 Materials and Methods

3 Results

3.2

3.2 Optimization parameter of Chitinase production

The effect of incubation time on Chitinase production is shown in Fig. 1. Its maximum production took place between 48 and 72 h of incubation. Thus, the incubation period of 72 h was chosen to test the effect of different parameters on the enzyme production. After the incubation, cells were harvested; cell-free supernatant was taken and assessed for Chitinase enzyme activity. The initial pH of the production media influences the availability of the metabolic ions because microorganisms are quite sensitive to proton concentrations in the media. Thus, Chitinolytic microbe was cultivated in the medium with different pH (5.5–9.0). The finding revealed that pH of the medium actively affected the growth and activity of the microbes (Fig. 2). pH 7.5 led to the maximum enzymatic activity. The incubation temperature for Chitinase production was investigated by culturing at different temperatures for instance 25 °C, 30 °C, 37 °C, 45 °C, 50 °C and 55 °C to achieve maximum Chitinase. The influence of temperature on Chitinase production exhibited maximum Chitinase activity at 45 °C. The obtained results have been depicted in Fig. 3. The extra-cellular Chitinases were produced by Bacillus licheniformis AG-W3, during different growth phases.

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Fig. 1

Screening of Chitinase activity in the supernatant of bacterial isolates with incubatio time (Bacillus licheniformis AG-W3).

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Fig. 2

Effect of pH effects on selected hyperchitinase-producing strain (Bacillus licheniformis AG-W3).

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Fig. 3

Comparative study of selected hyperchitinase producing strain (Bacillus licheniformis AG-W3) in different temperatures.

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3.3

3.3 Characterization of nanoparticles

SEM analysis of ZNPs shows the spherical shape of the particles but due to the high surface energy and being in dry form they are aggregated (Fig. 4A). The diameter of the particles calculated with XRD (Eq. (1) was 46 and it agrees with the direct observations in SEM (38–76 nm). The elemental composition of the nanoparticles was observed by EDS (Fig. 4B). The zinc, oxygen with traces of copper amounts have been presented in Table 1. Analysis of ZNPs by XRD showed the diffraction pattern produced from peak intensities, related to size and shape. The XRD 2 theta degrees signals were at positions; 31.66, 34.35, 36.19, 56.45, and 62.73 of the spectra respectively (Fig. 5), the d-spacing from this pattern confirm the formation of ZnO nanoparticles.

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Fig. 4

(A) SEM image of zinc oxide nanoparticles (B) EDX spectrum of a representative ZNPs.

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Table 1 Chemical composition of elements EDX of ZNPs.

Element	(%)	Atomic (%)
Zinc	71.85	38.97
Oxygen	27.33	60.57
Copper	0.82	0.46
Total	100	100

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Fig. 5

X-ray powder diffractogram of the prepared ZNPs.

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3.4

3.4 Inhibitory effect of NPs on Chitinase activity

The purified enzyme was pre-incubated with the ZNPs in phosphate buffer with a concentration ranging from 0.001 to 0.1 % (mass of ZNPs /mass media) for 1 h without substrate. The residual activity of the purified enzyme was estimated by adding colloidal chitin as substrate with standard assay protocol. In presence of 0.001 % ZNPs Chitinase showed ̴ 100 % residual activity, followed by a gradual decline as the concentration of nanoparticles increased. At concentrations <0.001 % ZNPs, it was observed that nanoparticles have no significant inhibitory effect on Chitinase activity. The residual activity of Chitinase was high and moderate in the presence of ZNPs with concentrations 0.001 %-0.01 %. Nonetheless moderate inhibition effect (∼9%) was observed at 0.1 % ZNP concentration as shown in Fig. 6.

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Fig. 6

Inhibitory effect of nanoparticles on Chitinase residual activity.

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3.5

3.5 In vitro antifungal bioassay

During the in vitro bioassay, it was observed that mycelial growth of Fusarium species was comprehensively controlled by 0.06 and 0.1 % concentrations of ZNPs keeping the enzyme (Chitinase) concentration either at 0.015 or 0.03 IU/ml. Mycelial growth inhibition was observed in Fusarium species exhibiting a dose dependency effect (Table 2). The maximum inhibitory effects (89.5 %) were recorded at 0.1 % ZNPs concentration with 0.03 IU/ml of Chitinase, while the minimum inhibitory effect was observed at 0.001 % concentration. ZNPs concentrations 0.001–0.01 % showed a gradual increase in growth inhibition. Thickening and rupturing of hyphal cell wall along with constriction of hyphal diameter were observed. Sporulation and low hyphal growth were also noticed with a compound microscope (Fig. 7aA & B). Rupture of the fungal cell wall was observed in SEM. (Fig. 7bC).

Table 2 Effect of ZNPs and Chitinase on in-vitro mycelial growth on mycopathogenic fungi.

NPs (%)	Mycelial growth inhibition%
	0	0.015 IU/ml Chitinase	0.03 IU/ml Chitinase
0	0	5.0	10.0
0.001	34.8 ± 2.2	45.8 ± 2.2	62.5 ± 2.1
0.005	48.5 ± 3.7	62.3 ± 1.7	76.8 ± 1.1
0.01	56.0 ± 0.8	72.2 ± 3.1	84.0 ± 1.8
0.02	61.3 ± 0.2	79.6 ± 0.2	87.3 ± 0.2
0.06	65.4 ± 0.1	81.1 ± 1.0	87.5 ± 0.4
0.1	69.3 ± 0.7	83.1 ± 3.2	89.1 ± 1.4

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Fig. 7a

A: micrograph of antifungal bioassay displaying the thickening and rupture of the hyphal cell wall of the fungi and constriction of the hyphal diameter (Fig. 7aA), while (Fig. 7aB) shows sporulation and low hyphal growth.

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Fig. 7b

Antifungal bioassay presenting rupture of the fungal cell wall designated with arrows (Electron microscopic studies).

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4 Discussion

In the current study, the Chitinolytic organisms were isolated from water and soil samples of diverse habitats, such as agricultural fields, springs, fish markets, and domestic samples. Though Chitinolytic microbes are ubiquitous in distribution, nevertheless, different studies showed their isolation from particular sources such as cotton fields (Jha et al., 2016), from potato fields (Servin et al., 2015), freshwater ponds for shrimp (Hsu and Lockwood, 1975), degraded stalk of mushrooms (Kiyani et al., 2019)coastal soil enriched with crab shells (Kanwal et al., 2010), lily plant (Jha et al., 2016), from marine environment Marine bacterial Chitinase as sources of energy, eco-friendly agent, and industrial biocatalyst (Jahromi & Barzkar, 2018), red palm weavil’s gut (Khiyami & Masmali, 2008) and intestine of the South American sea lion (Konagaya et al., 2006).

The profile of enzyme production of the isolates during the present research was assessed for five days, by cultivating in a minimum salt medium along with colloidal chitin, incubated under shaking conditions. During this study, the maximum enzyme production was observed on 3rd day of incubation. A rise in Chitinase production was obtained during the exponential growth phase to the stationary growth phase. Previous investigation results reflect that the incubation time is governed by cultural characteristics. Such type of investigation reported that enzyme production depends on the growth rate of microbial culture (Chakrabortty et al., 2012). As observed from the level of protein, it was evident that Chitinase production was indeed correlated to the growth of the microbial culture in the selected medium. Previous reports demonstrated that a rise in Chitinase production by Streptomyces hygroscopicus is shown from the exponential growth phase to the stationary growth phase (Priya et al., 2011). Similar results were obtained from Bacillus circulans (n°4.1) on the 4th day (Wiwat et al., 1999). Maximum Chitinase production from Streptomyces spp. NK1057 was been reported after the 5th day of incubation (Nawani & Kapadnis, 2004) and Beauveria bassiana (Suresh & Chandrasekaran, 1998).

The pH of the medium affects the growth and activity of the microorganisms. The results of an earlier investigation found that Bacillus subtalis produce maximum Chitinases at a pH 7–8. Previous studies reflect the hypothesis that most bacterial species produce Chitinase at neutral pH or slightly alkaline such as certain Bacillus strains and Pseudomonas aeruginosa K-187, these produced maximum Chitinase at pH 7 (Ghorbel-Bellaaj et al., 2012). Previous studies suggested that A. xylosoxydans (Vaidya et al., 2001), Micrococcus sp. AG84 (Kuddus & Ahmad, 2013), Serratia marcescens XJ-01 (J.-L. Xia et al., 2011) and Aeromonas spp. JKI (Al-Ahmadi et al., 2008), produced maximum Chitinases under alkaline conditions. B. pabuli K1 produced maximum Chitinase at pH 8 (Frändberg & Schnürer, 1994). B. laterosporus produces Chitinase at pH 8 (Shanmugaiah et al., 2008).

During this investigation, the influence of temperature on Chitinase production exhibited maximum Chitinase activity at 45 °C. Temperature also influenced protein denaturation, cell growth, and enzyme inhibition, thus it plays a significant role in biological processes (Chakrabortty et al., 2012). B. licheniformis show maximum Chitinase activity at 50 °C (Toharisman et al., 2005) whereas Tsujibo et al. have reported the maximum Chitinase activity at 50 °C by Streptomyces thermoviolaceous OPC-520 (Tsujibo et al., 1993), which is same as that B. Licheniformis. Strain JS. Bacillus p. BG-11 produces Chitinases at 50 °C (Bhushan, 2000). On the other hand, Chitinase productions from Serratia marcescenses at 30 °C were reported by Kannan and co-workers (Kannan et al., 2010). It is unclear why some mesophiles have evolved thermostable enzymes, but the such strategy may enable energy conservation through a decreased need for enzyme synthesis due to increased enzymatic stability (Yeoman et al., 2010). Thermostable enzymes can be obtained from thermophile as well as mesophilic organisms. Thermostable enzymes used by industry are still produced from mesophiles and commercial enzymes thermophiles are still scarce (Coolbear et al., 1992).

Chitinase with alkaline pH is considered to have major potential in the biological control of insect pests. Chitinases with better stability and activity in these conditions can be used in synergism with other biocontrol agents. Although alkaline Chitinases are also considered useful in the management of Chitinous wastes, generated by seafood manufacturing industries (Nawani & Kapadnis, 2003). Metals ions may result in enhancing the enzyme activity by acting as a binding between enzyme and substrate, combing with both, thus holding the substrate at the active site of the enzyme. The effect of metal ions may also be due to the participation of sulfhydryl groups in the active site of the enzyme (Tsujibo et al., 1993).

During the present study, the application of isolated Chitinase enzyme as an antifungal agent showed the results of approximately10% mycelial growth inhibition. While ZNPs application with different concentrations revealed a maximum fungal mycelial growth inhibition of 69.3 % by using the ZNPs concentration (0.1 %). The results of ZNPs used as an antimicrobial agent during earlier studied, revealed a significant inhibitory effect on the growth of B. cinerea by disturbing cell features and causing mat deformity while showing the inhibitory effect on P. expansum conidiophore and conidia development (He et al., 2011). ZNPs are far less toxic to plants and plant beneficial soil micro-organisms, could be better than AgNPs in N mediated plant safety against fungal pathogens (Dimkpa et al., 2013).

The ZNPs along with the Chitinase enzyme (0.03 IU/ml) exhibited a maximum of 89.9 % mycelial growth inhibition, which was recorded during this project. Nanoformulations can advance prevention of pest defense, stability of pesticides in the field, and be benign to both plants and animals, while killing the pests with new modes of action. This formulation might be prepared with low cost (Athanassiou et al., 2018; Benelli et al., 2016).

ZNPs along with Chitinase enzyme exhibit a maximum 89.5 % mycelial growth inhibition. Previous work demonstrated that ZNPs inhibited the growth of B. cinerea by disturbing cell features and causing mate deformity ultimately resulting in the death of fungal hyphae (He et al., 2011; Nuruzzaman et al., 2016). Similar results were obtained by using a 0.1 % concentration of NCPs, against A. alternate, M. phaseolina and R. solani, that showed 89.5 %, 63.0 %, and 60.1 % growth inhibition, respectively (Saharan et al., 2013). Copper-chitosan nanoparticles inhibited mycelial growth and scolorotia formation in R. solani and S. rolfsii (Rubina et al., 2017). Zataria multiflora (Essential Oil) encapsulated in chitosan nanoparticles with a controlled release reduced the occurrence of B. cinerea (Mohammadi et al., 2015). The study therefore confirms the fungicidal nature of the nanoformulations and the potential uses of these nanoformulations as an alternative to chemical fungicides for management of mycopathogenic Fusarium species. However, further studies should be conducted to evaluate the efficacy under field conditions.

5 Conclusions

This work has developed zinc oxide nanoparticles in combination with a natural biologically active substance (Chitinase) as a hybrid composite against phytopathogens. This is a new alternative of formulating pesticides. In this case, an inhibition of up to 89.1 % mycelial growth on mycopathogenic fungi and 18.3 % inhibition of Fusarium species were achieved. Future work should address the impact of such formulation in the ecosystem, and study its time-controlled released and stability of the effect and changes in the composite with temperature and solar irradiation. There is a need to discover eco-friendly hybrid nanofungicides as an alternative to conventional fungicides. New green nanobiofungicide can be produced with a suitable synergistic approach, to protect the soil and surrounding water, control their dispersion and targeted toxicity, to protect crops and enhance their yield. For future recommendation, it is significant to examine the synergistic influence of one of these mixtures in terms of antimicrobial growth, lowering pesticide utilization and delaying development of resistance. Required extensive research on larger scale to establish potential ecofriendly nano-based products.