Synthesis of gold nanoparticles from Metarhizium anisopliae for management of blast disease of rice and its effect on soil biological index and physicochemical properties

doi:10.21203/rs.3.rs-2080559/v1

Aims: The aim of this experiment was to synthesized gold nanoparticles from entomopathogen fungi Metarhizium anisopliae, an indigenous biocntrol agent and its antifungal activity against Magnaporthe grisea and its effect on soil biological index and physicochemical properties.

Methods:Biosynthesis of gold nanoparticles was done by following standard protocol and characterized by different equipments. soil biological and ph
ysicochemical properties was carried out by following standard protocol.

Results:Formation of gold nanoparticles were confirmed by UV-VIS spectroscopy study with absorption peaks at 550 nm. FTIR study showed that synthesized gold nanoparticle has all the required functional groups like OH, N-H, C-H and COO-. Study on surface properties of nanoparticles by using zetasizer resulted that gold nanoparticle from Metarhizium anisopliae was found to be negative and were stable in nature with zeta potential value of -20.7 mV. DLS analysis showed that the average size of the biosynthesized gold nanoparticles is 32.54 nm with polydispersity index of 0.560. TEM study showed that shape of the biosynthesized nanoparticle is from triangular to quasihedral and the size range from 9-54nm. Antifungal activity of gold nanoparticles at 150 ppm significantly inhibit the mycelia growth of the pathogens as compared to the Tryclozole @ 600 ppm

Conclusions : A positive effect was found on soil biological index and physicochemical properties of soil along with reduction of disease incidence when rice seedling was treated as seedling dip treatment + foliar spray + Soil application @ 150ppm of biosynthesized gold nanoparticles.

Chloroauric acid

Gold nanoparticles

Metarhizium anisopliae

Magnaporthe grisea

Rice (Oryza sativa L.) is an important cereal crop where it is considered to be the stable source of food for most of the human population. Kole, 2006 reported that rice is grown in mostly in Asia where more than 90% produced and 60% consumed. According to Boumas, 1985 it is the second worldwide production after maize. Rice grain contains on an average 7% protein, 62-65 % starch, 0.7% fat and 1.3% fibre and rice is a main source of vitamin B₁ (thiamin), B₂ (riboflavin), B₃ (niacin) and B₅ (pantothenic acid). The biological value of rice is 63% whereas biological value of wheat and maize is 49% and 36%. The domesticated rice consists of two species in the Poaceae family: Oryza sativa and Oryza glaberrima (Linscombe, 2006). These plants are native to Tropical and Subtropical Southern Asia and South eastern Africa, respectively (Linares, 2002).

Rice is mainly cultivated in asian continent which includes country like India, China, Indonesia, Bangladesh, Vietnam, Thailand, Myanmar, Japan, Philippines and Brazil. In terms of production China ranks the first followed by India, Indonesia and Bangladesh. But in terms of export India ranks first followed by Thailand, Vietnam and USA. It is found that 95% of the total production, belongs to the developing cuntries with China and India alone responsible for nearly half of the world output. It is estimated that 20% of the world’s dietary energy supply, is from rice and 19% and 5% is from wheat and maize.

Rice crop suffers from many diseases caused by fungi, bacteria, viruses, phytoplasma, nematodes and other non-parasitic disorders. Among the fungal diseases, blast caused by Magnaporthe grisea (Habert) Barr is proved to be a devastating one because of its variability and widespread distribution and its destructiveness under favorable conditions. This disease was recorded from 85 countries (Hawksworth, 1990) and it is estimated to cause 14-18% grain yield losses worldwide (Mew and Gonzales, 2002). The yield losses due to pests and diseases are estimated to be around 37% (IRRI, 2014) of which blast accounts to 14-18 per cent.

Besides rice this pathogens has been reported in Banlgadesh which caused blast in wheat which is an emerging disease (Bhattacharya and Pal, 2017). Approximately 30% of rice production losses globally by the blast diseases (Nalley et al., 2016)..Many integrated disease management strategies with biocontrol agents, botanicals etc have also been tried for this but with no effect. For the management of rice diseases largely depends upon synthetic chemical fungicides and to some extent upon conventional management practices. Recently environmental hazards caused by excessive use of pesticides have been discussed widely and putting concentration on emerging and re-emerging pests. Unjudicious applications of fungicides and other chemicals have created a situations that has become more tolerance to this chemicals by the pathogens . As an another option the role of nanotechnology qand nanoparrticles will play an important role in disease management. Nanomaterial potential agro-biotechnological applications for alleviation of these problems. In recent years, toxic effects and residue levels of pesticides has been discussed widely therefore, scientist in the agricultural field are searching for alternative measures against pesticides. Development of reliable and eco-friendly process for synthesis of nanoparticle is an important step in the field of application of nanotechnology. The increasing interest for safer and better process of biosynthesis has become a important aspects in isolating the nanoparticle. Various methods of isolation of nanoparticle is possible by different methods such physical, chemical and biological. Out of which biological methods seems to be the best ones as it is eco-friendly, cheap and easy way of synthesizing large production of various nanoparticle. In biological synthesis various plant extracts and microorganisms like fungi (Syed et al., 2013), bacteria (Shivaji et al., 2011), plant extract (Akhtar et al., 2013), etc. are used for synthesizing various nanoparticle. Application of nanotechnology in crop protection holds significant promise in plant disease management. Nanoparticle synthesized from biological agents such as plants and microbes are biodegradable (Chowdappa and Gowda, 2013). Thus they have advantages over conventional methods associated with ecotoxicity.

Source of microorganism

Metarhizium anisopliae, was collected from culture collection maintained at Mycology Research Section, Assam Agricultural University, Jorhat, Assam.

Production of biomass of microbes

Biomass production of Metarhizium anisopliae, was done in potato dextrose broth (PDB) supplemented with peptone (Hi Media Laboratories Ltd.) @ 1% and carbon source Carboxy methyl cellulose (Hi Media Laboratories Ltd.) @ 0.05%. Inoculam of all the microbes was grown freshly in potato dextrose agar (PDA) and was inoculated in PDB in aspetic condition and incubated (R.E.I.C.O BOD Incubator) for 7 days at 26±1º C for mass culture.

Preparation of 1mM Chloroauric acid (HAucl₄) solution

Molarity is moles per liter. Since the molar mass of HAucl₄ is 393.86 g/mol, a 1 M solution of HAucl₄ is 393.86 g (1 mole of HAucl₄) in 1 liter. For preparation of 100 ml of solution 0.039 g of Chloroauric acid was taken and added in 100 ml of deionised water.

Synthesis of gold nanoparticle from Metarhizium anisopliae

For synthesis of gold nanoparticle microbes was harvested and centrifuge at 5000 rpm for 10 minutes at 4°C. From this 50 ml of microbes supernatant was treated with 50 mL aqueous solution of 1 mM HAuCl₄ solution in a 250 mL erlenmeyer flask (pH adjusted to 10). The whole mixture was treated at rotary shaker (R.E.I.C.O, Horizontal shaker) for 5 days and maintained in the dark by wrapping the conical flasks with aluminium foil of cartoon box. Control experiments were conducted with uninoculated set (Amersan et al., 2016).

Characterization of biosynthesized gold nanoparticles

Characterization of gold nanoparticles was done by instruments like UV-VIS Spectrophotometer, DLS, XRD, Zeta sizer and Transmission Electron Microscopy study.

UV-Vis Spectrophotometer

The synthesized gold nanoparticles was characterized with the help of a UV-vis Spectrophotometer (Eppendorf Biospectrophotometer) to established the synthesis of nanoparticles. The sample was run in the range 200-800 nm. Water blank was used for the calibration of the equipments. For the data analysis purpose 2 ml of gold nanoparticles was taken in a cuvette. The UV-Vis absorption spectra of the biosynthesized gold nanoparticles was recorded and the data generated was plotted in the “Origin 8.5”.

Dynamic Light Scattering (DLS)

The prepared sample was dispersed in deionised water followed by ultra-sonication. Then solution was filtered and centrifuged for 15 min at 4°C with 5000 rpm and the supernatant was collected. The supernatant was diluted for 4 to 5 times and then the particle distribution in liquid was studied in a computer controlled particle size analyzer (ZETA sizer, Nanoseries, Malvern instrument Nano Zs, 2000).

Zeta potential

For the analysis of Zeta potential 2ml of sample was taken in a cuvette and then the particle distribution in liquid was studied in a computer control charge analyzer (ZETA sizer, Nano series, Malvern instrument Nano Zs, 2000).

Transmission Electron Microscope (TEM)

TEM study was done at North East Institute of Science and Technology, Regional Research Laboratory, Jorhat, at accelerating voltage of 20kv with magnification of 20,000x. The shape and size of the resultant particles was elucidated with the help of TEM. Solutions of biosyhthesized gold nanoparticle solution was placed on a carbon-coated copper grid and allowed to dry under ambient conditions and TEM image were recorded.

Fourier Transform Infrared Spectrometer (FTIR)

Infra red (IR) spectra of biosynthesized gold nanoparticles was recorded with Fourier Transform Infrared Spectrometer (Shimadzu, Japan). The spectral region between 400 to 4000 Cm^-1 was scanned. The spectra were the average of 50 corded at a resolution of 4 Cm^-1

Isolation of the pathogens

The pathogen Magnaporthe grisea was isolated from the freshly infected sample showing typical symptoms of a disease in oat meal agar media. Pure cultures of the pathogens was maintained by transferring in fresh slants of oat meal medium.

In vitro study of bio synthesized gold nanoparticle against Magnaporthe grisea

An in vitro assay was performed to test the efficacy of synthesized gold nanoparticle against M. grisea at three different concentration that is 50,100 and 150 ppm and the comparison was made with a recommended chemical i.e., Tricylazole @0.06%. For Food Poison Technique, PDA was poisoned at the desired concentration and was poured in sterilized petriplate and was allowed to solidify. After the solidification agar plugs cut with a sterilized cork borer containing the fungi was inoculated at the centre and a control was taken with a normal PDA medium. All the plate were incubated at 28±1°C in a B.O.D. incubator (R.E.I.C.O. BOD. Incubator) till the full growth was observed in the control (Kim et al., 2012). Mycelial growth inhibition was calculated when growth of mycelia in the control plate reached the edge of the petridish. The following formula used for calculation of the inhibition rate (%).

Inhibition rate (%) = R – r/R

Where, R is the radial growth of fungal mycelia on the control plate and r is the radial growth of fungal mycelia on the plate treated with gold nanoparticle.

In vivo study of bio synthesized gold nanoparticle against Magnaporthe grisea

A pot experiment was conducted in green house conditions to see the effect of biosynthesized gold nanoparticles on soil biological index and soil physicochemical properties with the following treatment combination:

T₁: Uninoculated Control (No pathogen)

T₂: Control (Only Pathogen)

T₃: Chemical spray Tricylazole @0.06% (Recommended)

T₄: Seedling dip treatment (SDT) with Metarhizium anisopliae mediated Au NPs @ 150ppm for 15-20 minutes

T₅: Foliar spray (FS) with M. anisopliae mediated Au NPs @ 150ppm

T₆: Soil application (SA) with M. anisopliae mediated Au NPs @ 150ppm

T₇: SDT with M. anisopliae mediated Au NPs @ 150ppm + FS with M. anisopliae mediated Au NPs @ 150ppm

T₈: SDT with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm

T₉: FS with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm

T₁₀: SDT with M. anisopliae mediated Au NPs @ 150ppm + FS with M. anisopliae mediated Au NPs @ 150ppm+ SA with M. anisopliae mediated Au NPs @ 150ppm

Effect of biosynthesized gold nanoparticle on soil parameters

The following soil biological index and soil physicochemical properties was conducted as per the protocol given by the scientist.

Sl No	Soil biological index and soil physicochemical properties	Method followed
1	Soil p^H	Jackson, 1973.
2	Organic carbon	Walkley and Black (1934).
3	Microbial Biomass Carbon	Vance et al. (1987).
4	Nitrogen content	Jackson, 1973
5	Phosphorus Content	Jackson, 1973
6	Potassium Content	Jackson, 1973
7	Exchangeable Cation	(Nagornyy, 2013).

Statistical analysis

Completely randomized design was performed for the statistical analysis of the data. The data collected were subject to statistical analysis by Fisher’s method of analysis of variance. Significance of variance among the data was analysed by calculating the “F” value and comparing it with the tabulated value of “F” at 5% level of probability.

The treatment means were compared among themselves by calculating critical difference (CD) as followed:

C.D. at 5%= S. Ed ×‘t’ 5% (at error d.f.)

The standard error of differences (S. Ed) was calculated by using the following formula:

Where, S. Ed. = Standard error of difference

‘t’ 5% = “t” for error d. f. at 5% level of probability

The significance and non-significance of the treatments at 5% level of probability were calculated out by multiplying the S. Ed. with appropriate tabulated value for error degrees of freedom.

Synthesis of gold nanoparticles

For the synthesis of gold nanoparticles microbes like Metarhizium anisopliae, (Plate 1). For green synthesis of gold nanoparticles, when supernatant of the bioresources was allowed to exposedto 1 mM aqueous solution of chloroauric acid the colour of supernatant changes from original colour of the extract to red wine to purple colour 192 hrs of reaction [Plate 2]. The supernatant of the selected bioresources without chloroauric acid retained its original colour. The colour change from original colour of the extract to purple confirms the synthesis of gold nanoparticles.

Characterization of effective biosynthesized gold nanoparticles

UV-Vis spectrophotometer analysis

Surface Plasmon Reasonance (SPR) absorption band occurs due to the combined vibration of electrons of metal nanoparticles. In the present study characteristics SPR absorption band was observed in supernatant of Metarhizium anisopliae 550 nm (Fig. 1).

Dynamic Light Scattering (DLS)

Dynamic light scattering is a technique used in material physics for determining the size distribution profile of nanoparticles in suspension or polymers in solution. This technique was used in the present study to determine the size distribution profile of nanoparticles present in the final solution after ultrasonication. DLS also determines polydispersity, hydrodynamic sizes and aggregation of particles in the suspension. Average size of biosynthesized gold nanoparticle was found to be 32.54 nm with Polydispersity Index of 0.560 (Fig. 2).

Zeta Potential

Charged of biosynthesized gold nanoparticle was found to be -20.7mV (Fig. 3) when the sample was subjected in the ZETA sizer, Nano series, Malvern instrument Nano Zs, 2000

Transmission Electron Microscopy (TEM)

Transmission Electron Microscopymicrograph was carried at an accelerating voltage of 200kv with 20,000X magnification and found that the nanoparticles are formed in the size range of 9-54 nm (Plate 7). The shape of the biosynthesized gold nanoparticles was found to be triangular to quasihedral.TEM micrograph also indicates that nanoparticles are relatively uniform in nature and also shows that particles were well separated from each other having no agglomeration.

Fourier transform infrared spectroscopy (FTIR)

FTIR study of synthesized nanoparticles was done to confirm the formation of biosynthesized gold nanoparticles by identifying the required functional groups of gold nanoparticles The main functional groups of gold nanoparticles are hydroxyl group (OH), amide group (NH), C-H stretching, C-N and C-O stretching etc.

The FTIR spectrum of the nanoparticles indicates the presence of various chemical groups, one of which is an amide. The presence of COO^–, possibly due to amino acid residues may indicate that protein co-exists with the gold nanoparticles. An amide I band was observed at 1630 to 1650 cm-1. This is further confirmed by the band at 3459.0cm-1. The band at 1684 cm-1 corresponds to amide I due to carbonyl stretch in proteins (Fig. 4).

Identification of the Pathogen

Identification of the pathogen was done through cultural and microscopic observations. Colony colour was found to be grayish black to black colour, smooth to irregular margin was observed {Plate 3 (a and b)}. Microscopic study revealed that conidia are pyriform, almost hyaline to pale olive, 2-septate and 3 celled (Plate 3c).

Pathogenicity

Results of the pathogenicity test revealed the appearance of symptoms after spraying of inoculums of M. grisea (Plate 4a) by maintaining the humidity with a polythene cover (Plate 4b). Typical symptoms of leaf blast (Plate 5a) appeared after 25 days of inoculation of spore suspension of M. grisea. Nodal blast (Plate 5b) and Neck blast (Plate 5c) symptoms appeared after 72 days and 125 days of inoculation of spore suspension of M. grisea

Antifungal activity of gold nanoparticles

Antifungal activity of biosynthesized gold nanoparticle in suppressing the M. grisea at different concentrations that is 50, 100 and 150 ppm and recommended dose of chemical Tricylazole @600ppm was carried out In Vitro. With the increase in concentration of gold nanoparticle the radial growth inhibition (%) was increased and found maximum at 150 ppm of gold nanoparticles. Mycelial growth inhibition of 100%, 75.99%, 52.51%, 81.67%, 59.32%, 49.03% was recorded at (150ppm, 100ppm and 50ppm) concentration of biosynthesized gold nanoparticle from M. anisopliae and O. gratissimum against M. grisea (Plate 6) (Table 1 and Fig. 1). At 600ppm of tricylozole mycelial growth inhibition was recorded as 100% against M. grisea which was found to be at par with M. anisopliae mediated gold nanoparticle. In this study we have found that gold nanoparticles synthesized from M. anisopliae was found to be effective in inhibiting mycelia growth of M. grisea.

Effect of different method of application of effective biosynthesized Au NPs on percent disease incidence

A pot experiment (Plate 11) was conducted to see the effect of different method of application of Au NPs on percent disease incidence. It was found that lowest percent disease incidence was found in treatment ten that is seedling dip treatment with M. anisopliae mediated Au NPs @ 150ppm + foliar spray with M. anisopliae mediated Au NPs @ 150ppm + soil application with M. anisopliae mediated Au NPs @ 150ppm (12.13%). But percent disease incidence with recommended dose of chemical (15.13%) was found to be at par with treatment ten. This was followed by treatment four that is seedling dip treatment with M. anisopliae mediatedAu NPs @ 150ppm for 15-20 minutes (19.43%). Highest percent disease incidence was observed in inoculated control with a percent disease incidence of (71.41%) (Table 2, Fig. 5).

Effect of effective biosynthesized Au NPs on soil physicochemical and biological properties

Effect of biosynthesized Au NPs on soil pH

Highest soil pH was found to be in treatment ten that is seedling dip treatment with M. anisopliae mediated Au NPs @ 150ppm + Foliar spray with M. anisopliae mediated Au NPs @ 150ppm+ Soil application with M. anisopliae mediated Au NPs @ 150ppm (6.5). This was followed by treatment four i.e. seed treatment with M. anisopliae mediatedAu NPs @ 150ppm. Lowest soil pH was found to be in inoculated control (4.213) with percent disease incidence of (15.13%) (Table 3, Fig. 6).

Effect of biosynthesized Au NPs on macronutrient

Data presented in Table 3 showed that highest per cent of macronutrient was found to be in treatment ten i.e. seedling dip treatment with M. anisopliae mediated Au NPs @ 150ppm + Foliar spray with M. anisopliae mediated Au NPs @ 150ppm+ Soil application with M. anisopliae mediated Au NPs @ 150ppm viz., N (0.282%), P₂O₅ (0.313%) and K₂O (2.712%). This was followed by treatment four i.e. seed treatment with M. anisopliae mediatedAu NPs @ 150ppm. Lowest macronutrient content was found to be in inoculated control N (0.167%), P₂O₅ (0.363%) and K₂O (1.66%). with percent disease incidence of (15.13%) (Fig. 7).

Effect of biosynthesized Au NPs on cation exchange capacity

Highest content of cation exchange capacity is found in treatment ten which is a combination of seedling dip treatment with M. anisopliae mediated Au NPs @ 150ppm + Foliar spray with M. anisopliae mediated Au NPs @ 150ppm+ Soil application with M. anisopliae mediated Au NPs @ 150ppm with a value of Ca⁺⁺ [0.085 Cmol (P⁺)kg^-1}], Mg⁺ [0.099 Cmol (P⁺)kg^-1}], Na⁺[0.098 Cmol (P⁺)kg^-1}], K⁺ [0.098 Cmol (P⁺)kg^-1}]. This was followed by treatment four i.e. seed treatment with M. anisopliae mediatedAu NPs @ 150ppm. Lowest macronutrient content was found to be in inoculated control Ca⁺⁺ [0.075 Cmol (P⁺)kg^-1}], Mg⁺ [0.067 Cmol (P⁺)kg^-1}], Na⁺[0.079 Cmol (P⁺)kg^-1}], K⁺ [0.068 Cmol (P⁺)kg^-1}] with percent disease incidence of (15.13%) (Table 3, Fig. 8).

Effect of biosynthesized Au NPs on organic carbon and microbial biomass carbon

Similarly organic carbon and microbial biomass carbon was found to be 0.488% and 1.583 in treatment ten (seedling dip treatment with M. anisopliae mediated Au NPs @ 150ppm + Foliar spray with M. anisopliae mediated Au NPs @ 150ppm+ Soil application with M. anisopliae mediated Au NPs @ 150ppm) followed by treatment four (seed treatment with M. anisopliae mediatedAu NPs @ 150ppm). Lowest organic carbon and microbial biomass carbon content was found to be in inoculated control with a value of 0.289 % and 1.183 with percent disease incidence of (15.13%) (Table 3, Fig. 9).

The colour change from original colour of the extract to purple observed in this study is due to Surface Plasmon Resonance (SPR) phenomenon. Generally the metal nanoparticles have free electrons, which helps in the formation of the Surface Plasmon Resonance absorption band. It happens due to the united vibration of the electrons of metal nanoparticles in resonance with light wave. The extracellular synthesis of nanoparticles involves trapping the metal ions on the surface of the cells and reducing ions in the presence of enzymes. A possible mechanism for the presence of gold nanoparticles in fungal biomass could be the extracellular reduction of the gold ions in the solution followed by precipitation on to the cells. We observed gradual increase in colour development from the original colour of extract to purple. This may due to Surface Plasmon Reasonance phenomenon (Sarkar et al., 2012). The colour change observed in this study may be due to the deposition of gold nanoparticle as reported by Bhambure et al. (2009) reported that gold nanoparticles shows red wine to purple colour in water due to excitation of surface plasmon vibration in metal nanoparticles.

The result of UV-Vis spectroscopy observed in the present study is in agreement with the earlier report of (Shelar and Chavan, 2014) who reported that Surface Plasmon Resonance (SPR) band for gold nanoparticles occurs at 300–700 nm. They also observed that Surface Plasmon Resonance band of gold nanoparticles at 540nm by using the fungus Aspergillus terreus at 540 nm. Mukherjee et al. (2001) also found the SPR band of biosynthesized gold nanoparticles from Verticillium spp. at an wavelength of 540 nm. Workers like Jha and Prasad (2012) also observed characteristic SPR band of gold nanoparticles synthesized from Mentha, Ocimum and Eucalyptus at an wavelength of 539, 536 and 537 nm, respectively.

It is said that polydispersity index is a scale in which we can know the monodispersed and polydispersed nature of gold nanoparticles. the scale is such that if the polydispersity index with values with 0.10 or less are considered highly monodispersed and above 0.10 are polydispersed as stated by Hughes et al. (2015). For calculation of size of nanoparticle, earlier worker (Nakkala et al., 2016 and Ahmed et al., 2016) also used DLS for determination of average size of nanoparticle. Nakkala et al. (2016), also determined the mean particle size of synthesized gold nanoparticles using Piper longum extract by DLS and the size was found to be in the range of 20–45 nm. Ahmed et al. (2016) synthesized gold nanoparticles using Azadirachta indica aqueous leaf extract and average size was determined as 34 nm by using DLS.

Negative zeta potential (-20.7 mV) observed in this study indicates that the biosynthesized gold nanoparticle from Metarhizium anisopliae are highly stable at dispersion. Zhang et al. (2009) reported that the large negative zeta potential value (above − 0.50 mV) indicates higher electrostatic repulsion among gold nanoparticle and stable on their dispersion. They also observed that gold nanoparticles synthesized from Penicillum possessed negative zeta potential and were stable at different pH due to electrostatic repulsion.

Roy et al. (2013) did TEM study on gold nanoparticle synthesized biologically from Aspergillus foetidus and TEM study showed the size of 10–58 nm with triangular shape. Selvi and Sivakumar (2012) synthesized gold nanoparticle from Fusarium oxysporium and TEM analysis revealed that gold nanoparticles are of spherical shape in the range of 5–50 nm and uniformly distributed without significant agglomeration.

The shape, septation and colour characters of the M. grisea are in agreement with those described by Nishikado (1926) and Hossain (2000).

The results are in agreement with the workers like Aoki (1935) and Srivastava et al. (2011) who also found the appearance of typical blast symptoms after 20 days of inoculation of spore suspension of Pyricularia grisea.

Maximum inhibiotory effect of gold nanoparticle at 150 ppm observed in the present study may be due to the smaller size of gold particle which help in rapid accumulation on the fungal mycelium and then penetration inside it as observed by Stroz and Imlay (1999). Hwang et al. (2008) reported that nanoparticle on entering inside the fungal mycelium, it causes interruption in the metabolic process and respiration by reaction with cellular molecules. Storz and Imlay, 1999 reported that gold ions are known to produce reactive oxygen species (ROS) via their reaction with oxygen, which are detrimental to cells, causing damage to proteins, lipids, and nucleic acids. Au NPs were much more genotoxic than Au chlorides (Shukla et al., 2014; Taylor et al., 2014). Jo et al. (2009) reported that various forms of silver ions and nanoparticles were found to have antifungal activity against two plant-pathogenic fungi, Bipolaris sorokin and Magnaporthe grisea at 100ppm concentration. Nano Green’ a product prepared by mixing several bio-based chemicals was reported to eliminate blast disease (Magnaporthe grisea) from infected rice plant. The test was conducted in University of Georgia and the product was found to outperform any other pesticide or fungicides currently in use in agriculture (Gogoi et al., 2009). Joshi et al. (2019) reported that selenium nanoparticle has a antifungal activity against Pyricularia grisea and inhibited the infection of Colletotrichum capsici and Alternaria solani on chili and tomato leaves at concentrations of 50 and 100 ppm, respectively.

Elamawi et al. (2013) s tudied, the effect of Ag NPs (20–30 nm) against rice leaf blast fungus and was evaluated under in vivo. Under greenhouse conditions, Ag NPs was sprayed in concentrations 0, 25, 50, 100 and 200 ppm on rice seedling leaves at three times (3 hours before inoculation, 1 and 5 days after artificial inoculation with spore suspension). Damaged Leaf Area Percentage (DLA %) indicated that the application of 100 ppm Ag NPs was highly efficient before and after inoculation (26.7, 15.3 and 20%), respectively compared to the untreated plants of 80%. Similarly Jo et al (2009) studied the various forms of silver ions and silver nanoparticles was tested to examine the antifungal activity on two plant-pathogenic fungi, Bipolaris sorokiniana and Magnaporthe grisea. Growth chamber inoculation assays further confirmed that both ionic and nanoparticles forms significantly reduced the two fungal diseases on perennial ryegrass (Lolium perenne) at 100ppm of concentration (Kim et al., 2012).

Soil pH was found to be increased slightly after application of gold nanoparticle. This increased in pH might be due to the precursor used for the synthesis of gold nnaoparticles, and during the reduction process gold cations (Au⁺) are formed from the chloroauric acid.

Higher content of macronutrient observed in the present study might be due to the smaller sized of nanoparticle that are absorbed easily and efficiently by the plants because of their higher surface area (Brackhage et al., 2013). Moreover this have the ability to enter into the cells directly thereby by passes the energy intensive mechanisms of their uptake/delivery into the cell.

Increased in cation exchange capacity might be due to the negative charge contributed by HClO⁴⁻ from nanoparticle. Increased negative charge will increased the ability of soil colloids to bind to cations. This is in line with the revelation proposed by Devinta, 2010 and Milah, 2013. Workers like (Shoults-Wilson et al., 2011; Benoit et al., 2013; Mishra and Singh, 2015; Klitzke et al., 2015; Schlich and Hund-Rinke, 2015) observed that lower range of soil pH, organic matter content and cation exchange capacity obstructs sorption of Ag to soil resulting into enhanced risk of mobility, toxicity and bioavailability of Ag NPs. In contrary, higher range of soil pH, organic matter content and cation exchange capacity facilitates Ag sorption to soil that prevent mobility, bioavailability and further toxicity of Ag NP. Likewise, Wang et al. (2013) reported reduction in the toxicity of ZnO-NPs in soil system and the reduction is mainly attributed to a range of soil characteristics like pH and cation exchange capacity.

The increase of soil organic carbon and microbial biomass carbon observed in the present study might be due to the application of green synthesized gold nanoparticle using Metarizium anisopliae

Table 1

*In vitro* efficacy of biosynthesized gold nanoparticles at different concentration
Treatment combination	Mycelial Growth Inhibition (%)
T₁: Control (Only pathogen)	0.00
T₂: Metarhizium anisopliae Au NPs @ 150ppm	90.00 (71.60)
T₃: Metarhizium anisopliae Au NPs @ 100ppm	75.99 (60.69)
T₄: Metarhizium anisopliae Au NPs @ 50ppm	52.51 (46.46)
T₈: Tricylazole @ 600ppm	100.00 (90.04)
SE. d (±)	0.49
CD(P = 0.05%)	1.31

Data in parenthesis are angular transformed value

Table 2

Effect of different method of application of effective biosynthesized gold nanoparticles on percent disease incidence
Treatment	PDI
T₁ : Uninoculated control (No Pathogen)	0.00
T₂ : Inoculated control (0nly pathogen)	74.41 (59.64)
T₃ : Chemical spray Tricylazole @600 ppm (Recommended)	15.13 (22.87)
T₄ : Seedling dip treatment (SDT) with Metarhizium anisopliae mediated Au NPs @ 150ppm for 15–20 minutes	19.43 (26.16)
T₅ : Foliar spray (FS) with M. anisopliae mediated Au NPs @ 150ppm	17.24 (24.54)
T₆ : Soil application (SA) with M. anisopliae mediated Au NPs @ 150ppm	23.02 (28.68)
T₇ : SDT with M. anisopliae mediated Au NPs @ 150ppm + FS with M. anisopliae mediated Au NPs @ 150ppm	18.32 (25.35)
T₈ : SDT with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm	19.98 (26.56)
T₉ : FS with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm	20.69 (27.06)
T₁₀ : SDT with M. anisopliae mediated Au NPs @ 150ppm + FS with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm	12.13 (22.90)
SE.d (±)	0.53
C.D.(P = 0.05%)	1.40

Data in parenthesis are angular transformed value

Data are mean of four replication

Table 3

Effect of biosynthesized gold nanoparticle on soil physicochemical and biological properties on rice
Treatment	Soil pH	Avail N (%)	Avail P₂O₅ (%)	Avail K₂O (%)	Ca⁺⁺ {Cmol (P⁺)kg^-1}	Mg⁺ {Cmol (P⁺)kg^-1}	Na⁺ {Cmol (P⁺)kg¹}	K⁺ {Cmol (P⁺)kg^-1}	OC (%)	MBC micro gram /gram soil
T₀ (Initial)	4.576	0.186 (2.473)	0.175 (2.398)	1.753 (7.612)	0.065	0.067	0.078	0.071	0.332	1.343
T₁	4.727	0.192 (2.512)	0.177 (2.412)	1.803 (7.720)	0.086	0.076	0.077	0.077	0.333	1.283
T₂	4.213	0.167 (2.343)	0.163 (2.315)	1.666 (7.420)	0.075	0.067	0.079	0.068	0.289	1.183
T₃	4.833	0.170 (2.364)	0.188 (2.486)	1.880 (7.884)	0.095	0.077	0.082	0.076	0.294	1.323
T₄	6.200	0.258 (2.912)	0.301 (3.146)	2.520 (9.138)	0.075	0.097	0.095	0.097	0.447	1.583
T₅	5.670	0.224 (2.714)	0.204 (2.590)	2.047 (8.229)	0.064	0.083	0.085	0.087	0.388	1.430
T₆	5.663	0.226 (2.726)	0.228 (2.738)	2.136 (8.408)	0.064	0.086	0.091	0.092	0.392	1.477
T₇	5.943	0.228 (2.738)	0.255 (2.895)	2.227 (8.586)	0.067	0.089	0.094	0.095	0.394	1.507
T₈	5.827	0.248 (2.855)	0.284 (3.056)	2.377 (8.873)	0.069	0.094	0.093	0.096	0.430	1.547
T₉	5.150	0.195 (2.532)	0.195 (2.532)	1.973 (8.076)	0.063	0.078	0.083	0.083	0.337	1.373
T₁₀	6.500	0.282 (3.045)	0.313 (3.208)	2.712 (9.483)	0.085	0.099	0.098	0.098	0.488	1.583
Se. D (±)	0.750	0.014	0.011	0.490	0.014	0.094	0.005	0.082	0.045	0.076
C. D (P 0.05%)	2.02	0.037	0.029	1.32	0.037	0.253	0.014	0.221	0.121	0.205

Data in parenthesis are angular transformed value

Data are mean of four replication

T₁– Uninoculated control (No Pathogen), T_2- Inoculated control (0nly pathogen) , T₃- Chemical spray Tricylazole @0.06% (Recommended) , T₄: Seedling dip treatment (SDT) with Metarhizium anisopliae mediated Au NPs @ 150ppm for 15-20 minutes, T₅: Foliar spray (FS) with M. anisopliae mediated Au NPs @ 150ppm , T₆: Soil application (SA) with M. anisopliae mediated Au NPs @ 150ppm , T₇: SDT with M. anisopliae mediated Au NPs @ 150ppm + FS with M. anisopliae mediated Au NPs @ 150ppm, T₈: SDT with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm, T₉: FS with M. anisopliae mediated Au NPs @ 150ppm + SA with M. anisopliae mediated Au NPs @ 150ppm, T₁₀: SDT with M. anisopliae mediated Au NPs @ 150ppm + FS with M. anisopliae mediated Au NPs @ 150ppm+ SA with M. anisopliae mediated Au NPs @ 150ppm

Ethics approval and consent to participate : Not Applicable

Consent for publication : Not Applicable

Availability of data and materials: Not Applicable

Competing interests: There is no conflict of interest between the authors for publications

Funding: Not Applicable

Authors' contributions:

Dr. Pranjal Kr Kaman- Performed the whole experiment and prepared the manuscript

Dr. Pranab Dutta- Arranged the manuscript as per the guidelines and helped in conducting the experiment

Dr. Ashok Bhattacharyya- Checked the manuscript

Acknowledgements: Not Applicable

Ahmed, S.; Ahmad, M.; Swami, L.B. and Ikram, S. (2016). Green synthesis of gold nanoparticles using Azadirachta indica aqueous leaf Journal of Radiation Research and Applied Sciences, pp. 1-7.
Akhtar, S.; Panwar, J. and Yun, Y.S. (2013). Biogenic synthesis of metallic nanoparticle by plant extracts ACS Sustainable Chemical Engineering, 1(6): 591-602.
Amerasan, ; Nataraj, T.; Murugan, K.; Panneerselvam, C.; Madhiyazhagan, P.; Nicoletti, M. and Benelli, G. (2016). Myco-synthesis of silver nanoparticles using Metarhizium anisopliae against the rural malaria vector Anopheles culicifacies Giles (Diptera: Culicidae). Journal of Pest Science, 89(1): 249-256
Benoit, R.; Wilkinson, K.J. and Sauvé, S. (2013). Partitioning of silver and chemical speciation of free Ag in soils amended with Chemistry Central Journal, 7(1): 75.
Bhambure, ; Bule, M.; Shaligram, N.; Kamat, M. and Singhal, R. (2009). Extracellular biosynthesis of gold nanoparticles using Aspergillus niger–its characterization and stability. Chemical Engineering & Technology: Industrial Chemistry‐Plant Equipment‐Process Engineering‐Biotechnology, 32(7): 1036-1041.
Bhattacharya, R. and Pal (2017). Deadly wheat blast symptoms enters India through the Bangladesh border, Bengal govt burning crops on war footing. Hindustan Times.
Boumas, G. (1985). Rice Grain Handling and Storage. Elsevier Science Publishers, V. 9-10.
Brackhage, C.; Schaller, J.; Bäucker, E. and Dudel, E.G. (2013). Silicon availability affects the stoichiometry and content of calcium and micro nutrients in the leaves of common reed. Silicon, 5(3): 199-204.
Chowdappa, P. and Gowda, S. (2013). Nanotechnology in crop protection: Status and Pest Management in Horticulture Economics, 19(2): 131-151.
Devnita, R. (2010). Characteristic of soil chemistry and soil physics of sole Andisols at West Research Report Faculty of Agriculture, Universitas Padjadjaran,
Elamawi, M.A. and Shafey, R.A.E.El. (2013). Inhibition effects of silver nanoparticles against rice blast disease caused by Magnaporthe grisea. Egypian Journal of Agriultural Research, 91(4): 1271-1282.
Gogoi, R.; Dureja, P. and Singh, P.K. (2009). Nanoformulationsa safer and effective option for agrochemicals. Indian Farming, 59(8): 7-12.
Hawksworth, D.L. (1990). CMI Descriptions of Fungi and Bacteria. Mycopathologia, 111(2): 109-115.
Hossain, (2000). Studies on blast disease of rice caused by Pyricularia grisea (Cooke) Sacc. in upland areas. M.Sc.(Ag) Thesis, University of Agricultural Sciences, Dharwad, pp. 52-53.
Hughes, M.J.; Budd, M.P.; Grieve, A.; Dutta, P.; Tiede, K. and Lewis, J. (2015). Highly monodisperse, lanthanide-containing polystyrene nanoparticles as potential standard reference materials for environmental ―nano‖ fate analysi Journal of Applied Polymer Science, 132(24).
Hwang, T.; Lee, J.H.Y.J.; Chae, Y.S.K.; Kim, B.C.; Sang, B.I. and Gu, M.B. (2008). Analysis of the toxic mode of action of silver nanoparticles using stress- specific bioluminescent bacteria. Small, 4(7): 46-50.

IRRI (2014). Rice blast. International Rice Research Institute. www.knowledgebank. irri. org/factsheetsPDFs/.Rice Fact Sheets.
Jackson, M.L. (1973). Soil Chemical Analysis. Prentice – Hall, Inc, Englewood Cliffs, New Jersey
Jha, A.K. and Prasad, K. (2012). Biosynthesis of gold nanoparticles using common aromatic plants. International Journal of Green Nanotechnology, 4(3): 219-224.
Jo, K.; Kim, B.H. and Jung, G. (2009). Antifungal activity of silver ions and nanoparticles on phytopathogenic fungi. Plant Disease, 93(10): 1037-1043.
Joshi, M.; De Britto, S.; Jogaiah, S. and Ito, S.I. (2019). Mycogenic Selenium Nanoparticles as Potential New Generation Broad Spectrum Antifungal Molecules. Biomolecules, 9(9): 419-423.
Kim, S.; Jung, H.J.; Lamsal, K.; Kim, S.Y.; Min, S.J. and Lee, S.Y. (2012). Antifungal effects of Silver Nanoparticles (AgNPs) against various plant pathogenic fungi. Mycobiology, 40(1): 53-58.

Klitzke, S.; Metreveli, G.; Peters, A.; Schaumann, G. E. and Lang, (2015). The fate of silver nanoparticles in soil solution—Sorption of solutes and aggregation. Science of the Total Environment, 535: 54-60.
Kole, C. (2006). Cereals and millets 1: Springer. http://dx.doi.org/10.1007/978-3-540-
Linares, O. F. (2002). African rice (Oryza glaberrima): history and future potential. Proceedings of the National Academy of Sciences, 99(25), 16360- 16365.
Linscombe, L.S. (2006). Rice cultivar designated cheniere. Available at http//www.freepatentsonline.com/741725.htm. Accessed 03/03/10.
Mew, T.W. and Gonzales, P. (2002). A Handbook of Rice Seedborne Fungi. International Rice Research Institute, Los Banos, Philippines. 83.
Milah, O.S. (2013). The effect of Steel slag and bokashi of rice husk on P retention, P- availability, Al-dd and H-dd at Andisol Lembang, West Java. Faculty of Agriculture, Universitas Padjadjaran
Mishra, and Singh, H.B. (2015). Preparation of biomediated metal nanoparticles. Indian Patent Filed 201611003248
Mukherjee, ; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S.R.; Khan, M.I.; Ramani, R.; Parischa, R.; Ajayakumar, P.V.; Alam, M. and Sastry, M. (2001). Bioreduction of AuCl₄⁻ ions by the fungus, Verticillium sp. and surface trapping of the gold nanoparticles formed. Applied Chemistry, 40(19): 3585-3588.
Nagornyy, V.D. (2013). Soil and plant laboratory analysis. Moscow, Russia: Peoples‘ Friendship University of
Nakkala, J.R.; Mata, R. and Sadras, S.R. (2016). The antioxidant and catalytic activities of green synthesized gold nanoparticles from Piper longum fruit extract. Process Safety and Environmental Protection, 100: 288-294.
Nalley, L.; Tsiboe, F.; Durand-Morat, A.; Shew, A. and Thoma, G. (2016). Economic and environmental impact of rice blast pathogen (Magnaporthe oryzae) alleviation in the United States. Plos one, 11(12) : 1-15.
Nishikado, Y. (1926). Studies on rice blast disease. Japanese Journal of Botany, 3: 239-244.
Sarkar, ; Ray, S.; Chattopadhyay, D.; Laskar, A. and Acharya, K. (2012). Mycogenesis of gold nanoparticles using a phytopathogen Alternaria alternata. Bioprocess Biosystem Engineering, 35: 637-643.
Schlich, K. and Hund-Rinke, K. (2015). Influence of soil properties on the effect of silver nanomaterials on microbial activity in five Environmental Pollution, 196: 321-330.
Selvi, K. and Sivakumar, T. (2012). Isolation and characterization of gold nanoparticles from Fusarium oxysporum. International Journal of Current Microbiology and Appllied Science, 1(1): 56-62.
Shelar, G.B. and Chavan, A.M. (2014). Biological Synthesis and Antibacterial Activity of Silver and Gold Nanoparticles produced by the fungus Aspergillus terreus. Inernational Journal of Science and Research, 3(6): 1679-1681.
S.; Madhu, S. and Singh. S. (2011) Extracellular synthesis of antibacterial silver nanoparticle using psychrophilic bacteria. Process Biochem. 46(9): 1800-1807.
Shoults-Wilson, W.A.; Reinsch, B.C.; Tsyusko, O.V.; Bertsch, P.M.; Lowry, G.V. and Unrine, J.M. (2011). Role of particle size and soil type in toxicity of silver nanoparticles to Soil Science Society of America Journal, 75(2): 365-377.
Shukla, ; Krishnamurthy, S. and Sahi, S.V. (2014). Genome wide transcriptome analysis reveals ABA mediated response in Arabidopsis during gold (AuCl⁻⁴) treatment. Frontiers in Plant Science, 5: 652-660.
Storz, G. and Imlay, J.A. (1999). Oxidative stress. Current Opinion in Microbiology, 2: 188-194.
Syed, A.; Saraswati, S.; Kundu, G.C. and Ahmad, A. (2013). Biological synthesis of silver nanoparticle using the fungus Humicola and evaluation of their cytoxicity using normal and cancer cell lines Spectrochim. Acta A Molecular Biomolecule Spectroscopy, 114: 144-147.
Taylor, A.F.; Rylott, E.L.; Anderson, C.W. and Bruce, N.C. (2014). Investigating the toxicity, uptake, nanoparticle formation and genetic response of plants to PLOS One, 9(4): 937-943.
Vance, D.; Brookes, P.C. and Jenkinson, D.S. (1987). An extraction method for measuring total soil microbial biomass carbon. Soil biology and Biochemistry, 19: 703-770.
Walkley, A. and Black, A. (1934). An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Science, 37 (1): 29-38.
Wang, P.; Menzies, N.W.; Lombi, E.; McKenna, B.A.; Johannessen, ; Glover, C.J. and Kopittke, P.M. (2013). Fate of ZnO nanoparticles in soils and cowpea (Vigna unguiculata). Environmental Science & Technology, 47(23): 13822-13830.
Zhang, X.; He, X.; Wang, K.; Wang, Y.; Li, H. and Tan, W. (2009). Biosynthesis of size-controlled gold nanoparticles using fungus, Penicillium sp. Journal of Nanoscience and Nanotechnology, 10: 5738-5744

Plate 1-7 are available in supplementary section.

P1.png
Plate 1. Mass culture of Metarhizium anisopliae,
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Plate 2. Centrifuge Tube containing the supernatant of Metarhizium anisopliae biomass in aqueous solution of 1 mM HClO4 the beginning of the reaction (A) and after 192 hrs reaction (B)
P3.png
Plate 3. (a) Pure culture of Maganporthe grisea in Oat meal agar slants (b) Pure culture of Maganporthe grisea in Petriplate (c) Microscopic view of Maganporthe grisea (40x)
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Plate 4. a. Spraying of Inoculum of M. grisea b. Maintaining humidity
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Plate 5. Development of typical blast symptom after application of M. grisea
(1x 1010cfu/ml) (a) Leaf Blast (b) Nodal Blast and (c) Neck Blast
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Plate 6. In vitro efficacy of biosynthesized Au NPs against M. grisea(Growth of in M. grisea a. Metarhizium anisopliae mediated Au NPs @ 150ppm, b. Metarhizium anisopliae mediated Au NPs @ 100 ppm, c. Metarhizium anisopliae mediated Au NPs @ 50 ppm, d. Tricylozole @ 600ppm, e . Control
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Plate 7. a (i and ii). TEM micrograph and (b). Electron diffraction pattern of biosynthesized gold nanoparticles from Metarhizium anisopliae

Synthesis of gold nanoparticles from Metarhizium anisopliae for management of blast disease of rice and its effect on soil biological index and physicochemical properties

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Findings

Discussion

Declarations

References

Plate

Supplementary Files

Status:

Version 1