Introduction

Rice is the primary food crop for societies world wide as well as in Indonesia (Ma, Wang, Cheng, Zhang, & Lyu, 2020; Panuju, Mizuno, & Trisasongko, 1961). Based on global data in 2017, Indonesia (81.4 million tons) are the third-largest producer after that China (427.1 million tons) and India (168.5 million tons) (FAOSTAT, 2020). One of the problems in rice production is rice blast disease, which caused by fungus of Pyricularia oryzae (Xiao et al., 2018). Globally, rice blast causes a decline in rice production estimated at around 30% (Nalley, Tsiboe, Durand-Morat, Shew, & Thoma, 2016). Therefore, the search for antifungal of P. oryzae is essential needed for improving rice production in agricultural management.

Recently, silver nanoparticles (AgNPs) have been significantly used as an antimicrobial agent in several area application such as cosmetics (Kokura et al., 2010), agricultural (Khaleghi, Khorrami, & Ravan, 2019; Torrent, Marguí, Queralt, Hidalgo, & Iglesias, 2019; Vijayabharathi, Sathya, & Gopalakrishnan, 2018), food storage (Bocate et al., 2019), and medicine (Khorrami, Zarrabi, Khaleghi, Danaei, & Mozafari, 2018). The AgNPs can be produced by the chemical and physical method and also the green synthesis routes, which uses microbial (yeast, algae, bacteria, and fungi) and plant extracts (Khodashenas & Ghorbani, 2019; Souza, Souza, & Franchi, 2019). For hazardous and toxic chemical reasons from the chemical and physical methods, the microbe is a crucial source of a biological system for the AgNPs biosynthesis. Several reports have investigated different microbe which produced AgNPs such as Lactobacillus (Gomma, 2016), Bacillus (Kalishwaralal, Deepak, Ramkumarpandian, Nellaiah, & Sangiliyandi, 2008; Saifuddin, Wong, & Yasumira, 2009), Enterobacter (Shahverdi, Minaeian, Shahverdi, Jamalifar, & Nohi, 2007), Escherichia (Maharani, Sundaramanickam, & Balasubramanian, 2016), Klebsiella (Müller et al., 2016), Saccharomyces cerevisiae (Korbekandi, Mohseni, Jouneghani, Pourhossein, & Iravani, 2016), and Caulerpa racemosa (Edison, Atchudan, Kamal, & Lee, 2016).

In this work, the biosynthesis AgNPs was studied using Lactobacillus delbrueckii subsp. bulgaricus culture. Furthermore, antimicrobial activity of AgNPs against P. oryzae was investigated.

Materials and Methods

Biosynthesis of AgNPs

The strain L. delbrueckiiwas purchased from the IPB culture collection, IPB University, Indonesia. The AgNPS was produced according to (Saravanan, Nanda, & Kingsley, 2011) method with some modification. Briefly, L. delbrueckiiwas newly inoculated with Man, Rogosa, and Sharpe medium. After incubated in 24 h for 150 rpm at 37°C, the culture filtrates were obtained using centrifugation at 8000 rpm for 10 min. The culture supernatant (100 ml) at pH sixwas mixed with 0.017 g AgNO3. Finally, the mixture was incubated in a dark room for 30 min. AgNPs characterized by changed color solutions from yellow to brown. The control was used without AgNO3 in solution.

Characterization

The characterization of AgNPs was performed using UV-vis spectrophotometer and particle size analyzer.

Antifungal activity of AgNPs

The biosynthesized Ag nanoparticles were evaluated against P. oryzae by agar well diffusion method, according to (Shanthi et al., 2016) with slight modification. P. oryzae was purchased from the IPB Collection Culture, IPB University, Indonesia. P. oryzae was sub-cultured newly in PDA (potato dextrose agar) plates. In the PDA plate, the well with a size of 8.00 mm was created using gel puncture. The sample AgNPs (0.1%, 0.25%, 0.5%, 10%, 25%, 50%, 75%, and 100%) of 20 ml loaded on to the wells. Nystatin and sterile water (control) were also investigated as controls treatment. After P. oryzae incubated at 25°C for five days, the diameter of the inhibition zone was measured, and the data presented in mm.

Results and Discussion

Biosynthesis and characterization of silver nanoparticle (AgNPs) from L. delbrueckii is shown in Figure 1. Generally, the formation of AgNPs is characterized by the change color from yellow to dark brown (Figure 1 a) in solution AgNPs with L. delbrueckii (Dakhil, 2017). Up to date, the mechanism of AgNPs synthesis by microorganisms is still unclear. Still, the mechanism through microorganisms’ enzymes such as nitrate reductase possible can contribute to reducing silver ions in AgNPs formation (El-Batal, Amin, Shehata, & Hallol, 2013; Wang et al., 2018). The nanoparticle of AgNPs was characterized by UV-Vis and PSA, data presented in Figure 1 (b and c). The maximum peak of the UV-Vis spectrophotometer was shown at 423 nm. (Mulfinger et al., 2007) identified absorption peaks of AgNPs in the range of 370 – 500 nm (Mulfinger et al., 2007).

This result indicated the colloid formation of AgNPs (Khaleghi et al., 2019). PSA results presented that the average size of AgNPs was 2 nm. Several reports demonstrated different particle sizes of AgNPs from different strain of Bacillus with range of 4 (Babu & Gunasekaran, 2009) to 94 nm (Das et al., 2014; Pourali & Yahyaei, 2016).

The antifungal effect of AgNPs was evaluated in P. oryzae. The inhibition zone diameter of the AgNPs on P. oryzae has been provided in Table 1 andFigure 2. The data clearly presented that AgNPs inhibited P. oryzae growth at different concentrations. The 0.75% AgNPs showed the highest for inhibiting P. oryzae. P. oryzae infected in rice which most destructive effect such as reduces of rice yield and the characteristic of rice (Xiao et al., 2018). This result indicated that potency of the AgNPs for bio-fungicide in agriculture area especially in rice blast management. Similar reports exhibited that AgNPs had antimicrobial activity on fungal pathogens (Bocate et al., 2019; Gulbagca, Ozdemir, Gulcan, & Sen, 2019; Vijayabharathi et al., 2018). Several researchers suggested mechanism antimicrobial of AgNPs through disrupting in DNA replications, damage cell membrane, disorderly respiration role, and creating reactive oxygen species (Khorrami et al., 2018; Shanthi et al., 2016; Singh et al., 2016).

Conclusions

The present work reports characterization silver nanoparticles (AgNPs) from Lactobacillus delbrueckii subsp. bulgaricus. Also, the potential of the AgNPs in the application antifungal activity against Pyricularia oryzae has been evaluated.

Based on the results, the AgNPs characterized by wavelength of absorbance and the average size of 423 nm and 2 nm, respectively.

The antifungal activity of AgNPs was shown with highest activity at 0.75% concentration.