Nano-emulsion of three essential oils as the potential eco-friendly control to Phytophthora capsici

Phytophthora capsici is a plant pathogenic oomycete that causes significant damage to many crops, including black peppers, chili, cucurbits, and tomatoes. This pathogen is widespread and can survive in soil and plant debris for several years, making it difficult to control. The aim of the research was to observe the effectiveness of three essential oils as nano-emulsion formulas (NS, NC, NE and NA) against the growth of P. capsici in vitro and in vivo. In vitro testing was carried out using 2 methods, namely the poison food and volatile disc assay. Testing of active ingredients using the GCMS method and measuring active ingredient particles using the DLS method. In vivo testing was carried out on chili seeds to determine their growth and germination. The result indicated that all nano-emulsions at 0.50% inhibited the growth of P. capsici which varied between 33,25% to 88,50%. The NC, NS, and NA formula caused complete mycelial growth inhibition at a concentration of 1.0%. Total inhibition of growth also occurred with volatile compounds produced by NS and NA formula. The NC, NS, NE and NA formulas are not phytotoxicity to chili seeds and their germination. Therefore, three essential oils (NC, NS, NE) and the mixture of them (NA) are potential to be used in managing P. capsici as eco-friendly control and possibly as an alternative to synthetic fungicides.


Introduction
Phytophthora capsici is the most devastating fungal pathogen, causing significant economic losses to many crops including pepper (Capsicum annuum) as annual crop and Black pepper (Piper nigrum) as perennial crop.Damping off on chili pepper and foot rot disease on black pepper occurs throughout the year, especially during rainy season.P. capsici is a soil borne pathogen, that can survive in the soil for many years in the form resting spore such as mycelia and oospore [1].
Farmers usually use the synthetic fungicide to control the disease.The application of synthetic fungicides to control soil-borne pathogens often does not provide benefits because it can cause air pollution, endanger human and animal health, and create an imbalance in soil microbes.[1].
Essential oils are secondary metabolites produced by aromatic plants.The characteristics of essential oils are their aromatic and volatile liquid, which have demonstrated anti-fungal activity [2].
Previous studies have confirmed that citronella oil, at a concentration of 750 ppm, could inhibit 75.95% of the growth of P. palmivora [3].
The objective of the research was to assess the effectiveness of three essential oils (Clove, Citronellal, Eucalyptus oils) as the source of nano-emulsion formulas namely Nano clove (NC), nano 1287 (2023) 012020 IOP Publishing doi:10.1088/1755-1315/1287/1/012020 2 citronella (NS), nano eucalyptus (NE) and nano essential (NA) which was the mixture of clove, citronella, and eucalyptus, against the growth of P. capsici both in vitro and in vivo.

Materials and Methods
The research was conducted at the Genomic Laboratory and Integrated Laboratory of the National Research and Innovation Agency (NRIA), in Bogor and field experiment was conducted in Pesisir Selatan West Sumatera from November 2022 to May 2023.The citronella oil, clove oil, and eucalyptus oil used was derived from West Java.

Preparation, Identification and Determining of Active Compounds on Nano-essential Emulsion
Formula.
Three essential oils namely clove oil, citronella oil, and eucalyptus oil were used as the source of nano essential emulsion formula.The nano formula consisted of Nano clove (NC), nano citronella (NS), nano eucalyptus (NE) and nano essential (NA) which was the mixture of clove, citronella, and eucalyptus.A spontaneous diffusion mechanism and an inversion phase with low energy were used to create the nanoemulsion formula.The modification method of [4] was used to create all essential oil nano-emulsion formulas.For the eucalyptus nano emulsion formulation using the addition of agrilan emulsifier (as a substitute for ethylan SP25).The stirring time to produce the nano-emulsion formula was 1.5 hours at a speed of 500-700 rpm.
Identification and determination of active compounds on the Nano-essential Emulsion formula were conducted by Gas Chromatography-Mass Spectroscopy (GC-MS), at Integrated Laboratory National Research and Innovation Agency, Cibinong Bogor.

Determining Antifungal of Nano Essential Emulsion Formula Against P. capsici
The isolate of Phytophthora capsici (K2) was used as the inoculum.Four nano formulas namely NC, NE, NS, and NA were tested for their antifungal potential against P. capsici using two methods: poison food/direct action and volatile disc assay as indirect action.

Poison food method of Nano Formula against P. capsici
Three concentration of each formula (0.5; 1; 1.5%) were tested.Each concentration was mixed into sterile PDA media, shake a while then pouring it into a Petri dish.The nano emulsion formula was not added to the PDA media (control treatment).The treatment consisted of 12 treatments arranged in a Completely Randomized Design (CRD) and 6 replications for each treatment.A disc of inoculum (5 mm in diameter) from five days inoculum culture of P. capsici was laid down in the centre of each Petri dish.All petri dishes were incubated in the dark at room temperature.The growth of P. capsici was IOP Publishing doi:10.1088/1755-1315/1287/1/0120203 monitored every two days.Completely randomized design with six replications of each treatment was carried out in this experiment.
The antifungal activity was assessed by calculating the percentage of growth inhibition (PGI) for each treatment [2]: Where, dC is the colony diameter (mm) in control and dT is the colony diameter (mm) in the treated Petri dish.

Volatile disc of Nano Formula against P. capsici
Four concentrations of each formula (0; 150; 250; and 450 µl) were tested.A disc of P. capsici inoculum (5 mm in diameter) was laid down in the centre of each Petri dish contained with PDA medium.A sterile filter paper disc (1 cm in diameter) was put down in the centre of the inside of the Petri dish cover and dripped with the concentration of each formula.As the control treatment, sterile distilled water was dropped on the filter paper.Petri dishes were sealed with parafilm to reduce loss of volatile through evaporation.All Petri dishes were put upside down and incubated in room temperature.
Completely Randomized Design (CRD) with four treatments and 6 replications of each treatment, was carried out in this experiment.The growth of P. capsici was measured every two days.Antifungal activity was evaluated by calculating the percent growth inhibition (PGI) of each treatment [2].

The effect of Nano formula against germination and seedling growth of chili pepper
Seeds of pepper (Capsicum annuum) namely Akar variety which is an indigenous seed of West Sumatera.Seeds treatments were conducted by soaking seeds in nano formulas for 30 minutes.The control consisted of soaking in fungicide solution, warm water (which was commonly used by farmers) and tap water.Nano formulas consisted of NC, NE, NS, and NA with three concentrations (0,5; 1,0; and 1,5%) of each.Germination and seedling were carried out using germinated trays which were fulfilled with a mixture of soil and manure (2:1).Randomized Block Design (RBD) with 12 treatments and 3 replications for each treatment and, 50 seeds per each was conducted in this experiment.
Five parameter observation according to [5], consisted of growth potential (Gp),
The chromatogram images of each formulation can be seen in Figures 1-4.

Determining Antifungal of Nano Essential Emulsion as the formula Against P. capsici
The poison food method proves as direct action of the antifungal activity of four nano formulas against P. capsici.It was calculated as the percent growth inhibition (PGI) of the colony.Formula NA, NC, and NS exhibited a stronger antifungal effect on the growth of P. capsici compared to formula NE.At a 0.5% concentration in the test, those three formulas exhibited a PGI ranging from 73.5% to 88.5%, while formula NE only showed a PGI of 33.25%.However, at a 1.0% concentration, formulas NA, NC, and NS displayed complete antifungal activity, resulting in the total inhibition of mycelium growth and the death of P. capsici.In contrast, formula NE inhibited growth by only 38.28% (Fig 5 and Fig 6).The death of the P. capsici was proven by transferring the inoculum to PDA media without adding the formula, apparently, there was no growth after three days incubated at room temperature.Four nano formulas had been in vitro tested, showing antifungal activity against P. capsici through both direct and indirect action.All formulas showed direct action, but the formula NA, NC, and NS were better than formula NE.The three formulas showed completely inhibited mycelial growth (100%) or fungicidal against P. capsici at a concentration of 1.0%.All formulas produced volatile compounds that acted as indirect inhibitors of mycelial growth.Eugenol, eugenol acetate, iso-eugenol, borneol, dipetene, limonene and β-caryophyllene, are the common compounds of essential oils that have an antifungal of P. parasitica mycelium [8].Essential oil of Clove and citronella inhibited P.capsici as direct and indirect action.Based on observation by SEM, the mechanism of inhibition was caused by the lyse of mycelial [2].
[9] stated that eucalyptol as the main compound in nano Eucalytus formula (NE) inhibited the growth of P. capsici in vitro test as direct and indirect action.Besides that, based on the in vivo test, NE inhibited the infection of P. capsici on the black pepper leaves.[10], stated that the eucalyptol as the major component in rhizome oil of Alpinia officinarum, showed totally inhibited the mycelia growth of P. parasitica at concentration 898.01 mg/L The formula NA and NS displayed totally inhibited growth of P. capsici at a concentration of 150 µl.Based on those results, the formula NA and NS have two actions antifungal, which means those formulas are better than NE and NC.The formula NS is a single compound of nano Citronella essential oil, whereas the formula NA is the mixture of the three nano essential oils that are clove, citronella, and eucalyptus.According to [11], the efficacy of mixed essential oils is better than one essential oil.The combination of some essential oils could have a synergistic action against the target.[12] proved that the combination eugenol and citral showed the antifungal activity against Penicillium roqueforti better than eugenol or citral alone.The synergistic effects of those compounds can reduce the dosage, the cost and improve the biological activity.

The Effect of Nano Formula against Germination and Seedling Growth of Chili Pepper
The effect of 12 treatments of essential nano formula against germination and seedling growth of chili pepper is presented in Table 1.All treatments are not significantly different in Germination percentage (Gp), Vigor index (Vi), and Growing speed (Gs) of chili pepper.There are a significantly different in Simultaneous growing seeds (Sgs) and Total 50% germination chili seeds (T50).The highest Sgs (93.33%) occurred with the treatment of NC 0,5% was significantly different in Sgs with NA formula in 1.5% but was not significantly different with Fungicide, warm water, and water (control).The same resulted by [2] that clove essential oil did not affect the seedling emergence of chili pepper.The same case also occurred in the Total germination chili seeds (T50).Seeds germination with NC 0,5% quicker than the treatment with 1.5% NA, but was not significantly different with Fungicide, warm water, and tap water (control).These results showed that all essential oils of nano emulsion are not phytotoxicity to chili seeds and their germination.[2] stated that phytotoxicity must not occur if the essential oil is applied to plants or soil, therefore it can apply as a fumigant for soil.According to [1], the compounds in essential oils which have antifungal activity, do not leave residues in the environment, have low toxicity against mammals, and are degradable under natural conditions.

Conclusion
The antifungal activity of the formulas NA, NC and NS is stronger than NE when assessed using the poison food method/direct action.The complete antifungal activity of these formulas occurred at of 1% concentration.The totally antifungal activity of NA and NS when exposed to volatile compounds at concentration 150 µl was found to be the same.When all nano essential oil formulas were treated as a seed treatment, they did not cause any phytotoxicity in chili seeds and their germination and growth were not unaffected.

Acknowledgments
The authors would like to thank the Research Centre for Horticulture and Estate Crops, National Research, and Innovation Agency (NRIA) for their research equipment facilities and the laboratory supervisor for helping with sample analysis.Then, would like to thanks to PT Nuryon and the team for providing Agrilan®AEC145 and Ethylan@SP25.

Figure 1 .Figure 2 .
Figure 1.Chromatogram of the active ingredient of Essential Nano emulsion (NA)

Figure 5 .
Figure 5.The antifungal activity of four nano formulas against the growth of P. capsici on the PDA medium after 7 days of incubation.

Figure 7 .
Figure 7.The volatile antifungal activity of four nano formulas against the growth of P. capsici on the PDA medium after 7 days of incubation.

Table 1 .
The effect of essential oil of nano emulsion against germination and seedling of chili pepper.
Means followed by the same letters were not significantly different, determined by Fisher's least significant difference test (α = 0.05).