Biopriming of cayenne seed (Capsicum frutescens L.) using Indole Acetic Acid (IAA) - producing fluorescent pseudomonads to increase germination and growth of seeds

Capsicum frutescens L. is one of the horticultural commodities that plays an important role as food and industrial raw materials. The amount of chili consumption continues to increase, so its production requires quality seeds. The physiological quality of seeds can be improved, one of which is by using fluorescent pseudomonads as biopriming. The fluorescent pseudomonads LAHLS1, LAHT1, LAHCS2, and PfPb3 produced Indole Acetic Acid (IAA) with concentrations between 5.37 to 20.31 ppm, and were significantly different. Biopriming of C. frutescens L. seeds using different fluorescent pseudomonad isolates affected the percentage of germination, wet weight and dry weight of C. frutescens L. seedlings. Fluorescent pseudomonads LAHLS1, LAHT1, LAHCS2, and PfPb3 can increase the percentage of C. frutescens L. seed germination up to 100%. The highest seedling wet weight (43.52 mg) was found in seed biopriming using fluorescent pseudomonad LAHLS1 and the lowest was control (12.33 mg). The highest seedling dry weight (4.40 mg) was found in seed biopriming using fluorescent pseudomonad LAHLS1, and the lowest was control (0.93 mg). The results showed the potential use of IAA-producing fluorescent pseudomonads in increasing the percentage of seed germination and influencing the growth of C. frutescens L. seedlings based on observations of wet weight and dry weight of seeds.


Introduction
Chili is one of the horticultural commodities whose role is quite important as food and industrial raw materials.Cayenne pepper (Capsicum frutescens L.) or also known as bird's eye chili is rich in β carotene (pro-vitamin A), ascorbic acid (vitamin C), and contains tocopherol (vitamin E) which acts as an antioxidant [1].
Increased production of cayenne pepper can be done by using quality seeds and controlling plant pests and diseases.The physiological quality of seeds can be improved by seed treatment, one of which is priming.Priming is a slow hydration activity before the seeds are germinated, which aims to balance the water potential of the seeds to activate metabolic activities in the seeds [2].A lot of research has been done on the priming combination using biological agents, and it is called biopriming.Biopriming using Pseudomonas fluorescens, P. putida and Bacillus subtilis has been reported by [3].While Anhar et al. [4] and Entesari et al. [5] used Trichoderma sp. as biopriming.
The principle of priming is to activate the resources owned by the seed (internal: viability, growth strength vigor, storage vigor, and other seed-forming materials), and external resources (external: optimum germination conditions) which together function to maximize growth.Proper priming treatment can control the rate of seed need for water during the germination process and increase the metabolic rate.This will provide physiological improvements, such as: seeds germinating more quickly and simultaneously and increase the percentage of germination [6].
Fluorescent pseudomonads are a group of bacteria that live in colonies around plant roots.Some bacteria included in the fluorescent pseudomonad group are Pseudomonas fluorescens, P. aeruginosa, and P. putida [7].Fluorescent pseudomonads are a group of rhizobacteria that can be used as an alternative to increase growth, and plant health, and suppress several soil-borne diseases [8].
Fluorescent pseudomonads originating from the rhizosphere of various plants have been successfully isolated.Test results on the fluorescent pseudomonad isolates LAHLS1 (from Singgalang cabbage roots), LAHT1 (from eggplant roots), LAHCS2 (from chili roots), and PfPb3 (from banana roots) show their ability to produce the plant hormone Indole Acetic Acid (IAA), siderophores, phosphate solubilization, and cyanide acid [9].Pseudomonas fluorescens isolate Pf1 can inhibit the growth of Macrophomina phaseolina root rot pathogen of Coleus forskohlii Briq.This is due to its ability to produce antibiotics, siderophores, and lytic enzymes (chitinase, β -1,3-glucanase) that degrade fungal cell walls [10].
Biopriming of sunflower seeds using P. fluorescens effectively controls seed-borne disease infection by Alternaria helianthi and increases seedling growth [13].Priming seeds can increase the resistance of rice plants to environmental stress [14], suppress weeds and increase rice yields [15].Priming rice seeds using growth regulators (ABA, GA3, and IAA) is the best in rice seed germination [16].
C. frutescens L. can be propagated by seeds.The hard seed coat can inhibit germination because it is impermeable to water or oxygen, so that it becomes a mechanical barrier against the emergence of radicles [17].Seed germination can be accelerated by providing biopriming treatment of seeds.The interest in using fluorescent pseudomonads as an ingredient for seed priming is due to its potential to increase the growth and yield of various plants.There is very little information about the use of fluorescent pseudomonads as biopriming.This research was conducted to test the ability of fluorescent pseudomonad isolates LAHLS1 (from Singgalang cabbage roots), LAHT1 (from eggplant roots), LAHCS2 (from chili roots), and PfPb3 in enhancing the germination and growth of chili pepper seedlings, including their wet and dry weights.

Material and Method
The type of research conducted was descriptive research for the detection of IAA and an experiment for the germination and growth of seedlings.The experiment was repeated four times.

Fluorescent pseudomonads preparation
The isolates used as biopriming are fluorescent pseudomonad LAHLS1 (from Singgalang cabbage roots), LAHT1 (from eggplant roots), LAHCS2 (from chili roots) and PfPb3.While the control of the study is the priming of seeds using sterile distilled water (hydropriming).All of the isolates used are Advinda's collections in plant physiology laboratories, Biology majors, FMIPA UNP.All isolates are rejuvenated using the Nutrient Agar (NA) medium and reproduced using Nutrient Broth (NB) medium.After being incubated for 2x24 hours at room temperature, the observation of IAA production and treatment of cayenne seeds was carried out.

C. frutescens L. seed preparation
The C. frutescens L. seeds used are Dewata 43 F1 varieties.Each suspension of fluorescent pseudomonad isolates (population of 3x108 cell/ml) is used to soak 10 C. frutescens L. seeds.Biopriming is done for 24 hours.Each biopriming treatment is repeated 3 times.The seeds that have been treated are sowed in the tray pot that has been filled with sterile land.

IAA detection
The fluorescent pseudomonad suspension is diluted to a population of 3x108 cell/mL.0.1 mL of suspension is taken, then inoculated on the nutrient agar medium which has been added by tryptophan 200 g/mL on a spread plate.The sample was then incubated at room temperature for 2x24 hours [18].
One loop culture was inoculated at 10 mL of medium nutrient broth which had been added by tryptophan 200 μg / mL and was incubated on a shaker at a speed of 150 rpm for 3x24 hours.Furthermore, the culture is centrifuged at a speed of 13,000xg for 10 minutes, and its pellets and supernatants are separated.1 mL of supernatants are inserted into 2 ml of Salkowski reagents and incubated for 30 minutes.If the resulting color is Pink it shows a positive sign of IAA, while yellow shows a negative [19].The existence of a quantitative IAA was observed using a spectrophotometer (OD 530 Nm).The concentration of IAA from the sample was calculated based on the Pure IAA standard curve [20].

Percentage of germination
The percentage of germination is the average number of seeds that germinate and counted on the 5th day after seedling.In this study, the data was calculated on day 5.The percentage of germination was calculated using the formula submitted by [21].Germination (%) = (Number of seeds that germinated)/(Number of seeds on the tray) x 100

Wet weight of seeds
Wet weight of seeds is calculated 14 days after seedling.Determining the wet weight of seeds is done by taking the seeds carefully, and then washed using running water.Then dried on the newspaper, and then weighed.

Dry weight of seeds
Dry weight seeds are calculated 14 days after seedling.Dry weight measurements are done by weighing the seeds that have been dried in the oven at a temperature of 60-70 o C until it is constant.

IAA detection
Detection of IAA in the four fluorescent pseudomonad isolates LAHLS1, LAHT1, LAHCS2, and PfPb3 produced various colors, ranging from dark pink to light pink (Figure 1.).The presence of IAA was quantitatively observed using a spectrophotometer (OD 530 nm).The IAA concentration of the sample was calculated based on the standard curve with pure IAA.The use of the Salkowski reagent is a fast and easy method to detect the presence of indole compounds.Iron from the Salkowski reagent reacts with IAA, forming a complex compound.The color change becomes more intense as the produced IAA content increases [22].For accurate quantitative testing and identification of IAA, a spectrophotometer (OD 530 nm) is used.
The results of the quantitative test using a spectrophotometer (OD 530 nm), the highest IAA production from the fluorescent pseudomonad isolate was found in the LAHCS2 isolate with a concentration reaching 20.31 ppm, while the lowest concentration was produced by the LAHT1 isolate at 5.37 ppm as shown in Figure 2. The pink color produced by fluorescent pseudomonads from the IAA test will turn darker as the concentration of IAA increases [19].Several genera of growth-promoting bacteria derived from the rhizosphere of rice plants can produce IAA with different concentrations [23].In addition, [24] stated that IAA is a hormone that plays a role in plant growth and development, so that synthesis by certain microorganisms that interact with plants can lead to increased plant growth.

Percentage of germination
The physiological quality of the seed will affect the ability of the seed to germinate.In this study, biopriming of C. frutescens L. seeds was carried out using fluorescent pseudomonads to activate the metabolic activities of the seeds.Observations were made on the percentage of germination of seeds treated with fluorescent pseudomonads LAHLS1, LAHT1, LAHCS2, PfPb3 and control (hydropriming) (Figure 3.).The test results showed that biopriming using fluorescent pseudomonads LAHLS1, LAHT1, LAHCS2, and PfPb3 100% could increase the percentage of C. frutescens L. seed germination.While the percentage of germination in the control treatment (hydropriming) was slower at 80%.The results of this study were supported by [25] who stated that plant growth-promoting rhizobacteria can provide beneficial effects, such as increasing the percentage of germination.The increase in the percentage of germination and seedling growth was caused by an increase in the synthesis of growth hormones that would trigger the activity of specific enzymes that promote early germination.Sasirekha et al., [26] stated that the production of plant growth hormones, especially IAA, has long been considered an important feature of plant growth-promoting rhizobacteria.Each different bacterial isolate will produce IAA with different concentrations as well.According to Bharathi [27] the best synthesis of IAA by growth-promoting rhizobacteria can increase seed vigor.

Seed wet weight
The average wet weight of cayenne pepper seeds produced by biopriming with fluorescent pseudomonad isolates and control (hydropriming) can be seen in Figure 4.The highest seed wet weight (43.52 mg) was observed in seed biopriming using fluorescent pseudomonad LAHLS1, followed by PfPb3 (39.83 mg), LAHT1 (31.97 mg), and LAHCS2 (27.83 mg).While the lowest seed wet weight (12.33 mg) was found in the control (hydropriming).In this study, the studied fluorescent pseudomonad isolates produced IAA concentrations between 5.37 to 20.31 ppm.This shows that IAA produced by fluorescent pseudomonad isolates can affect the wet weight of C. frutescens L. seeds.As stated by [28], 11 isolates of Pseudomonas isolated from the rhizosphere produced IAA with concentrations between 10.2 to 31.2 ppm, and could increase germination and wet weight of chickpea seedlings.Jaiman et al., [29] stated that the increase in plant vegetative growth parameters was associated with the biopriming role of Pseudomonas fluorescens and Trichoderma harzianum in fixing nitrogen, producing growth hormones such as gibberellins, cytokinin, and auxins that can stimulate nutrient absorption and play a role in the photosynthesis process.

Seed dry weight
The average dry weight of C. frutescens L. seeds produced by biopriming with fluorescent pseudomonad isolates and control (hydropriming) can be seen in Figure 5.It can be seen in Figure 5 that there was a significant difference in dry weight of C. frutescens L. seeds produced by biopriming of fluorescent pseudomonad isolates with control (hydropriming).The highest seed dry weight (4.40 mg) was observed in seed biopriming using LAHLS1 fluorescent pseudomonad and was significantly different from the other 4 treatments.Biopriming using fluorescent pseudomonads LAHCS2, LAHT1, and PfPb3 resulted in seedling dry weight, which was not significantly different, but significantly different from the control.This is in line with what was stated by Ali et al., [30] that inoculation of IAA-producing bacteria can induce plant growth which has an impact on increasing plant dry weight, root length, number of roots, and root surface area.Riyanti et al., [31] stated that of the 19 isolates tested, Pseudomonas fragi isolates Aj 3.7.1.14had a moderate concentration of IAA (14.77 ppm), and could increase root length, plant dry weight, and tomato fruit yield.

Conclusions
The results of this study showed that the fluorescent pseudomonads LAHLS1, LAHT1, LAHCS2, and PfPb3 could produce Indole Acetic Acid (IAA) with different concentrations.Biopriming using fluorescent pseudomonads LAHLS1, LAHT1, LAHCS2, and PfPb3 can increase the percentage of seed germination and growth of C. frutescens L. seeds based on observations of wet weight and dry weight of seeds.Finally, this biopriming becomes remarkable because it can prevent farmers from using chemicals.

Figure 3 .
Figure 3. Percentage of cayenne pepper seeds germination

Figure 4 .
Figure 4. Wet weight of C. frutescens L. seeds produced by biopriming with fluorescent pseudomonads

6 Figure 5 .
Figure 5. Dry weight of C. frutescens L. seedlings produced by biopriming with fluorescent pseudomonads