Enhancing Melon Growth and Nutrient Uptake with Plant-Growth-Promoting Microorganisms from Cucumis melo

2.1 Sampling site

Sampling activities were conducted on farmers’ melon plantations in three locations Wonogiri Regency, Sragen Regency, and Karanganyar Regency (Table 1). Isolation and isolate characterization were conducted at the Laboratory of Soil Biology and Biotechnology, Faculty of Agriculture, Sebelas Maret University. The selection of sampling locations was based on interviews that had been conducted with farmer groups and local farmers.

Table 1. Location of soil sampling

The first location is located in Mojosari, Ngadirejo Village, Eromoko District, Wonogiri Regency with soil ordo Vertisols (Table 1). Sampling was done when the land was being prepared for the next melon-growing season. The cultivation system uses a non-organic farming system with an irrigation system and land is only for melon cultivation.

The second location chosen is located in Dawungan Village, Masaran District, Sragen Regency with soil ordo Vertisols (Table 1). The soil samples were conducted when the melon plant was ready to be harvested, about 2 weeks before harvest. The cultivation system is like the first location with the cropping pattern used is paddy-shallot-melon.

The third location chosen for soil sampling is located in Sukosari Village, Jumantono District, Karanganyar Regency with soil ordo Alfisols (Table 1). Melons are cultivated by Agrimaga Farmhouse in a screen house the system used is a non-organic farming system with a drip irrigation system. Soil sampling was carried out when the plants reached the initial stage of flower emergence.

2.2 Soil Sampling, isolation and purification of soil microorganisms

Rhizosphere soil samples were collected from three melon fields (Table 1). Isolation was conducted using the dilution method on nutrient agar (NA) for bacterial growth and potato dextrose agar (PDA) for fungal growth. After 7 days of isolation at room temperature, colony forming unit (CFU) or viable cells/gram soil was calculated by the following formula:

$\mathrm{CFU}=\frac{ { Mean \,plate \,count } \times { Dilution \,factor }}{ { Amount \,of \,dilution }}$         (1)

Morphologies of the colony were observed by Bergeys’ Manual Systematic Bacteriology. The selected microorganisms were then purified three times on the media to obtain pure isolates using the streak method.

2.3 Screening of microbial potency to provide soil nutrients, produce IAA and antagonism assay

2.3.1 Screening of N-fixing ability, P and K solubilization

N-fixing ability was identified by inoculating purified microbial cultures on Jensen's N-free medium and incubating for 3 days at room temperature. Microbes that have the potential to fix N are characterized by the growth of clear colonies like water droplets on the surface medium.

P and K solubilization ability was identified by inoculating purified microbial culture on Pikovskaya medium for P and Aleksandrow medium for K and incubating 3 days at room temperature. The microorganisms were characterized by the presence of a halo zone in the media around the growing colonies. The ability of mineral solubilization is then calculated by the formula of the Solubilization Index below:

$\mathrm{SI}=\frac{ { halozone \,diameter }}{ { colony \,diameter }}$           (2)

2.3.2 Quantitively assay for IAA production

The production of IAA by microorganisms was quantitatively measured using the colorimetric method by adding the Salkowski reagent (150 ml of H₂SO₄ and 7.5 ml of 0.5 M FeCl₃.6H₂O in 250 ml of distilled water). Bacterial isolates were cultured on 10 ml nutrient broth (NB) and fungal isolates were cultured on 10 ml potato dextrose broth (PDB). One hundred g.ml^-1 L-tryptophan was added as a precursor to producing IAA. The culture was then shaken at 100 rpm for 48 h at room temperature. Then the culture was centrifuged at 5000 rpm for 30 minutes. Two ml of the supernatant was taken and put into a sterile flask, then mixed with 2 ml of Salkowski's reagent and then incubated for 60 minutes at room temperature. Next, the absorbance of the solution was measured using a spectrophotometer with λ = 530 nm and quantified in an IAA standard curve.

2.3.3 Biocontrol potential against F. oxysporum

The biocontrol potential analysis was conducted by a bi-culture antagonistic test by placing the purified fungi isolates and the F. oxysporum isolates side by side on the petri dish. The F. oxysporum isolates were obtained from the Laboratory of Plant Pests and Disease, Faculty of Agriculture, Gadjah Mada University, Indonesia. The media used to place the isolates was PDA and mixed PDA and NA. After the isolates were incubated for 7 days, observations were made by observing the growth direction of the isolate and F. oxysporum, if is there an obstructed growth. The percentage of antagonism potency was calculated using the formula below:

F. oxysporum inhibition $\%=\frac{(a-b)}{b} \times 100$          (3)

where,

a = diameter of F. oxysporum on control

b = diameter of F. oxysporum on bi-culture

2.4 Sequence analysis for selected isolate

The sequence analysis was conducted to investigate the species taxa of each potential isolate based on the molecular assay. The analysis was conducted by PT. Genetika Science Indonesia using PCR and sequencing of 16S rDNA for bacteria and rDNA ITS region for fungi. A similar sequence was obtained by BLAST search at the NCBI Database.

2.5 Pot experimental test for selected isolate

2.5.1 Inoculum formulation

Three selected isolates were then cultured until the cell density reached 10⁸ CFU.ml^-1. The liquid media were used in the form of NB for bacterial cell culture and PDB for fungal spore cultures. Each isolate was cultured in each media with a stirring speed of 180-200 rpm for 3×24 hours at room temperature and then we enumerated the cell density. The inoculant was applied before planting with a dosage of 10 ml.pot^-1 and twice a week with a dosage of 5 ml.pot^-1 using a sprayer.

2.5.2 Experimental design

An experiment was conducted at the Greenhouse of the Faculty of Agriculture, Sebelas Maret University to examine the impact of specific inoculum on the growth of melon and the nutrient status of the soil. The research design used a CRD with a single-factor, formulation of inoculum. The formulation consists of 7 levels, viz control+ (NPK fertilization, 35 g urea, 50 g TSP, and 40 g KCl per plant), control- (without inoculum and NPK), F1 (BK 1), F2 (BS 1A), F3 (FW 2B), F4 (BK 1 + BS 1A), F5 (BK 1 + FW 2B), F6 (BS 1A + BW 2B), and F7 (BK 1 + BS 1A + FW 2B). Each treatment was repeated 3 times. C+ was chosen to see whether the effect of the inoculated isolates could be as good as the use of NPK fertilizer and C- was selected to be a comparison when there’s no treatment at all. The NPK fertilizer for C+ was applied on the day before planting and the 10th, 20th, and 35th days after planting.

2.5.3 Greenhouse condition

The Greenhouse of the Faculty of Agriculture Sebelas Maret University is located at 7°33’41” S and 110°51’32” E with an average daily temperature of 30-33^oC and humidity of 63-78%, monitored every day. We used the soil from Entisols soil ordo. Soil initial test was conducted to analyze the characteristics of the initial soil. The results of the initial soil analysis that the soil used had a neutral pH (7.3), very low organic C content (0.65%), medium total N in the soil (0.46%), very low available P content (0.56 ppm), and moderate exchangeable K levels (25.23 mg/100g).

2.5.4 Melon cultivation and growth observation

We used the “Pertiwi” variety with a germination rate of 85% and a high germination vigor level. Seeding was conducted until the seedlings were ± 14 days old. Then, the seeds are transferred to polybags/planting pots.

The soil to be used is sifted so that it is smooth and there are no lumps. The soil is then sterilized to remove indigenous microbes. The dose of manure used is 20 tons.ha^-1. Initial fertilizer analysis was carried out to determine the nutrient content of the manure. The results of the analysis showed a pH value of 7.71, a total N of 0.23%, a P of 0.23%, and a K of 0.59%.

The cultivation treatment for these plants involves four main steps: watering, pruning, stake installation, and pesticide spraying. Watering is done once a day either before 7 a.m. or after 4 p.m. until the soil is sufficiently moist. To promote stronger growth, pruning is performed to remove excess branches and retain only one main stem. A week after planting, bamboo stakes are installed to support the plants. We use a botanical pesticide made from crushed garlic soaked in water overnight and mixed with cooking oil. This mixture is sprayed twice a week in the morning before 7 a.m. or after 4 p.m. Plant height measurements were taken once a week until the maximum vegetative period.

2.5.5 Plant harvesting and nutrient uptake analysis

Plant harvesting activity was conducted after the melon plants were ± 65 days after planting. Melon plants were taken and weighed to determine the fresh weight of the plants. Melon plants were then stored in an oven at 70°C for 48 hours to determine the dry weight.

Dried whole plant tissue was ground for the analysis of nutrient uptake including, N (H₂SO₄ wet ashes), P, and K (HNO₃ and HClO₄ wet ashes) based on the procedure from the Indonesian Soil Research Centre (2005) [32].

2.6 Statistical Analysis

An ANOVA analysis was performed with a confidence level of 95% to evaluate the impact of the treatment on the observed variables. If the treatment showed a significant effect, the DMRT was conducted to compare the different inoculum formulations. The analysis was carried out using the SPSS 26 software.

4.1 Exploration of soil microorganisms from melon rhizosphere

The isolation of microorganisms from three melon fields showed that there are various densities of microorganisms (Table 2). The melon field from Wonogiri had the highest bacterial density, while Karanganyar had the highest fungal density (Table 2). The amount of organic matter in the soil can impact the diversity and activity of microorganisms in the soil [33, 34]. These may explain the variation in colony density and morphological diversity (Table 3).

The results of the screening to provide soil nutrients, only one of the isolates found in the rhizosphere of melon plants was able to fix nitrogen, and it was identified as BS 1A (Table 3). Applying this isolate to melon growth is expected to help reduce the use of inorganic N fertilizer. Nitrogen-fixing bacteria have benefits such as increasing root surface, seed production, and fruit ripening [35]. Using a consortium of nitrogen-fixing bacteria was shown to reduce the use of inorganic fertilizers in plants by up to 25% [36].

The high P solubilization activity is expected to enhance the P availability. A higher value of PSI indicates a higher activity of phosphatase enzymes and the production of organic acids [37]. For the greenhouse experiment, PSMs BS 1A and BK 1 were selected (Table 5). The results showed that the inoculation of PSM increased the uptake of P by plants (Table 8). PSM is an alternative way to maintain soil health and minimize its degradation [38]. The use of PSM as a biological fertilizer is expected to increase the yield of fruits on melon plants, help these plants resist pathogen attacks, and reduce the excessive use of chemical fertilizers [39-41].

The K solubilization index (KSI) was used to quantify the ability to solubilize K. The results showed that BK 1 and FW 2B were suitable for inoculant application in the greenhouse experiment (Table 5). KSM produces organic acids that can solubilize K directly or chelate silicate ions first to absorb K and become available to plants [42, 43]. Utilizing KSM increased K uptake in plants (Table 8), indicating its potential as a biofertilizer. The use of KSM in agriculture as a biofertilizer can reduce the need for K fertilizer by up to 50% [44].

Plant growth is largely influenced by microbes, which produce phytohormones such as IAA that stimulate growth. IAA is a natural auxin and its production and concentration are affected by various factors such as species/strain, pH, carbon source, nitrogen source, and tryptophan availability. Microorganisms can produce varying amounts of IAA, as observed in studies using L-tryptophan as a precursor (Tables 4 and 5) [45, 46]. The use of microorganisms that can produce IAA will improve plant tolerance to pathogens by regulating growth [47]. Plants use IAA for various growth and physiological processes, including embryogenesis, organogenesis, root and shoot development, growth, and fruit development [48].

Several selected microorganisms showed the potency of F. oxysporum inhibition with the highest inhibition activity shown by BS 1A (Table 4) and FK 1C (Table 5). The inhibition occurs because of the mechanization of antibiotics or anti-microbials from isolates to fight pathogens [49, 50]. Several compounds synthesized by plant growth-promoting microorganisms (PGPM) such as HCN, chitinase, and gluconate make PGPM have antagonistic abilities [51]. This is expected to reduce the use of chemical products in fighting F. oxysporum in melon cultivation. Biocontrol agents can prevent pathogen infection, improve plant growth, and increase resistance to pathogens [21, 47].

We selected 3 potential isolates for greenhouse experiments, namely BS 1A, BK 1, and FW 2B based on the results of the screening that had been conducted. The selection was based on the outstanding characteristics of each isolate to complement each other when formulated into a biofertilizer in the future. Utilizing the PGPM with a beneficial effect for providing nutrients and plant growth offers environmentally friendly and sustainable alternatives to chemical fertilizer and disease control methods in melon cultivation.

4.2 The effect of inoculation soil microorganisms on plant growth and nutrient uptake

The use of a microorganism formula on melon plants had a positive impact on their growth, as indicated in Table 7. This can be attributed to the presence of inoculum, which provides necessary nutrients for the plants [52]. The addition of inoculum leads to an increase in nutrition and subsequently, plant height [53]. Furthermore, the application of microorganism inoculum resulted in an increase in plant biomass - both fresh and dry weight (Table 8). Results from the Pearson’s correlation test indicate that factors affecting plant fresh weight were N uptake (r=0.849**), K uptake (r=0.590**), and exchangeable K value in the soil (r=0.547**). On the other hand, plant dry weight was influenced by fresh weight (r=0.839**), N uptake (r=0.919*), P uptake (r=0.419*), and K uptake (r=0.740**). This demonstrates the significant impact of nutrition on plant biomass. When plants receive adequate nutrients, they can grow optimally and produce good biomass [54].

The inoculation of microorganisms that provide nutrients can effectively improve plant growth and nutrition. In this study, a single inoculation of B. cereus resulted in the highest increase in both fresh and dry plant biomass compared to other formulas (Table 8). B. cereus has been proven to promote plant growth in various studies involving sunflowers, Phaseolus vulgaris, and tomatoes. The ability of B. cereus to enhance plant growth is directly related to its production of the growth hormone IAA (Table 4). IAA is a known promoter of plant growth, and plants utilize IAA produced by microorganisms to encourage root elongation, increased root coverage, and mineral uptake, as well as root exudation [55, 56]. Furthermore, IAA contributes to plant tolerance against stress and pathogen attacks. Microbe-produced IAA enhances the number of root hair and plant lateral roots, which in turn improves plant mineral uptake and root exudation. Overall, IAA improves plant tolerance, growth, dry mass, and photosynthetic pigments [57].

The application of microorganisms in melon cultivation in greenhouses can increase NPK uptake by plants (Table 10), this is related to the ability of the applied microorganisms to fix N and dissolve P and K (Tables 4 and 5). Single inoculant B. cereus gave the highest N uptake results. Farmers commonly use inorganic nitrogen fertilizers to meet plant needs, but up to 65% of applied minerals are lost through gas emissions, runoff, erosion, and leaching [58]. Utilization of N-fixing bacteria (NFB) has been widely used to reduce the use of N fertilizers in plants. NFB can convert N₂ in nature into NH₃ because it produces the enzyme nitrogenase. The addition of microorganisms can increase nitrogen through several mechanisms such as N fixation, enzyme activity such as N-acetylglucosaminidase and urease in soil N mineralization, ammonification, and nitrification [59, 60].

Formulation F5 which is a combination of B. cereus and Trametes polyzona gives the highest K uptake value compared to the others (Table 10). The inoculum of the two species used is known to have the ability to solubilize K (Table 10). KSM can increase the availability of K in soil solution thereby increasing the absorption of K for plant growth and development [40]. KSM can dissolve K in the soil through the mechanisms of acidolysis, chelation, exchange reactions, and complexation of the organic acids produced [14, 61, 62]. Likewise, the presence of PSM can make more P available for plant absorption [63]. PSM has been widely accepted as an environmentally friendly and easy-to-obtain P fertilizer for increasing soil orthophosphate concentrations and the geochemical P cycle since the early 20th century [41]. PSM produces organic acids and enzymes such as phosphatase and pyrophosphatase which can dissolve P to become available [64, 65]. PSM has many roles for plants, including growth boosters [39], biocontrol and bioremediation activities [40], and increasing plant growth and fruit yields [41]. The F7 formulation consisting of a combination of the three isolates, in which the three isolates could produce organic acids in dissolving K and P, was able to give the highest P absorption results.

The use of PGPM in this research plays a vital role in enhancing nutrient uptake and plant growth. This ensures the long-term health, and productivity of melon, and offers a promising path forward for more eco-friendly and efficient agricultural practices. Beyond the promotion of plant growth, PGPM also supports biocontrol and bioremediation activities, reducing the reliance on chemical products in agriculture.