MATERIALS AND METHODS

Location and time duration: Pseudomonas putida IFO 14796 (Osaka, Japan collection) was accessed from Food and Nutrition Laboratory of Gadjah Mada University, Indonesia. The experiment was under taken for 90 days from September 7th, 2018-December 5th, 2018 in the green house, the center for integrated laboratory of Dayanu Ikhsanuddin University, Baubau, Southeast Sulawesi-Indonesia.

Experimental materials: The proliferation of bacteria was carried out in potato extract- sucrose¹⁶ broth (PESB) at 28°C. Carrier made from combination green compost and rice bran in size particle <0.5 mm was sterilized in an autoclave at 121°C, a pressure of 15 pounds¹⁷ for 1 h. The top soil for the growth media was taken from the agriculture area in particulate size <1.5 mm. Pseudomonas putida IFO 14796 (5.1×10⁸ CFU g^–1) in carrier 3:1 proportion was used for treatment.

Design experiment: The research was undertaken in the randomized group design, where one group for treatment and one group for control. A poly bag (25×25× 40 cm) contained with unsterilized soil media and it is the top mixed with 5% (v/v) of the carrier enriched P. putida IFO 14796 (5.1×10⁸ CFU g^–1) for the treatment and carrier without P. putida control. Each group of the experiment was used about 200 of poly bag which was grown one sterilized corn seed (0.36±0.06 g), respectively. Randomized, 12 of the experiment units were chosen to observe the stem size after 55 days of cultivation as well as maize grain samples (g) were taken from all grain in 12 sample unit after was harvested at 90 days in cultivation or 30 days after pollination (DAP).

Data analysis: Stem size data were analyzed by the paired t-test using the software package SPSS 15.0. While the iron concentration in maize grain was analyzed by the atomic absorbance spectrophotometer (Indonesia national standard 19-286-1992 point 3.3.2) method at the Laboratory of center plantation based industry, Makassar, Indonesia. The percentage of improving iron contents in maize grain was analyzed by comparing the difference iron contents in the treatment and control then multiplied by 100 percent.

RESULTS

Corn plant growth: The mean stem size of the plant with the treatment of P. putida IFO 14796 was 65.09 cm higher over the control (52.39) after 55 days of cultivation. Based on the t- test, the treatment was significantly different over the control at p<0.05 (Table 1).

Iron contents in maize grain: The iron contents in maize grain that inoculated by P. putida IFO 14796 was 10.1117 mg kg^–1 higher than uninoculated or control (8.5130 mg kg^–1) (Fig. 1).

Table 1:	Average stem size of the corn plant after the intervention of P. putida IFO 14796 for 55 days of cultivation
*Means difference is significant at p<0.05

Fig. 1:	Iron concentration in maize grain after root inoculating with P. putida IFO 14796 for 90 Days

Thus, there were improved iron contents in maize grains up to 18.79% after 90 days in cultivated or 30 DAP.

DISCUSSION

Measurement of the corn stem size and mineral content in maize grain intended to provide an overview of the ability of P. putida IFO 14796 to enhance the growth and improving the iron ingredient in maize grain product. Average of stem size of corn plant significantly increased after 55 days in cultivated. This phenomenon has increased the expectation that applied P. putida IFO 14796 in agriculture able to support corn production. Although the growing corn plant in shoot and root weight, plant height and plant diameter were significantly different from bacterial consortium than over mono-inoculum treatments for blue maize plant¹⁸. However, the increased stem size of corn plants that were intervened of P. putida IFO 14796 was an important outcome to enhance transport minerals from root to leaf and maize grain. Meanwhile, the iron concentration of maize grain with P. putida IFO 14796 treatment was 10.1117 mg kg^–1 higher than control (8.5130 mg kg^–1). This illustration revealed that P. putida IFO 14796 intervention has caused increasing iron contents in maize grains up to 18.79% after 90 days in cultivated. The previous study also has shown the ability of the Pseudomonas plecoglossicida SRI-156 for increasing the iron content in chiaspeak and pigeonpea grains up to 18 and 12%, respectively¹⁹. However, the transgenic method has increased iron content²⁰ in rice 3.4-fold and organic and chemical fertilizers intervention has increased iron concentration in wheat grain up to 1.4 fold²¹. The ability of P. putida to enhance plant growth and iron content was thought caused by phytohormone and other beneficial compounds production, such as indole-3-acetic acid (IAA), gibberellins (GAs), cytokinins (CTKs), ethylene, extracellular enzymes, antibiotics, cyanide and siderophore which gave a positive effect on plant growth^{11, 22}.

Biofortification with PGP for increasing the growth and improving iron content in maize grain is very important because iron content in maize grain was 6.2 mg kg^–1 only²³. However, the growth and variation of the iron concentration in the grain product was influenced by many factors such as plant genetic, harvest age and mineral content in soil and environment²⁴, such as iron content in maize grain in sub-saharan Africa-ranged from 15-159 ppm for mid-latitude and from 14 to 134 ppm for lowland maize inbred lines²⁵. In the present study, the roots space in the poly bag was thought to be a limiting factor for increasing the growth and iron mineral absorption. Thus, field experiments are needed to maximize the potential of the P. putida IFO 14796 to increase the growth and the iron content in the maize grain. The iron content in maize grain resulted from this biofortification process around 10.1117 mg kg^–1 has a suitable for human consumption as well as previously recommended in rice (6 -22 mg kg^–1), maize grain (10 -160 mg kg^–1) and wheat (15-360 mg kg^–1)²⁶.

The improved iron concentration in biofortified maize was become fundamental in further research for application the protection motivation theory (PMT) in developing dietary strategies to improve the iron status in iron deficiency anemia (IDA)²⁷. As previous study showed intervention of iron biofortified staple food significantly increased serum ferritin concentrations and total body iron²⁸, iron biofortified pearl millet significantly improved iron status in children²⁹, iron biofortified bean increased iron status in Rwandan women after 128 days³⁰ and positively affects cognitive performance in 18-27 year old Rwandan female college students in an 18 weeks³¹.

CONCLUSION

From this study it was concluded that Pseudomonas putida IFO 14796 with density 5.1 × 10⁸ CFU g^–1 can improve iron content in maize grain up to 18.78% after 90 days in cultivated. Thus, proposed to use iron biofortified maize to develop the research to reduce iron deficiency anemia (IDA) through community trial.

SIGNIFICANCE STATEMENT

This study discovered biofortification through P. putida IFO 14796 intervention has increased iron content in maize grain that can be beneficial to overcome iron requirement in human. This study will help the researchers to uncover the critical areas of the role important of P. putida IFO 14796 in biofortification for improving iron content in maize grain that many researchers were not able to explore. Thus a new theory on the protection motivation theory (PMT) to reduce the burden of iron deficiency anemia may be achieved.

ACKNOWLEDGMENT

The author would like to acknowledge to all of the staff of the Cereal Research Center-Maros, Indonesia, Food and Nutrition Laboratory of Gajah Mada University and Rector of Dayanu Ikhsanuddin University for the permission undertaken the study on Department of Public Health, Hasanuddin University, Makassar, South Sulawesi, Indonesia