Enhancing zinc uptake and grain yield of Ciherang and Inpari IR Nutri Zinc rice varieties using Zn-solubilizing bacteria

. In elevated atmospheric CO 2 conditions, a prominent greenhouse gas contributing to climate change, there is a notable reduction in zinc (Zn) concentration within lowland rice crops. Zn deficiency diminishes rice productivity and lowers Zn content in grains, impacting human health, particularly stunting. This study aims to assess the effects of applying isolated Zn-solubilizing bacteria (ZnSB) strains and ZnSO 4 on Zn uptake and yield in two rice varieties: Inpari IR Nutri Zinc (high Zn content) in the first trial and Ciherang (medium Zn content) in the second trial, conducted in an Inceptisol soil. The treatments were organized in a Randomized Block Design with three replications. The first factor involved ZnSO 4 fertilizer (Z0 = without ZnSO 4 ; and Z1 = with ZnSO 4 ). The second factor was ZnSB strains (B0 = without ZnSB; B1 = Klebsiella pneumoniae Zn2; B2 = Enterobacter cloacae CmA12; B3 = E. bugandensis CmD2; B4 = S. marcescens CmT13; and B5 = B. thuringiensis PuT1). The results indicated that applying ZnSB strains without ZnSO 4 significantly increased grain yields for both Ciherang and Inpari IR Nutri Zinc varieties (7.67% and 4.12%, respectively) and enhanced Zn uptake (28.05% and 13.58%, respectively). Moreover, combined ZnSB and ZnSO 4 application also significantly increased yields for both varieties (12.14% and 25.03%, respectively) and Zn uptake (33.29% and 13.99%, respectively), demonstrating the potential of ZnSB for enhancing Zn uptake and rice yield. These findings suggest promising strategies for addressing Zn deficiency in rice cultivation under elevated CO 2 conditions.


Introduction
The rise in average global air temperature or global warming, which causes climate change, has been accompanied by several reports from diverse regions of a substantial drop in food output by 17 to 40%, with food scarcity occurring during severe dry seasons.The concentration of carbon dioxide (CO2) in the atmosphere, a major greenhouse gas responsible for climate change, is quickly growing and is anticipated to exceed 550 ppm by the end of the century [1].It has risen by roughly 30% since pre-industrial times, and CO2 levels might double or treble by the end of the century.
As the CO 2 content in the atmosphere increases daily, it is important to understand the impact of increasing CO2 on plant growth and development and plant nutritional status.The negative impact of increasing atmospheric CO2 levels on the quality of cereal grains is widely known, especially in reducing micronutrient levels in rice grains, such as Zn and Fe [2].It is important to note that rice (Oryza sativa L.) is a staple food for more than half of the world's population and contributes more than 70% of total food energy in several developing countries, including Indonesia [3].Zn deficiency results in stunted child growth or stunting [4].
Zinc deficiency in paddy fields was identified in soil that was given NPK fertilizer intensively, causing the formation of compounds such as Zn-PO4, ZnCO3, and Zn(OH)2, or due to poor drainage resulting in the formation of insoluble ZnS compounds.Zn deficiency also occurs in rice fields with a pH > 7.0 or in waterlogged areas with high Fe, Ca, Mg, Cu, Mn, and P levels.Most of the paddy fields in Indonesia already contain high P nutrients (2.90 million ha) out of 7.54 million ha of paddy fields [5].Under these conditions, Zn is fixed with P, making it unavailable to plants.
Several previous studies have investigated the interaction between using of ZnSO4 fertilizer and plant growth-promoting rhizobacterial (PGPR) and the interaction between using of various rice varieties and Zn-solubilizing bacteria.[6] isolated native bacteria increased grain yield and Zn absorption by 17.0% and 52.5%, respectively.Most of the PGPR strains identified so far belong to the genera Pseudomonas, Ochrobactrum, Bacillus, Azospirillum, Azotobacter, Rhizobium, Stenotrophomonas, Serratia, and Enterobacteria [7].Meanwhile, [8] found that a Zn-solubilizing bacterial strain, namely Bacillus megaterium KY687496, could increase Zn solubility in soil.Therefore, inoculating this bacterial strain into paddy soil is a potential strategy to reduce the need for Zn fertilizer use.
Biofortification is one approach to increase the Zn content in rice grains.Inpari IR Nutri Zinc is rice with the highest Zn content in Indonesia, containing around 29-34 ppm Zn [9].The strategy to increase biofortification in Indonesia through the use of biofertilizers with Zn-solubilizing bacteria active ingredients in rice varieties, such as Ciherang, which has a moderate Zn content (around 24.70 ppm), seems to be an option for farmers to produce grain with higher Zn content.In addition, this approach is considered a new concept in plant biofortification because biofertilizers have various abilities to increase growth and production and Zn uptake.According to [6], adding Zn-solubilizing bacteria to rice plants increased dry weight, productive tillers, and panicle height compared to the treatment without inoculation.Bacterial inoculation significantly increased the Zn concentration in rice grains by 41.6% compared to the control treatment and ZnSO 4 fertilization.
Based on the previous studies, to obtain the right adaptation technology to the impacts of climate change, this study was designed to isolate native Zn-solubilizing bacterial strains (ZnSB) from the rhizospheres of reeds and bamboo.This strain has been tested in vitro to determine the potential for solubilizing Zn in paddy soil.The study results showed that these Zn-solubilizing bacteria were found to produce various organic acids and phytohormones indole acetic acid (IAA) [10].
So far, information on to which extent Zn-solubilizing bacterial (ZnSB) strains isolated from Imperata and Bamboo bushes (which represents the novelty of the initial results of this research) could enhance the Zn uptake and yield of Inpari IR Nutri Zinc and Ciherang is still very limited.Therefore, this study aimed to examine the impact of ZnSB strains and ZnSO4 fertilizer application on the yield and Zn uptake of rice varieties, specifically Inpari IR Nutri Zinc (with high Zn content) and Ciherang (with medium Zn content), especially in Inceptisols.

Bacterial culture
The Zn-solubilizing bacteria used in this study were non-pathogenic bacterial strains previously isolated from the rhizospheres of reeds and bamboo.All strains were selected based on their growth promotion capability and ability to solubilize insoluble Zn in a modified Pikovskaya agar medium (amended with 0.1% ZnO).Based on partial 16S rRNA sequencing, these bacterial strains were identified as Klebsiella pneumoniae Zn2, Enterobacter cloacae CmA12, Enterobacter bugandensis CmD2, Serratia marcescens CmT13, and Bacillus thuringiensis PuT1.All the bacterial strains were checked previously for their plant growth-promoting rhizobacteria properties, such as N2-fixing, Zn, P, Ksolubilization, production of organic acids and phytohormones IAA [10].These activities demonstrated the potential of these bacteria to be used as biofertilizers.

Site description and experimental design
Two experiments were installed, one using the rice variety of Inpari IR Nutri Zinc (high Zn content), the other using the rice variety of Ciherang (medium Zn content) in the next glasshouse at the Indonesia Soil Research Institute (ISRI), Bogor (6 o 34'32"S -106 o 45'15"E) in August 2021.The bulk sample of soil collected from Sukaresmi Village, Megamendung District, Bogor Regency (6 o 42'12"S -106 o 54'27"E), taken from an area of 3 x 3 m 2 with a depth of 20 cm and cleared of branches and roots.The soil is classified as Inceptisols [11], having selected chemical and physical properties that can be seen in Table 1.
Based on analysis using the Kit PUTS, the N, P, and K content were classified as moderate, high, and low, respectively.The recommended dosages of fertilizers are 250 kg/ha of Urea, 100 kg/ha of SP-36, and 100 kg/ha of KCl.The total Zn content of soil used in the experimental pot was 119 ppm, classified as moderate.Whereas the available Zn concentration was only 1.1 ppm, classified as low, indicating that the Zn concentration in the soil solution was insufficient for optimal growth of rice plants.The low available Zn concentration in the soil of this study could be related to the high P availability [12] or the formation of insoluble franklinite (ZnFe2O4) in acidic soils [13].Before the soil was put into the plastic pots, the soil was air-dried and passed through a 5 mm sieve.Each pot contained 5 kg of soil.All pot soils were given basal fertilizers, calculated based on the Paddy Soil Test Kit (PUTS) recommendations.In addition, compost was added at the rate of 5 g/pot or equivalent to 2 t/ha.The pot was watered and flooded for seven days to get the equilibrium.The experiments tested the effects of the isolated ZnSB strains applied with ZnSO4 fertilizer.The treatments were arranged factorially in a Randomized Block Design with three replications.The first factor was fertilizer of ZnSO4 (Z0 = without ZnSO4; and Z1 = with ZnSO4).The second factor was ZnSB strains (B0 = without ZnSB; B1 = Klebsiella pneumoniae Zn2; B2 = Enterobacter cloacae CmA12; B3 = E. bugandensis CmD2; B4 = S. marcescens CmT13; and B5 = B. thuringiensis PuT1).
The fertilizer of ZnSO4 was applied seven days before planting at the rate of 10 kg ZnSO 4 /ha (as recommended).Nitrogen, phosphorus, and potassium were applied as a basal application at 57.5 mg N, 18 mg P2O5, and 30 mg K2O per kg soil.

Preparation of bacterial inocula
Bacterial inocula for plant inoculation were prepared by cultivating Zn-solubilizing bacteria in Nutrient Broth by placing the inoculated broths on a rotary shaker at 100 rpm and 30°C for 24 to 48 h.Then, finally, the concentration of the bacterial suspension was adjusted to 10 7 CFU/ml.

Application of bacterial on rice seeds
The rice seeds were surface sterilized using 2% NaOCl for 15 minutes, followed by rinsing with sterilized water.Subsequently, the seeds were inoculated by soaking them for 24 h in different bacterial inocula maintained at 10 7 CFU/ml.Five seedlings were transplanted per pot and grown until the grain yield.Two top dressings of Urea were applied at 21 and 35 days after rice transplantation.

Harvesting of plants and Zn analysis
All the pot plants were harvested by cutting the shoots approximately 1 cm above the soil surface.After initial air drying, plant samples were placed in paper bags, dried at 60⁰C in an electric oven for 48 h, and weighed to determine the dry matter and grain yield.The analysis of Zn uptake was performed using atomic absorption spectrophotometry in the Soil Chemistry Laboratory, ISRI, Bogor.

Data processing and statistical analysis
The observations included grain weight and grain Zn uptake.An analysis of variance (ANOVA) for a factorially randomized block design was performed.The Duncan's Multiple Range Test (DMRT) at P < 0.05, unless otherwise stated, was used to separate the means when the ANOVA results indicated that there were significant treatment effects [14].

Grain yield
The results in Table 2 showed that the inoculation of Zn-solubilizing bacteria on Ciherang rice seeds significantly increased grain yield compared to control.Likewise, Inpari IR Nutri Zinc rice seed inoculation significantly increased grain yield.Whereas the application of ZnSO4 fertilizer did not significantly affect grain yields of both rice varieties.Meanwhile, the interaction between the inoculation of ZnSB and the application of ZnSO4 fertilizer significantly affected grain yields of Ciherang and Inpari IR Nutri Zinc varieties.For the Ciherang variety (medium Zn content) at without the addition of ZnSO4 but with inoculation by B1 (K.pneumoniae Zn2), B3 (E.bugandensis CmD2), and B4 (S. marcescens CmT13), higher grain yields were observed compared to the control by 22.2%, 16.6%, and 14.3%, respectively.Meanwhile, at the addition of 10 kg ZnSO4/ha, the inoculation of B1 (K.pneumoniae Zn2), B2 (E.cloacae CmA12), and B3 (E.bugandensis CmD2) resulted in higher grain yields compared to the control by 5.8%, 5.7%, and 5.2%, respectively.
For Inpari IR Nutri Zinc variety (high Zn content) without the addition of ZnSO4, the inoculation of B2 (E.cloacae CmA12), B1 (K.pneumoniae Zn2), and B3 (E.bugandensis CmD2) gave higher grain yields compared to the control by 39.6%, 29.4%, and 28.0%, respectively.Meanwhile, with the addition of 10 kg ZnSO 4 /ha, the inoculation of B5 (B.thuringiensis PuT1), B3 (E.bugandensis CmD2), and B1 (K.pneumoniae Zn2) gave the higher grain yields compared to the control by 16.9%, 9.4%, and 8%, respectively.The result of the present study follows the findings of other studies.[6] conducted glasshouse research to investigate the effects of isolated indigenous bacteria and Zn fertilizer on the growth promotion and Zn absorption of Zn responsive (NDR 359) and Zn non-responsive (PD 16) rice cultivars.They found that these three isolates boosted grain yield by 17.0%.
The effects of the inoculation of ZnSB and the interaction between ZnSB and ZnSO4 were significant on the grain yields.However, adding ZnSO4 fertilizer 10 kg/ha did not result in any difference in grain yield when compared to the control.These results indicate that applying Zn-solubilizing bacterial strains on rice field soils may reduce or eliminate the need for ZnSO4 fertilizer, as there was no grain yield response to ZnSO4 fertilizer.In line with this result, [8] reported that B. megaterium KY687496 was a potential candidate for enhanced Zn solubilization in soil, which would allow for lower inorganic Zn application rates after isolating an array of bacteria from soils of virgin forest sites and evaluating them for Zn solubilization.
The insignificant effect of applying ZnSO4 fertilizer on the grain yield could be related to the total Zn content of the soil used in this study, which is classified as moderate.However, the concentration of plant-available Zn is low.Therefore, the removal of the readily available form of Zn by the crops in the soil will be replenished from exchangeable Zn or less soluble Zn pools (fractions).It explains why there was no grain yield response to the ZnSO4 fertilizer.Following this result, a study conducted by [15] in Subang showed that ZnSO4 had no significant impact on the yield of 24 rice genotypes despite its influence on the concentration of Zn in rice grains.Furthermore, [16] revealed that Zn fertilizer did not cause a significant increase in grain yield on soils with sufficient to high Zn availability.

Zn uptake
Inoculation of Zn-solubilizing bacteria on Ciherang rice seeds significantly increased Zn uptake by the rice crops.Similarly, Zn uptake by the Inpari IR Nutri Zinc variety was increased because of the inoculation of the rice seeds compared to the control treatment (no inoculation of ZnSB).Generally, due to the inoculation treatments, the increase in Zn uptake by the Ciherang variety was greater than that by the Inpari IR Nutri Zinc variety.However, the total uptake of Zn by the Inpari IR Nutri Zinc variety was relatively greater than that by the Ciherang variety (Table 3).
Table 3.The effect of the application of ZnSO4 fertilizer and the inoculation of Zn-solubilizing bacteria on Zn uptake by Ciherang and Inpari IR Nutri Zinc varieties in an Inceptisol rice field.

Zn-solubilizing bacteria
Zn uptake (g/pot) Regarding the difference in the magnitude of Zn uptake increase between the Ciherang and Inpari IR Nutri Zinc varieties, [17] noted that rice genotypes exhibit varying responses to zinc application in zinc-deficient soils, emphasizing the role of genetic factors in this response.

Ciherang
Furthermore, the application of ZnSO4 fertilizer significantly affected the Zn uptake of both varieties.The interaction between the inoculation of ZnSB and the application of ZnSO4 fertilizer also significantly affected Zn uptake by Ciherang and Inpari IR Nutri Zinc varieties.For the Ciherang variety (medium Zn content) at the addition of 0 kg ZnSO4/ha, the inoculation of B2 (E.cloacae CmA12), B3 (E.bugandensis CmD2), and B1 (K.pneumoniae Zn2) gave higher Zn uptake in grain over control treatment by 31.4%, 29.2%, and 25%, respectively.Meanwhile, with the addition of 10 kg ZnSO4/ha, the inoculation of B2 (E.cloacae CmA12), B5 (B.thuringiensis PuT1), and B4 (S. marcescens CmT13) also gave higher Zn uptake in grains when compared to the control by 35.8%, 31.6%, and 30.7% respectively.In other words, the effect of interaction between the inoculation of E. cloacae CmA12 and the application of either without or with 10 kg ZnSO4/ha gave the highest Zn uptake in rice plant of Ciherang variety, suggesting that E. cloacae CmA12 strain had a greater potential to solubilize Zn than other strains.
The results of the present study demonstrated that the Zn-solubilizing bacteria strains employed could enhance the Zn uptake by the two rice varieties.This capability is most likely related to the ability of these strains to produce various organic acids, such as acetic, citric, malic, lactic, and oxalic acids [10].Moreover, they could also produce phytohormone IAA, which influences the number and length of lateral roots, leading to improved nutrient absorption in the soil [10].This result follows the findings of [6], who found that the isolated indigenous bacteria were able to enhance total Zn absorption (52.5%) and grain yield (17.0%).Furthermore, [18] reported that the Zn-solubilizing bacteria strains, identified as Bacillus cereus by 16S rRNA gene analysis, enhanced Zn uptake in grains and increased super basmati rice variety yield by 18-47%.

Changes in Zn uptake
The data regarding changes in Zn uptake in rice grains due to different treatments, including Zn-solubilizing bacterial strains (ZnSB), ZnSO4, ZnSB + ZnSO4, and the control, generally followed this order of decrease: ZnSB + ZnSO4 > ZnSO4 > ZnSB > control for both Ciherang and Inpari IR Nutri Zinc varieties (see Figure 1).The increase in Zn uptake in the Ciherang variety was greater than in the Inpari IR Nutri Zinc variety.However, the total Zn uptake by the Inpari IR Nutri Zinc variety was relatively higher than that by the Ciherang variety.In line with this result, [17] reported variations in the response of rice genotypes to Zn application in Zn-deficient soils, highlighting the influence of genetic factors in this response.Comparison of the effect of Zn-solubilizing bacterial strains and ZnSO4, and their interaction on the changes in Zn uptake by Ciherang and Inpari IR Nutri Zinc varieties Figure 1 showed that the Zn uptake in rice grains at the three treatments (ZnSB, ZnSO4, and ZnSB + ZnSO 4 ) was relatively similar.Studies suggested that applying Zn fertilizers to soils is a general strategy to cope with Zn deficiency and enhance grain Zn concentration [19].However, this approach is not always optimal from an economic perspective [20].It is one reason why people are now starting to switch to using more biofertilizers than it was.

Conclusions
The results of the present study have demonstrated in a glasshouse that the Zn-solubilizing bacteria strains employed could enhance the grain yield and Zn uptake in rice grains of Ciherang and Inpari IR Nutri Zinc varieties, indicating their Zn solubilization potential in rice Zn deficient soils.In turn, they may reduce the need for ZnSO4 fertilizer.However, further research under field conditions is required to see the possibility of reducing the cost incurred on Zn fertilizers in rice cultivation and their effectiveness in augmenting the Zn nutrition of rice.The increase in Zn uptake in the Ciherang rice grains was greater than in the Inpari IR Nutri Zinc variety.However, the total uptake of Zn by the Inpari IR Nutri Zinc variety was relatively greater than that by the Ciherang variety.Concerning this result, further research is also required to study the possible compatibility of a strain with a particular rice variety.

Fig 1 .
Fig 1.Comparison of the effect of Zn-solubilizing bacterial strains and ZnSO4, and their interaction on the changes in Zn uptake by Ciherang and Inpari IR Nutri Zinc varieties

Table 1 .
The selected chemical and physical properties of the rice paddy soil of Megamendung District, Bogor Regency.

Table 2 .
The effect of the application of ZnSO4 fertilizer and the inoculation of Zn-solubilizing bacteria on the grain yield of Ciherang and Inpari IR Nutri Zinc varieties in an Inceptisol rice field.