Rhizo-inoculation of phosphate solubilizing bacteria strains to improve rice (Oryza sativa L. var. FARO 44) growth under ferruginous ultisol conditions.

The research investigated the possibility of phosphate solubilizing bacteria (PSB) with plant growth-promoting (PGP) capabilities to improve growth properties of rice plant under ferruginous ultisol (FU) condition through rhizo-inoculation strategy. The PSB with PGP properties used in this research were Bacillus cereus strain GGBSU-1, Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 that were previously isolated and characterized following the 16S rRNA gene sequencing. The rice seeds were sown in a composite FU soil sample and a humus soil (control) and then rhizo-inoculated along the root region of the growing rice seedling at 16 days after sowing. The rice plant was studied for differences in morphological, physiological and biomass parameters for 16 weeks after rhizo-inoculation. Results showed that the FU soil used in the study had high pH, low bioavailable phosphorus, low water holding capacity and high iron levels which has led to a low growth properties of rice seeds sown in FU soil without rhizo-inoculation. After rhizo-inoculation, a signicant increase in plant height and physiological parameters were observed in the rice plant grown in the FU soil as against the control and the rice plant in FU soil without inoculation except for terpenoid which is usually known to signify biotic stress and as part of plant defense mechanism. Generally, rhizo-inoculation of rice seedling with the three PSBs under FU soil condition signicantly improved growth properties of the rice plant. This suggest the ability of the PSBs to solubilize and mineralize soil phosphate and improve its availability for plant use in phosphate stressed soil, thereby improving plant growth properties. sowing. Results showing similar superscripts on same column did not differ from each other at (p>0.05). FA-FD= Ferruginous soil, AVR= Average ferruginous soil, NF= Non-ferruginous soil.


Introduction
Rice (Oryza sativa L.) is an important grain that is consume by over half of the world population. (Maclean et al., 2002). In African, it is the second most consumed food after maize (Ajala and Gana, 2015) and accounts up a large component of the food consumed in most Nigerian families (Ikhajiagbe and Musa, 2020). It is a signi cant starchy grain for human consumption since it supplies 23% of world human per capita calories and 16% of global human per capita protein (Ojo and Adebayo, 2012). Additionally, it increases national income. Rice consumption per capita in Nigeria is predicted to be more than 100 kg per year (Udemezue, 2018). Rice demand is rising, and more production is required to feed more people while reducing costly imports. Rice production in southern Nigeria has been severely hampered since the 1970s. High soil pH, low nitrogen, phosphorus de ciency, leading to a ferruginous conditions are some of the variables that have been related to this (Doyou et al., 2017). Unfortunately, the majority of soils in southern Nigeria, particularly in Edo State, are ferruginous (Daramola, 2005;Obayelu, 2015). According to Musa and Ikhajiagbe (2021), iron toxicity leading to phosphorus de ciency and acidic pH has been linked to reasons why ferruginous soils are not supporting rice growth. To sustainably improve rice productivity in these P-de cient soils, there is need to consider the use of biological agents such as microorganisms as inoculant, as against the widely used chemical fertilizers (Adnan et al., 2018;Joshis et al., 2007).
Ferruginous soil also known as red soil are iron-rich soil. This kind of soil is usually observed in warm and humid climates (Yu et al., 2016), or in some tropical regions as seen in some parts of Africa (Zhao, 2014).
Ferruginous soils have been documented to account for about the average percentage (45.2%) of the Earth's landscape. In Nigeria for instance, it is transcendent in some southern States, for example, Edo state, possessing several regions, including northern region and Benin central (Doyou et al., 2017). Ferruginous soils are known for its unique properties such as high iron levels; which creates complexes with soil phosphate and making it low available for plant use (Gyaneshwar et al., 2002). Consequently, this condition now brings about low biotic and abiotic properties needed for plant growth (Wang et al., 2014). Hence to enhance soil fertility, some local farmers have tried to use synthetic chemicals which had negative in uence on the soil and plants as a results of insu cient bene cial microorganisms to fuel the nutrient cycling and release important metabolites that can improve the soil properties and plant growth (Sharma et al., 2013). Musa and Ikhajiagbe 2021 following 16S rRNA gene sequencing successfully isolated and identi ed Bacillus cereus strain GGBSU-1, Klebsiella variicola strain AUH-KAM-9 and Proteus mirabilis strain TL14-1 as e cient in solubilizing insoluble phosphate in soils (Musa and Ikhajiagbe, 2020b; Plate 1, 2 and Supplementary table 1). These isolates have signi cantly improved germination and yield parameters in rice in in vitro setup. In a bid to investigate the e cacy of these isolates to in uence the iron-phosphate ux in soil and as biofertilizer, there is need to consider a eld experiment. According to Saneya and Muhammad (2017), con rmation of e cacy in vivo is very important because in vitro studies may not necessarily give the real situation when introduced to the ecosystem or may not consider the real environmental conditions. This research considered rhizo-inoculation of rice seedlings with PSB strains in ferruginous ultisol conditions. Rhizo-inoculation involves introduction of microorganism into the root region of plants to stimulate growth . Rhizo-inoculation of PGPs are known to enhance plants' survival by releasing important plant nutrients as well as plant growth hormones (Glick, 1995). Gupta et al. (2000) and Biswas et al. (2000) have reported signi cant enhancement in growth and yield of important crops in response to rhizo-inoculation with PGPR. Previous research by Musa and Ikhajiagbe (2021) con rmed the use of PSB with PGP to improve the growth parameters of rice seeds. Increase in physiological parameters of sweet pepper (Capsicum annum) as response to rhizo-inoculation with PGP have been severally reported by Backer et al. (2018;. Therefore, the present study aim to investigate the possibilities of these PSBs (Bacillus cereus strain GGBSU-1, Klebsiella variicola strain AUH-KAM-9 and Proteus mirabilis strain TL14-1.) that were previously obtained from FU soils and a control soil to improve the growth parameters of rice plant under FU conditions. The current study will bring about another sustainable strategy of improve agricultural productivity and food security.

Preparation of soil used in the experiment
The experiment was carried out at the experimental garden of the Department of Biology and Forensic Science, Admiralty University of Nigeria, Delta State Nigeria. Ferruginous soils that was previously obtained by Musa and Ikhajiagbe (2020a) from six locations around Benin City, Edo State of Nigeria were pooled to obtain a composite sample, whereas non-ferruginous soil (control) was obtained from rich-humus region at the deep underground root of a banana tree at the Botanical garden, University of Benin as reported by Musa and Ikhajiagbe (2020a). The ferruginous soil and the control soil were prepared in experimental bowls (30 x 25 cm) and made in ve replicates.

Soil physiochemical parameter
The ferruginous soil sample and the control soil were air-dried at temperature of 22-25 o C and then analyzed for soil organic matter levels (SOM), soil available phosphorus, cation exchange capacity (CEC), pH of the soil, total nitrogen, organic carbon (OC), exchangeable acidity (EA), available potassium, available micronutrients such as sodium (Na) and Aluminum (Al), electrical conductivity, soil texture class and maximum water holding capacity following Musa and Ikhajiagbe (2020a). The iron levels of the soil was analyzed following the method of Cheng et al. (2013) by using concentrated perchloric acid to digest the soil sample and subjecting it to titration with versanate solution.

Sowing of rice seeds
An improved rice variety (FARO 44) previously obtained from the Center for Dryland Agriculture, Bayero University, Kano was used in this study. The rice seeds were analyzed for viability according to (AOSA, 2000) and sown at the rate of 10 seeds per pot. The experimental design was at an open environment and as such relied entirely on rainfall. The experimental region experiences a moderate rainfall and humidity (< 120-200 cm) during this study. However, the soil moisture content was maintained periodically as described by USDA (2010) method. This set up was weeded at every two days maintained for 16 days to allow seedling formation.

Bacterial species
Three phosphate solubilizing bacteria species (Bacillus cereus strain GGBSU-1, Klebsiella variicola strain AUH-KAM-9 and Proteus mirabilis strain TL14-1) that were isolated from FU soil and humus soil in an earlier study in Benin City by Musa and Ikhajiagbe (2021) were prepared in stock cultures for this study. The bacteria species were previously identi ed using molecular tool of 16S rRNA after biochemical test involving catalase, indole, citrate, nitrogen xing activity and bromotyhmol blue test and pH tolerance level test with HCL following (Mondala et al., 2016). PGP capabilities of the isolates were determined by IAA and siderophores production

Preparation of inoculum
The pure PSB having PGP traits (Bacillus cereus strain GGBSU-1, Klebsiella variicola strain AUH-KAM-9 and Proteus mirabilis strain TL14-1) were prepared by streaking on to agar plates and incubated at 28 o C for 48 hours. After 48 hours growth, the isolates were inoculated in Nutrient broth and then prepared into 0.5 McFarland Standard with Cat. No (TM50) to standardize the approximate number of bacteria in the suspension. Following this process 500 mL of each bacteria isolate was prepared to obtain an average microbial suspension of 1.5x10 8 cfu/mL.

Rhizo-inoculation of rice seedlings
After 16 days of seedling growth, the prepared McFarland standard (McFarland and Nephelometer, 1944) of 500 mL bacteria inoculum were made in to 10 mL of each bacteria (Bacillus cereus strain GGBSU-1, Klebsiella variicola strain AUH-KAM-9 and Proteus mirabilis strain TL14-1). To obtain the control, 10 mL of distilled water was prepared. All setup were made in ve replicates. On to each seedling, the calculated inoculum volume was introduced into the root region of the growing seedling using 10 mL syringe following Etesami et al. (2014). The three bacteria numbered as (A= Bacillus cereus strain GGBSU-1, B= Proteus mirabilis strain TL14-1 and C= Klebsiella variicola strain AUH-KAM-9). The setup was further observed for 16 weeks using randomized blocked design and wetted with 5 mL distilled water every 3 days. The experimental pots were weeded at every 2dyas. Plant growth parameters were measured and recorded.

Morphological parameters
Morphological parameters that are related to growth and yield of rice were investigated. Fresh shoot length, fresh root length, panicle length and length of the rst leaf were calculated in (cm) by using a transparent ruler that was mounted on a white calibrated paper throughout the study. To obtain the dry shoot length and dry root length (cm), seedlings were air dried for 24 hours and the root were measured from day 3 to 16 weeks after rhizo-inoculation. The length of internodes (cm) was measured from the coleoptile to the rst node using a sample of 5 best seedlings from all treatments weekly, while the number of secondary roots were carefully observed and counted daily.

Physiological parameters
Total soluble sugar of fresh leaves were estimated a day before rhizo-inoculation (16th day) and at interval of 2 weeks after rhizo-inoculation till 16th week by drying the tallest leaf in oven at 70 o C for 24h as described by Nelson (1944) with some modi cation by Sankar and Selvaraju (2015). Growth enzymes such as alpha amylase (AA) of the seedling and growing plant extract was determined at day 16 after sowing (before rhizoinoculation) and at 2 weeks interval (after rhizo-inoculation) till 16th week (harvest day) by DNS method of  at a pH of 7.5. Terpenoid and lycopene of fresh leaves were determined at similar days as AA following the method of Moran (1982). Chlorophyll content index (CCI) of old and fresh leaves were determined using a non-destructive method by Apogee chlorophyll concentration meter. The CCI were measured as average of the mesocotyl, mid seedling and top seedling at similar days as AA. Chlorophyll a and b levels were determined following Arnon et al (1949); Maxwell and Johnson (2000).

Biomass parameters
Leaf area (cm 2 ) was determined using an android application (Leaf-IT) following Julian et al. (2017) at 16th day after sowing (before rhizo-inoculation) and at from 2weeks after rhizo-inoculation to the 16th week with 3 weeks intervals. Number of leaves were measured by counting at 16th day after sowing (before rhizoinoculation) and at from 2weeks after rhizo-inoculation to the 16th week with 2 weeks intervals. Leaf tip necrosis was calculated as the percentage of the total number of leaves produced by plants that showed signi cant signs of necrosis following Ikhajiagbe et al. (2017) at similar days as the number of leaves. Weight of fresh leaf (g) was calculated using analytical weighing balance at similar days as the number of leaves.

General growth characteristics at harvest
Time at which the rice plant matured was measured as the period when more than half the total number of seed began to turn dry following Ikhajiagbe et al., 2021 with slight modi cation. Average number of panicle per pot, number of tillers, number of reproductive tillers, and number of seeds per panicle were measured by counting. Average panicle weight, weight of husked rice, weight of de-husked rice, weight of peduncle without rice were measured using analytical weighing balance. 100 grains were counted from ve plants of each replicate and weighed (g). Plant tissue water content was calculated at the harvest day as:

Statistical analysis
Data obtained from the analysis were presented as means and standard errors of ve replicates. Data were analyzed following two-way analysis of variance on GENSTAT (8th edition). Signi cant p-values were obtained, differences between means were separated using Student Newman Keuls Test (Alika, 2006). The ferruginous soil used in the current experiment was homogenized.

Morphological performance of rice after rhizo-inoculation
After rhizo-inoculation of the rice seedling with the PSBs at day 16th after sowing, the setup was allowed to grow for two weeks. At the two weeks, signi cant increases were observed in fresh shoot length, dry shoot length, fresh root length, dry root length, length of internodes, number of secondary roots and stem girth in the rhizo-inoculated setup. The seedling inoculated with Bacillus cereus strain GGBSU-1 was observed to show highest morphological yield (80%) compared to the non-ferruginous soil (65%) ( Fig. 2 and Plate 3). The ferruginous soil with no bacteria inoculation was observed to show lowest yield. More increase in morphological properties were observed in the inoculated seedling with increasing days after rhizo-inoculation (Supplementary table 2) however, the ferruginous soil without inoculation was observed to show no morphological improvement since week 6 after rhizo-inoculation.

In uence of inoculated PSBs on rice physiological parameters
3.3.1 Physiological performance of rice seedling before rhizoinoculation Physiological parameters relating to rice seedling yield at 16th DAS (before rhizo-inoculation) was investigated. Signi cant differences (Fig. 3) were witnessed in total soluble sugar (TSS), alpha amylase (AA) and chlorophyll content index (CCI) between the ferruginous soils and the non-ferruginous soil. The nonferruginous soil showed greater physiology yield than the ferruginous soils. Terpanoids activity in rice seedling from the non-ferruginous soil was observed to be signi cantly lower (p> 0.5; 24%) than from the ferruginous soil before rhizo-inoculation, while lycopene content was observed to be signi cantly higher (p> 0.5; 20%) in the non-ferruginous soil compared to the ferruginous soil.
Chlorophyll-a and Chlorophyll-b content of rice seedlings at day 8 and 16 after sowing (Table 3) also showed signi cant difference with the non-ferruginous soil, showing 50% greater than the chlorophyll levels in the ferruginous soils.

Physiological performance of rice after rhizo-inoculation
Results of the rice yield physiology at 2 weeks after rhizo-inoculation are presented in Fig. 4. Plants inoculated with the three bacterial species were observed to show signi cant increase (Fig. 4a) in TSS, AA, old and new leaf CII as compared with the seedling without rhizo-inoculation. The seedling rhizo-inoculated with Bacillus cereus strain GGBSU-1 was observed to show improved physiological yield properties, even though a not signi cant difference was witnessed in the both leaf CCI with the control.
Furthermore, Fig. 4b showed that terpenoid was higher in the growing rice seedling under ferruginous soil without inoculation, while the rice seedling from the non-ferruginous soil and those rhizo-inoculated with Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 showed no signi cant difference. Lycopene; a protein that protects plants from excessive light damage was observed to show signi cant increase in all rhizo-inoculated seedlings as compared to the non-inoculated seedlings (Fig. 4b). Also, the lycopene level at 2 weeks after rhizo-inoculation was observed to show no signi cant difference between the Bacillus cereus strain GGBSU-1 inoculated seedling and the control. However, the rice seedlings from noninoculated ferruginous soil was observed to show lowest lycopene levels.
Supplementary table 3 showed the physiological performance of rice plant from 5 weeks after rhizoinoculation to 16 weeks after rhizo-inoculation. The TSS and AA of rice plants inoculated with bacterial isolates were observed to show signi cant increases as compared to the non-inoculated plant. These metabolites were observed to keep increasing with increasing weeks till harvest day (16WAI). The CCI of the old and new leaf were also witnessed to follow similar trend. However, the old and new leaf CCI of the ferruginous soil without inoculation was observed to remain the same with no signi cant increase. There was no signi cant difference between the old and new leaf CCI between the Bacillus cereus strain GGBSU-1 inoculated rice plant (FA) and the control (NF). However, a signi cant difference was witnessed across the three species of bacteria inocula.
The chlorophyll content of rice leaves from 5WAI to 16 WAI as observed in the (Supplementary table 3) showed signi cant changes in the chlorophyll pattern. In chlorophyll a, the plant from the ferruginous soil without inoculation was observed to be lowest as compared to the one from inoculated ferruginous soil and the control. A similar observation was seen in chlorophyll b. The rice leaves from the seedling rhizo-inoculated with Bacillus cereus strain GGBSU-1 (FA) in a ferruginous was seen to show highest levels of chlorophyll contents.
Results of terpenoids levels in the growing rice plants were analyzed and presented in (Supplementary table 3

In uence of inoculated PSBs on rice biomass parameters
3.4.1 Biomass performance of rice seedling before rhizoinoculation Table 4 showed the performance of rice seedling at 16th DAS (before rhizo-inoculation). Rice seedlings from the non-ferruginous soil were observed to have higher weight of fresh leaf (0.09), leaf length area (2.0) and no leaf tip necrosis was observed as against the seedlings from ferruginous soils, where all the seedlings showed signs of necrosis.

Biomass performance of rice after rhizo-inoculation
At 2 weeks after rhizo-inoculation, the biomass performance of rice seedling (  The time at which the rice matured was observed to be the 13th week after sowing for plants inoculated with bacterial seedlings in ferruginous soil. However, the non-ferruginous soil was observed to mature at 14th week. For the ferruginous soil without bacterial inoculation (FD) growth and yield seized at the 3rd week after sowing (Fig. 6). Figure 7 showed the number of tillers and reproductive tillers observed at harvest day. A signi cant difference was observed among all the rice plat. The FA was observed to show highest NT and NRT while the FC was observed to show the least. In the FA, only 3 of 23 tillers did not produce panicle and seed while, while in the FC, 6 of 15 tillers did not produce panicle and seed. Similarly, the FA was observed to show improved number of panicle, straw yield and seed per panicle (Figure 7 and Fig. 8). However, there was no signi cant difference in the straw yield and number of seed per panicle between the FA and the non-ferruginous soil (NF).
The 100-grain weight at harvest was observed to be signi cantly lower in the FC than other inoculation treatments. About 15% increase in the 100-grain weight was observed in the FA which showed no signi cant difference with the NF (p>00.05). A similar results was obtained in the plant tissue water content ( Table 5). The FA was observed to have highest yield in terms of weight of rice panicle, weight pf peduncle without rice, weight of husked and de-husked rice seed (Table 5 and Fig. 9). Root development and architecture at harvest showed a more developed nature at the FA compared to FB, FC and NF, even though the NF showed more root hair (Fig. 10)

Discussion
The in uence of bacterial inoculum on growth parameters of rice in FU soil comparative to the control (nonferruginous soil) have been analyzed. Ferrugenicity is de ned most especially by the high iron, acidity and low phosphorus levels in soils (Musa and Ikhajiagbe, 2020a) as observed in Table 1.
Before rhizo-inoculation, the non-ferruginous soil (NF) was observed to show higher morphological parameters than the ferruginous soil. This clearly showed the anti-growth properties of the ferruginous soil (Wang et al., 2014). At 2 weeks after inoculation, the morphological properties were improved especially in the Bacillus cereus strain GGBSU-1 inoculated seedling, even more than the control. This has avail the ability of bacterial to improve plant morphology. This set up was continuously monitored for 16 weeks and similar trend was observed. This is likely as a result of the ability of these bacteria to interact with the root architecture and improving water use e ciency and physico-chemical parameters of the rhizospheric soil, thereby improving rice plant morphology. This is consistent with the work of Gupta et al. (2012b) who observed that treatment of plants with certain PSB bacteria consequently improved plant morphological parameters.
Plant productivity can be determined using total sugars. Before inoculation, a low plant physiological parameters was observed in the ferruginous soil as against the non-ferruginous soil. This shows the low productivity of ferruginous soil. Terpenoid is usually known to signify biotic stress and as part of plant defense mechanism , the high level of terpenoid observed in the seedlings from ferruginous soil showed some possible biotic stressors, this stress may be linked to the poor physico-chemical conditions of the soil (Table 1)  The FA showed fastest time at which the rice matured. This signifying the effectiveness of the Bacillus cereus strain GGBSU-1 strain as against the other isolates. This effect is also observable when higher tillers and reproductive tillers were produced in the FA compared to others. All harvest parameters showed signi cant increase in the FA as against other isolates. Root systems in uences plant tness, health and productivity (Adnan et al., 2018). A more developed root architecture that was observed in the FA showed the positive in uence of the PSB, leading to more establishment of new micro-environments and ecological niches for different microbial species, in order to bring about bene cial plant-bacteria interaction at the rhizospheric regions.

Conclusions
The current study has been established that rhizo-inoculation of rice seedling with phosphate solubilizing bacteria having plant growth-promoting capabilities have the ability to improve growth parameters of rice plant grown in ferruginous soil. Results of this study showed the ferruginous soil to be having low nutrients with high iron as against the non-ferruginous soil. Before rhizo-inoculation, rice growth parameters were observed to be low in the ferruginous soil setup, which increase signi cantly after rhizo-inoculation with the three PSB strains. The three PSB bacteria showed different levels of growth and yield properties on the rice plant.
Generally, the setup inoculated with Bacillus cereus strain GGBSU-1 (FA) was observed to show the highest growth in uence on the rice seeds, followed by Proteus mirabilis strain TL14-1 (FB) while Klebsiella variicola strain AUH-KAM-9 (FC) was observed to show the least rice growth parameters. Since these bacteria proved effective in improving growth and yield parameters of rice plant in ferruginous ultisol, further studies should be conducted to trail the effectiveness of these bacteria on other important crops in nutrients de cient soils. This would provide a sustainable way of enhancing crop production to serve increasing world population.  Morphological parameters of rice seedling at 2 weeks after rhizo-inoculation (30 days after sowing). Here, FA= rice seedling in ferruginous soil, rhizo-inoculated with Bacillus cereus strain GGBSU-1; FB = rice seedling in ferruginous soil, rhizo-inoculated with Proteus mirabilis strain TL14-1; FC = rice seedling in ferruginous soil, rhizo-inoculated with Klebsiella variicola strain AUH-KAM-9; FD = rice seedling in ferruginous soil without rhizoinoculation; Control = rice seedling in non-ferruginous soil without rhizo-inoculation.      Here, FA= rice plant in ferruginous soil, rhizo-inoculated with Bacillus cereus strain GGBSU-1; FB = rice plant in ferruginous soil, rhizo-inoculated with Proteus mirabilis strain TL14-1; FC = rice plant in ferruginous soil, rhizo-inoculated with Klebsiella variicola strain AUH-KAM-9; FD = rice seedling in ferruginous soil without rhizo-inoculation; NF = (control) rice seedling in non-ferruginous soil without rhizo-inoculation.

Supplementary Files
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