Organic soil amendments using vermicomposts under inoculation of N₂-fixing bacteria for sustainable rice production

Introduction

The use of organic and biologic fertilizers in rice production is one of the most important methods used for soil fertility in a sustainable system (Wang et al., 2015; Amanullah, 2016; Chen et al., 2018). Among various organic fertilizers, vermicompost superiority has been proved in maintaining the long-term soil fertility (Lim et al., 2012; El-Haddad et al., 2014; Guridi et al., 2017; Sharma & Garg, 2018). Actually, soil amendment with vermicompost is an agronomically interesting practice as well as an attractive waste management strategy (Ludibeth, Marina & Vicenta, 2012; Baghbani-Arani & Modarres-Sanavy, 2017). Production of vermicompost as an easy and nature-friendly technology is a semi-aerobic process carried out by a specific group of earthworms (Eisenia fetida) and some soil microorganisms, especially bacteria and actinomists used for production of organic fertilizers from the waste and stabilization of these materials (Mahanta et al., 2012; Lim, Lee & Wu, 2016). As a result, stabilization reduces the environmental problems associated with manure (such as salinity) by transforming it into safer and more stabilized material for soil amendment (Mahanta et al., 2012). In addition to less time for bioconversion, the presence of higher content of minerals, hormones, enzymes, humic acids and bioavailable form of nutrients has been reported for vermicompost (Campitelli, Velasco & Ceppi, 2012; El-Haddad et al., 2014; Khan, 2018). Vermicompost contains nutrients in such forms that are readily available to the crops, such as nitrates, exchangeable P, soluble K, iron (Fe), calcium (Ca), magnesium (Mg), etc. (Tejada & González, 2009; Ludibeth, Marina & Vicenta, 2012). Guridi et al. (2017) reported that, vermicomposts contain biologically active substances such as plant growth regulators, and have great potential in maintaining the soil fertility. If vermicomposts are integrated in nutrient management in agricultural fields, the costs of crop production may be reduced significantly (Lim, Lee & Wu, 2016; Sharma & Garg, 2018). On the other hand, some composts and vermicomposts (especially those made from chaff of wheat or rice straw straw)with high value of carbon –to- nitrogen (C/N) may increase the time of bioconversion of the crop or stimulate N fertilization in the soil, and face the plant with N deficiency in some growth stages (Zhu et al., 2013; Li et al., 2015; Eo & Park, 2019).

The use of Azolla pinnata (a free-floating weed) is a solution for increasing the N composts and vermicomposts. In terms of nutrients, the amount of Azolla nutrient has been shown to vary in different periods of time and has an average of 5.3% N, 8.3% K and 0.6% Mg, and is free from lead, mercury or arsenic (Sreenivasa, 2012). It has been reported that, vermicomposting of the straw is mixed with Azolla along with cattle dung to obtain a better quality crop suitable for agricultural applications (Arora & Kaur, 2019).

The use of bacteria with the capability for Biological N Fixation (BNF), such as Azospirillum, Herbaspirillum, and Azotobacter , as an effective strategy is another solution for increasing N amount in paddy soil, under nutrient management for production of safer and cheaper rice (Ladha & Reddy, 2003; Sanati et al., 2011; Zhang et al., 2017). In this regard, Bgattacharjee, Singh & Mukhopadhyay (2008) showed an increase in host-plant N content, up to 30–45 mg of N plant⁻¹ (6-week-old seedlings) resulted from N₂-fixing. This group of bacteria, in addition to the N₂-fixing capability in a cooperative way, contributes in solving the nutrients such as P, K ,and iron (Fe), and also has the capability for producing Phytohomones, Vitamins and Siderophores (Zhang et al., 2018). Due to their positive effects on plant growth stimulation, these microorganisms are called as Plant Growth Promoting Rhizobacteria (PGPR) (Wu et al., 2005). Mahanta et al. (2012) revealed the superior performance of PGPR in increasing rice growth and grain yield and improving soil health in addition to saving 40–80 kg N ha⁻¹. In this regard, there is great interest in exploring the diversity of PGPR as substitutes for some chemical agricultural inputs (Rodrigues, Ladeira & Arrobas, 2018). Generally, the N-transformation processes in the rice rhizosphere include N mineralization, denitrification, N fixation, and ammonia volatilization. Microbial-mediated mineralization and BNF are very important to the level of available N content in the soil and N uptake by rice (Pattnaik et al., 1999; Nayak, Babu & Adhya, 2007; Kumar, Swain & Bhadoria, 2018; Zhang et al., 2018), so an increase in the abundance of microbial populations could result in a faster N mineralization rate and increased N availability for plants (Li et al., 2015; Chen et al., 2017; Khan, 2018).

In Iran, rice is the second source for supplying food after wheat, with an annual consumption of 39.4 kg per person (Ashoori et al., 2018). Since Iran is located in a warm and dry region, its soils have low organic matter, and it is noteworthy that, the organic matter of the soil as the most important factor in crop yield ranks after the water . On the other hand, 20 million tons of manure is produced by animals per annum in Iran. The rate of compost and vermicompost production is negligible compared to manure. Efforts have been taken in recent years to produce more vermicompost, but its application in farms, especially in rice farms has not been increased remarkably (Rezaei, 2013; Taheri Rahimabadi, Ansari & Razavi Nematollahi, 2018). In the present study, cow manure vermicomposting process was done in different mixtures with Azolla and rice straw under inoculation of N₂-fixing bacteria aimed at improving the agronomic value of the vermicomposts on promoting biological activity of paddy soil, nutrient uptake and grain yield of rice.

Materials and Methods

Experimental site and plant growth conditions

The field experiment was conducted on clay-loam soil at the Rice Research Institute Farm of Rasht, Guilan province, Iran during 2017–2018. The area is located at 37°22N latitude and 49°63E longitude and 15 m above the sea level. To simplify the comparison of the growing season weather, the monthly total precipitation and temperature were considered from May to September at the Rasht Agricultural Research Farm (Figs. 1A and 1B). To determine soil characteristics, soil sampling was performed before the experiment. To do this, field soil sampling was done from the depth of 0–30 cm in 8 spots. Then, the collected samples were sent to the laboratory to determine soil texture and chemical composition. Properties of experimental soil samples are given in Table 1.

Table 1:

Results of physio-chemical analysis of the experimental soil.

Year	Sand	Silt	Clay	EC	pH	Organic Carbon	N	P	K	Zn	Fe	Mn
		(%)		(dS/m)		(%)	(%)		(mg/kg)
2017	29.8	26.3	43.9	1.05	7.6	0.75	0.71	15.8	164	0.89	4.41	6.21
2018	28.5	32.2	39.3	1.24	7.1	0.86	0.66	12.4	194	1.23	5.82	7.51

DOI: 10.7717/peerj.10833/table-1

Results

Urease enzyme activity

Urease enzyme activity of soil increased up to stage T3 in most organic amendments in both years of the experiment, but then (in stage T4) its activity decreased, and under non-inoculation conditions (Figs. 2A and 2B), the urease activity trend lines of the control treatment collided with the urease activity trend lines of organic amendments while, such a collision was not seen under inoculation of Azotobacter (Figs. 2C and 2D) and Azospirillum (Figs. 2E and 2F). Therefore, the effectiveness of organic amendments on enzyme activity in rhizosphere of inoculated plants increased compared to non-inoculated plants. In both years, VAM showed the most urease activity and Azotobacter seemed to show more enzyme activity compared to Azospirillum in response to the application of organic amendments.

Soil bacterial account and microbial biomass–C

The interaction effect of bacteria × organic amendments × year on microbial carbon biomass was significant (P < 0.01) (Table 4). The mean comparison showed that, in the first year (2017), compared to control, organic amendments increased microbial carbon biomass (24–45%) in non-inoculated plants, plants inoculated with Azotobacter (42–78%) and Azospirillum (31–64%). While in the second year (2018), this increase was obtained as 29–41%, 68–82%, and 63–71%, respectively in non-inoculated plants, plants inoculated with Azotobacter and Azospirillum compared to control. In both years, the highest microbial biomass-C was obtained from VAM under Azotobacter inoculation, although it did not statistically different with some treatments (Fig. 3A and 3B).

The number of bacteria was influenced by interaction effect of bacteria × organic amendments (P < 0.01) (Table 4). The mean comparison showed that, organic amendments increased the number of bacteria in non-inoculated plants by 22–32%, and it increased the number of bacteria by 22–32% both in plants inoculated with Azotobacter and Azospirillum. Although, there was no significant difference between VAM and some organic treatments in non-inoculated plants, but in the plants inoculated with Azotobacter and Azospirillum, the highest number of bacteria was obtained from the VAM (Fig. 4).

Table 4:

ANOVA of the effect of organic amendment on soil bacteria count (SBC), microbial biomass-C (MBC), chlorophyll a (CA), chlorophyll b (CB), total chlorophyll (TC), No. of tiller hill⁻¹ (NTH), seed yield (SY), biological yield (BY), protein yield (PY), P uptake (PU), and K uptake (KU) under inoculation with PGPRs.

S.O.V	Mean square (MS)
	SBC	MBC	CA	CB	TC	NTH	SY	BY	PY	PU	KU
Y	14934ns	789*	3.4*	1.07**	0.65ns	0.31ns	1595781**	3771791**	5698**	35.9ns	1108**
Y(R)	12449ns	160ns	20.8**	0.24**	16.8**	0.86ns	3232ns	474140ns	790ns	50.6ns	93.9ns
B	81900**	73401**	63.8**	8.14**	116**	6.41*	4341781**	19679459**	236472**	660**	818**
M	30723**	26830**	12.0**	2.88**	25.3**	136**	5293349**	15884979**	37202**	790**	436.9*
Y ×B	1892ns	1124**	0.09ns	0.05ns	0.01ns	1.71ns	77762**	3324497**	25543ns	287*	484*
Y × M	5232ns	232ns	0.01ns	0.01ns	0.004ns	3.23ns	348570**	926332**	20414*	114ns	265ns
B × M	23592**	1111**	3.13**	1.36**	7.74**	18.5**	127978**	645059**	57888**	364**	795**
Y × B × M	8293ns	614**	0.01ns	0.008ns	0.40ns	5.54*	20420*	910690**	40842**	75.1ns	60.2ns
Error	4975	134	0.549	0.0376	0.552	1.48	7893	225514	9956	61.8	134

DOI: 10.7717/peerj.10833/table-4

Note.

ns

not significant

*significant at the 0.05 and 0.01 probability levels, respectively. S.O.V: source of variations, Y: year, R: replication, B: bacteria, M: organic amendments. The Y, B, and M are experimental factors whose main (individual) and interaction effects on the measured traits are included in this table.

**significant at the 0.05 and 0.01 probability levels, respectively. S.O.V: source of variations, Y: year, R: replication, B: bacteria, M: organic amendments. The Y, B, and M are experimental factors whose main (individual) and interaction effects on the measured traits are included in this table.

Chlorophyll content

Chlorophyll content of flag leaf was influenced by interaction effect of organic amendments × bacteria (Table 4). The plants treated by N₂-fixing bacteria increased the effect of organic amendments on chlorophyll content, so that the content range of chlorophyll a was obtained as 4.5–1.9, and 7.3–4.3 mg/L, respectively (Fig. 5) in non-inoculated plants and inoculated plants, and the content range of chlorophyll b was obtained as 1.0–0.4, and 0.7–2.8 mg/L, respectively (Fig. 6) in non-inoculated and inoculated plants. Under non-inoculation conditions, although organic amendments of total chlorophylls (chlorophyll a + b) increased by 16–52% compared to control, but this increase was obtained as 23–79% and 11–67%, respectively (except VM) under Azotobacter and Azospirillum inoculation conditions. N₂-fixing bacteria played a positive role in improving flag leaf chlorophyll content especially in vermicomposts containing Azolla. The highest content of total chlorophyll was obtained from VRAM, VRM, and VAM under Azotobacter inoculation, and from VRAM under Azospirillum inoculation (Fig. 7).

Protein yield

The interaction effect of organic amendments × bacteria × year on protein yield was significant (P < 0.01) (Table 4). In both years, organic amendments had significant superiority compared to the control. In both years, under non-inoculation and inoculation with Azotobacter the highest protein yield was obtained from plants treated with VAM, but under inoculation conditions with Azospirillum, there was no significant difference between VAM and VRAM. Among all treatments, in both years, the highest protein yield was obtained from Azotobacrer + VAM (438 kg/hm² in the first year and 404 kg/hm² in the second year). It is worth noting that, although grain yield of treatments was less in the first year compared to the second year, but the protein yield was higher in the first year (Table 5).

P and K uptake

K and P uptake by grain were influenced by interaction effect of organic amendments × bacteria (Table 4). The mean comparison of interaction effect of organic amendments × bacteria on P uptake showed highest P uptake was obtained under non-inoculation conditions by VAM and VARM (with a 76 and 79% increase compared to the control), under inoculation conditions with Azotobacter by VAM and VM (with a 44 and 35% increase compared to the control + Azotobacter), and also the under inoculation conditions with Azospirillum by VAM (with a 63% increase compared to the control + Azospirillum) (Fig. 8).

The mean comparison of the interaction effect of organic amendments × bacteria on K uptake showed highest K uptake under non-inoculation conditions, and under inoculation with Azospirillum by VAM (with a 51% increase compared to control), and VRAM and VAM (with a 80 and 74% increase compared to control + Azospirillum). But in plants inoculated with Azotobacter, there was no significant difference between vermicompost treatments including VM, VM, VAM, and VARM, but significantly increased K uptake compared to the manure treatment (M) and control (Fig. 9). Also, control + Azotobacter increased K and P uptake by 25 and 32%, respectively, and control + Azosperilum increased K and P uptake by 8.8 and 19%, respectively compared to control + non-inoculation.

Discussion

The results showed that, vermicomposting of manure with and without Azolla and rice straw and their application as organic fertilizers under inoculation with N₂-fixing bacteria, especially Azotobacter, and increased the number and biomass-C of soil microorganisms and urease enzyme activity. The number and biomass-C of soil microorganisms and urease enzyme activity were lowest in control plots due to high stress and inadequate nutrient supply, lack of using organic fertilizer and lower amounts of rhizodeposition (root exudates and root biomass). These results are consistent with the findings of the studies by Cooper & Warman (1997); Bhattacharyya, Chakrabarti & Chakraborty (2005); Kopecky et al. (2011); and Tu et al. (2017). An increase in the number and biomass-C of microorganisms in planted rice under flooded condition has been reported following the application of vermicompost (Nayak, Babu & Adhya, 2007). Albiach et al. (2000) and Cui et al. (2018) reported that, annual application of adequate amounts of some organic residues led to significant increase in soil enzyme activities.

The inoculation of plants with Azotobacter and Azospirillum increased the application efficiency of organic fertilizers in improving the number and biomass-C of soil microorganisms and urease enzyme activity. The positive role of N₂-fixing bacteria in increasing the population of Diazotrophic bacteria, improving enzyme activity and microbial biomass N in rice rhizosphere has been confirmed in the studies by Kumar, Swain & Bhadoria (2018) and Zhang et al. (2018). Mahanta et al. (2012) reported that, vermicomposting of manure increased the soil microbial activity by 79% and biomass-C by 68% compared to manure, resulting from improvements in soil physical and chemical activity by vermicompost.

In both years of experiment and in all treatments, the urease activity increased to stage T3 ($\frac{1}{4}$ panicle initiation stage), due to increased microbial activity of the soil, but decreased in the stage T4 ($\frac{1}{4}$ maturity). Although, the organic treatments increased the urease activity, but inoculation of plants with Azotobacter and Azospirillum increased the effect of organic amendments on urease activity and among the treatments, VAM + Azotobacter showed the most urease activity. In agreement with our results, Rodrigues, Ladeira & Arrobas (2018) reported that, the organic material containing Azotobacter increased the bioavailability of N over organ, by an additional N₂-fixing value of 11.4 kg/hm² estimated from the six crops of the field experiment. Nayak, Babu & Adhya (2007) reported increased urease activity with a compost made of residues of rice + urea in flooded rice, and Arora & Kaur (2019) reported about Aspergillus terreus-enriched organic manures. The increase of total organic carbon, water-soluble carbohydrate, water-soluble C, and proliferation and microbial activity of the soil are among the most important contributions of organic amendments (Srivastava, Aragno & Sharma, 2010) resulting in the release of elements in the soil and organic materials and making available roots by increasing the bacterial activity (Pérez-Piqueres et al., 2006). In fact, changes in the composition of microbial communities have been observed as a result of incorporating inorganic or organic amendments (Marschner, Kandeler & Marschner, 2003; Crecchio et al., 2004). But various properties of organic materials can have a different effect on soil microbiota and will strongly influence the microbial use of the C contained in these materials. Vermicomposting increased the number and biomass-C of microorganisms of the soil, especially under inoculation with Azotobacter, compared to the manure treatment (M) and control. Carbohydrates would act as sources of energy in respiration of microbiota (Zhong et al., 2010; Li et al., 2015) showing intense temporal changes due to their continuous process of synthesis and degradation. This is reflected by the changes in urease activity during the growing season of rice under various treatments. During the growing season, urease activity was higher in treatments under inoculation compared to non-inoculation treatments, especially VAM and VRAM treatments that showed more enzyme activity under inoculation with Azotobacter compared to Azospirillum. Higher urease activity under the application of VAM and VRAM treatments demonstrated higher N -fixing ability of Azotobacter, which increased the available N content in the rice rhizosphere.

Zhang et al. (2017) and Islam et al. (2012) also suggested that, wide variety of free-living N₂-fixing bacteria can be used as a feasible alternative to N fertilizer in rice ecosystems. The addition of organic material to soil results in an increase in the total organic carbon content increasing the proliferation and activity of microorganisms (Martinez-Balmori et al., 2013), therefore in the beginning of the growth, although the enzyme activity of microorganisms is high, but they take out the nutrients from the plant and spend them for their bodies (Rodrigues, Ladeira & Arrobas, 2018).

Therefore, the reason for the superiority of Azolla-containing organic treatments, namely VAM (vermicompost of manure + Azolla) and VRAM (vermicompost of manure + Azolla + rice straw), can be due to more amounts of N compared to other treatments. The application of Azolla compost in paddy rice has been studied in many countries and its positive effect in increasing the crop has been well proved (Razavipour et al., 2018). The preferred effect of VAM and VRAM on the microbial population of the soil and the urease activity, as well as the growth and yield of rice in inoculated plants is evident. In this regard, Zhang et al. (2018) reported that, inoculation of A. brasilense and P. fluorescens in the rice rhizosphere accelerated N transformations and improved the N-supplying capacity of the rhizosphere soil, and increased rice biomass. The most beneficial effects were observed with A. brasilense and P. fluorescens co-inoculation in the rice rhizosphere. Inoculation of microorganisms is influenced by agricultural practices (Velusamy, Immanuel & Gnanamanickam, 2013) improving both growth and nutrient acquisition (Khan, 2018) of rice grown under upland conditions (Rajeshkannan, Sumathi & Manian, 2009; Zhang et al., 2017).

The used treatments have improved the biological, physical, and chemical properties of the soil and increased the growth of morphological traits. So that in both years, under both non-inoculation and inoculation conditions, application of organic amendments compared to control increased plant and panicle height. The highest plant and panicle height was obtained from plants treated with VAM and VRAM under inoculation with Azotobacter. Kumar, Swain & Bhadoria (2018) and Lin, Zhu & Lin (2011) reported that, the number of functional leaves, leaf area and total number of tillers was higher in plants treated with organic nutrient, which increased the photosynthetic rate leading to higher plant height.

Moreover, the increase in photosynthesis of the plant depends on the content of leaf chlorophyll (Murchie et al., 2002; Singh, Singh & Sharma, 2013) as influenced by organic amendments and bacterial inoculation in this experiment, and the highest chlorophyll of a, b and total chlorophyll was observed in vermicompost treatments under inoculation with Azotobacter. In most studies, the increase in chlorophyll content of leaf has been reported to be directly associated with the absorption of N and Fe (Hosseinzadeh, Amiri & Ismaili, 2018). In addition to atmospheric N fixation, the solubility of minerals such as P, K, Cu, and Fe and the production of sidrophor are among the important effects of Azotobacter and Azospirillum (Yadav et al., 2014; Rodrigues, Ladeira & Arrobas, 2018). On the other hand, vermicompost is also rich in some macro and micro elements (García et al., 2014; Yadav et al., 2014; Eo & Park, 2019). Therefore, vermicompost involves desirable properties such as a high capacity for cation exchange, increased absorption of nutrients and other beneficial physical, biological, and chemical properties (Ludibeth, Marina & Vicenta, 2012) increasing the chlorophyll content and stability of the photosynthetic system of rice plants .The vermicompost application led to a decrease in the Reactive Oxygen Species (ROS) production, increased accessibility to nutrients and the required elements for biochemical activity (Baghbani-Arani & Modarres-Sanavy, 2017). Mahanta et al. (2012) and Shirkhani & Nasrolahzadeh (2016) reported increased chlorophyll content of leaves by organic fertilizers and N₂-fixing bacteria. Increasing the leaf chlorophyll content leads to an increase in photosynthetic capacity. The acceleration of photosynthesis leads to an increase in plant height, number of tillers and number of grain per panicle (Yi-hu et al., 2014). García et al. (2016) reported that, vermicompost increased the number of tillers and plant dry weight by producing humic acid and supplying nutrients, especially N and K.

Meena & Shivay (2010) introduced the improvement of rhizosphere in increasing the root activity as a factor increasing the number of tillers. In our experiment, in both years, bacterial treatments × organic amendments had a positive effect on the number of tillers. Although the number of tillers is dependent on environmental and genetic factors, but environmental conditions, especially nutrition have highest effect in the early stages of growth (Murchie et al., 2002; Nuemsi et al., 2018). In both years, vermicomposts containing Azolla under inoculation with Azotobacter produced greatest tillers. Previous studies revealed that, these management practices have amended the soil structure and function, as well as nutrient availability influencing the plant height, number of tiller, panicle length, biological and grain yield of rice by the synergistic effects (Simarmata et al., 2016; Tsujimoto et al., 2009; Thakur et al., 2010; Kumar, Swain & Bhadoria, 2018). The combined use of vermicompost + N₂-fixing bacteria is better than sole application of them for grain, straw and biological yields, because organic fertilizers can reduce N loss (Kumar & Singh, 2001) and maintain the supply of N to rice plants for a longer time (Nayak, Babu & Adhya, 2007; Li et al., 2015).

In the present experiment, in both years, the grain and biological yield of inoculated plants increased significantly by applying different vermicomposts compared to non-inoculated plants under similar treatments. The highest grain yield by VAM + Azotobacter treatment in the first and second years was obtained as 4,667 and 5,081 kg/hm², respectively, although there was no statistically significant difference in some of the treatments. In both inoculated and non-inoculated plants, VAM treatment produced the highest biological yield and VRAM treatment ranked the next, in addition, the maximum biological yield was observed under inoculation with Azotobacter. Eo & Park (2019) also stated the role of vermicompost in increasing rice growth and yield, so that the vermicompost application has altered the resource allocation and soil chemical properties leading to establishment of new interactions between root parameters and components. In most studies similar to our study, the main cause of increase in the growth and grain yield of rice by applying organic fertilizers under inoculation with N₂-fixing bacteria has been introduced due to the increase in the availability and supply of N in different stages of rice growth. In our experiment, due to the application of bacteria × vermicompost, the amount of fixed atmospheric N₂ and organic materials content was enhanced in the soil.

Marginal increase in N content of rice straw due to inoculation with Azotobacter and use of organic fertilizers has been reported (Rodrigues, Ladeira & Arrobas, 2018), resulting from an increase in the N availability through synchronized released from the inoculation of N₂-fixing bacteria, which increased the N concentration proportionately in grains and straw and finally led to higher N uptake with the highest amount of N (Li et al., 2015; Tu et al., 2017). In our study, in both years, protein yield significantly increased in plots treated with both vermicompost under inoculation and non- inoculation conditions compared to control and M treatments. In non-inoculated and inoculated plants with Azotobacter, the highest protein yield was obtained from VAM, but there was no significant difference between VAM and VRAM in plants inoculated with Azospirillum. Tejada & González (2009), Bejbaruah, Sharma & Banik (2013), Mengi et al. (2016) and Taheri Rahimabadi, Ansari & Razavi Nematollahi (2018) have reported the effect of vermicompost and organic amendments on increasing the grain protein of rice. The higher rice yield using the integrated N management might be due to the increase in number of filled grains per panicle, the number of panicles per plant, and 1,000-grain weight (Amanullah, 2016). The difference in nutrient uptake from different N source combinations significantly influences growth and yield potential (Gu et al., 2014). Myint et al. (2010) reported that, organic amendments can increase yield through the improvement of soil water holding capacity, physical and chemical conditions, reducing volatilization of nitrogenous fertilizers to NH₃ gas and the greater availability of plant nutrients for a longer time.

The P and K uptake of grain were influenced by interaction effect of organic amendments × bacteria. The highest P uptake in non-inoculated plants was obtained from VAM and VARM (with a 76 and 79% increase compared to control). In plants inoculated with Azotobacter, VAM and VM treatments showed the highest P uptake, but in plants inoculated with Azospirillum, VAM treatment showed the highest P uptake. The highest K uptake in non-inoculated plants was obtained from VAM treatment and it was obtained in plants inoculated with Azospirillum from VRAM and VAM. But in plants inoculated with Azotobacter, there was no significant difference between vermicompost treatments including VM, VRM, VAM, and VARM, but significantly increased K uptake compared to the manure (M) and control. Our results confirm the findings of the study by Yadav et al. (2014) who have reported 50% increase in content and uptake of N, P, and K in wheat using PGPR strains. An increase in P uptake over control may be due to the bacterial solubilization of insoluble phosphate in soil. These bacteria showed an effective role in P uptake and growth promotion of plants by dissolution of inorganic insoluble phosphate as reported in the study by Narula et al. (2000).

These results showed that, the application of organic amendments + growth stimulating bacteria leads to an increase in organic material pools and nutrient availability, as well as amendment of the physical environment of soil and rice yield (Chen 2017). Additionally, the application of vermicompost, especially VAM and VARM treatments, either alone or together with Azotobacter and Azospirillum, in addition to increasing the number and biomass-C of soil microorganisms, leads to improvement of the solubility of nutrients, especially N, P, K and some microelements.

In line with our results, Zhong et al. (2010), Li et al. (2015); Wang et al. (2015) and Cui et al. (2018) reported that, the application of organic fertilizers influences on soil physicochemical and biological properties, especially regarding soil pH and microbial biomass C. Nayak, Babu & Adhya (2007) showed that, soil urease activity had a significant negative relationship with Eh and had a positive relationship with the content of N, P, and Fe. Therefore, application of vermicompost and N₂-fixing bacteria by changing the state of redox and soil pH and catalytic efficiency may influence the accumulation and activity of enzymes, and importantly increases the availability of nutrients (Wlodarczyk, Stepniewski & Brzezinska, 2002). In this regard, Basha, Basavarajappa & Hebsur (2017) and Taheri Rahimabadi, Ansari & Razavi Nematollahi (2018) reported the effect of vermicompost application on increasing the biomass and grain yield of rice due to the increased absorption of essential elements of plant growth. Similarly, Yadav et al. (2014) reported that, Azotobacter and Azospirillum inoculated with rice seedling led to a 35–21% increase in the yield and yield components, respectively compared to control under field condition. There were significant interactions between nitrogen level and biofertilizer regarding yield, yield component, and protein content (Nosheen et al., 2016). In this study, Azotobacter and Azospirillum also acted complementarily regarding positive effects of vermicomposts on nutrients absorption and rice yield.

Conclusion

The results of the present study showed that, vermicomposting of cow manure, rice straw and Azolla led to improvement of physicochemical properties of used materials. In both years, the application of vermicomposts had a positive effect on the number and biomass-C of soil microorganisms and urease enzyme activity. It was found that, these changes led to the amendment of physical and biological properties of the soil and increased the chlorophyll content, biological and grain yield, and the uptake of N, P, and K by the availability of nutrients. In this study, vermicomposts containing Azolla (VAM and VARM) were more beneficial for increasing the rice productivity. Inoculation of the plants with Azotobacter and Azospirillum also showed significant superiority in the above mentioned traits compared to non-inoculated plants. On the other hand, the synergistic effect of bacteria with organic amendments, especially with Azotobacter was observed with vermicomposts containing Azolla, so that the highest grain yield in the first and second years with an average of 4667 and 5081 kg/hm², respectively was obtained from VAM + Azotobacter. The present study provides information on the effect of applying manure and various vermicomposts on the number and biomass-C of soil microorganisms and urease enzyme activity and finally on the absorption of elements and grain yield under inoculation with N₂-fixing. Our results suggested that, applying organic source for vermicomposting has a great effect on the enzymatic and biological activity of the soil under flooded conditions contributing to the sustainable development of agro-ecosystems. It is suggested to evaluate the vermicomposting effect on various organic sources weak and rich in nutrients, especially N, along with inoculation of plants or vermicompost enrichment with N₂-fixing bacteria in future studies.

Supplemental Information