Effects of Long-Term Fertilization Strategies on Soil Productivity and Rhizobial Diversity in Chinese Mollisol

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Title: Effects of Long-Term Fertilization Strategies on Soil Productivity and Rhizobial Diversity in Chinese Mollisol

Author: YAN Jun, HAN Xiao Zeng, CHEN Wen Feng, WANG En Tao, ZOU Wen Xiu, ZHANG Zhi Ming

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Please cite this article as: YAN Jun, HAN Xiao Zeng, CHEN Wen Feng, WANG En Tao, ZOU Wen Xiu, ZHANG Zhi Ming, Effects of Long-Term Fertilization Strategies on Soil Productivity and Rhizobial Diversity in Chinese Mollisol, Pedosphere (2017), 10.1016/S1002-0160(17)60470-3.

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ACCEPTED MANUSCRIPT PEDOSPHERE Pedosphere ISSN 1002-0160/CN 32-1315/P

doi:10.1016/S1002-0160(17)60470-3

Effects of Long-Term Fertilization Strategies on Soil Productivity and Rhizobial Diversity in Chinese Mollisol

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YAN Jun1, HAN Xiao Zeng1,, CHEN Wen Feng2, WANG En Tao3, ZOU Wen Xiu1, ZHANG Zhi Ming1 1 National Observation Station of Hailun Agro-ecology System, Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081 (China) 2 State Key Laboratory of Agrobiotechnology, College of Biological Sciences and Rhizobia Research Center, China Agricultural University, Beijing 100193 (China) 3 Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México 11340 (México)  Corresponding author. E-mail: [email protected] ABSTRACT

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Aiming to maintain and recover the productivity of Chinese Mollisol, a long-term fertilization experiment has been carried out for 29 years under the wheat-maize-soybean rotation system, including application of ROM (recycled organic manure), chemical fertilizers and their combinations. The effects of different treatments were evaluated by determining the soil physicochemical features, soybean production and soybean rhizobial diversity. The results showed that application of recycled organic manure (ROM)-combined with chemical fertilizer maintained or increased soil fertility, companying by higher production and higher diversity of soybean rhizobia. The negative association of Bradyrhizobium japonicum with N-fertilizer, positive association of B. diazoefficiens with soil acidification, and reducing of N-impression on the diversity of Bradyrhizobium by addition of ROM were recorded as new findings. Therefore, the ROM involved treatments, especially NPK+ROM could be a feasible strategy for maintaining and recovering the fertility of the Chinese Mollisol, while the rhizobial diversity could be an indicator for the soil fertility.

INTRODUCTION

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Key words: Bradyrhizobium, chemical fertilizer, housekeeping gene, multilocus sequence analysis, organic manure, rhizobial community

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Mollisols are characterized by the presence of mollic epipedon or fertile surface horizon (varied from 30 to 100 cm in depth) with high content of organic matter resulting from the long-term accumulation of vegetable materials. Based upon the high soil fertility of Mollisols in the provinces of Heilongjiang, Jilin, Inner Mongolia and Liaoning in China, most of these areas have been explored as agricultural fields (Han and Li, 2011). However, the serious erosion with loosing of 0.3-1.0 cm in depth of the mollic epipedon per year has been happening in Chinese Mollisols and the other Mollisols areas of world (Fenton, 2012) during the past 100 years of their agricultural exploration, which is caused by the loss of its natural covering vegetation and have resulted in the degradation of soil productivity (Miao et al., 2013). To maintain and remediating the high productivity of the Mollisols, some suggestions and laws for the Mollisols protection have been drawn up since the end of last century and the related scientific research was started even earlier (Miao et al., 2013). In the Hailun National Field Station, located in Heilongjiang Province, experiments for studying effects of long term different fertilizer application, land use, crop sequence and other agricultural strategies on the productivity of Mollisols have been established since 1985. From these experiments, variation of soil organic matter contents under different farming systems (Ding et al., 2014), increase of low-molecular-weight organic acids by repeated cattle manure addition (Miao et al., 2013), significantly greater aggregation for > 1 mm aggregates and significant SOC changes in this fraction at 0-5 cm depth (Liang et al., 2011), greater richness and abundance of bacteria and AMF (Arbuscular mycorrhizal fungus) (Zhang et al., 2015) in no tillage treatment were observed. Moreover, the effects of land use (bare, grassland, and cropland) and crop managements (maize/soybean/wheat rotation with or

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without fertilizer supply) on abundance and diversity of soybean-nodulating rhizobia (Yan et al., 2014) have been revealed. However, the effects of fertilization strategy on the crop productivity, the rhizobial diversity, and rhizobial community composition in the maize/soybean/wheat rotation Mollisols have not been fully studied previously. As the symbiotic nitrogen-fixing bacteria, the abundance and diversity of rhizobia are particularly important in agroecosystems, and could be an indicator for the soil fertility (Pastor J, 1998; Honsova et al., 2007; Chmelikova and Hejcman, 2014). Previously, genotype of host legumes (Saeki et al., 2008), soil conditions (like content of organic carbon, pH, and available phosphorus) (Kahindi et al., 1997; Zahran, 1999), and soil management practices (cropping rotation, tillage, and fertilization practices) have been reported as factors to regulate the rhizobial communities in soils (Ferreira et al., 2000; Depret et al., 2004; Grossman et al., 2011). For the fertilization strategies, previous study showed that the high organic carbon containing (38%) manure could offer some nutrients for rhizobia (Zengeni et al., 2006). Recently, application of fertilizer accompany to organic matter input has been improved the soil fertility and increased rhizobial population (Kimiti and Odee, 2010; Vanlauwe et al., 2010). In another case, the application of N fertilizer combined with maize residues decreased the rhizobial diversity (Herrmann et al., 2014). Therefore, the rhizobial diversity in a certain fields could be due to the interactions of the soil features, fertilization strategy, and cropping system (Li et al., 2012), and it also could be an indicator to estimate the soil productivity. However, the interactions among the rhizobial community, the chemical-organic manure fertilization, and the plant production have not been well documented in the Mollisol with long-term experiments. Considering the information mentioned above, we performed the present study to assess the effects of different long-term fertilization on rhizobial communities and to estimate the correlation among the fertility, production and genetic diversity and community structure of soybean-nodulating rhizobia in the wheat-maize-soybean rotation system in the Mollisol at northeast of China. MATERIAL AND METHODS Study site and experimental set-up

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The experiment was set up in 1985 at Hailun National Field Station (47°26′N, 126°38′E, and altitude of 240 m), located in Hailun Country, Heilongjiang Province, China. Eight fertilization treatments in triplications were included in crop rotation system of wheat, corn, and soybean. The eight treatments were (1) control (unfertilized treatment, CK), (2) recycled organic manure (ROM) supplied as decomposed pig manure which is a traditional form to supply nutrients for plants and to improve the soil fertility, (3) nitrogen fertilizer (N), (4) N fertilizers plus recycled organic manure (N+ROM), (5) nitrogen and phosphate fertilizer (NP), (6) NP fertilizers plus recycled organic manure (NP+ROM), (7) nitrogen, phosphate and potassium fertilizer (NPK) and (8) NPK fertilizers plus recycled organic manure (NPK+ROM). In all the treatments, the same crop was planted at the same time. K 2SO4 was used as the K source at a rate of 60 kg ha-1 year-1 for all the plants, respectively (Table I). (NH4)2HPO4 was used as the P source, but 17.9 kg N ha-1 was also supplied when (NH4)2HPO4 was applied at a rate of 20 kg P ha-1. Urea was used as the N source for the treatment without P fertilizer (i.e. N, N+ROM treatment), and it was the additional N source for the NP and NPK involved treatments. The plots for the treatments were randomly distributed in the fields and were ridged by rotary tillage in October after harvest. All above-ground crop residues were removed from CK, N, NP and NPK plots. While 80% of the harvested grains from ROM, N+ROM, NP+ROM and NPK+ROM plots were fed to pigs, and the corresponding straw was shredded into pieces to mat pigsty. Before sowing, the decomposed, mixed manure and residues were used as recycled organic manure (ROM), applied on the soil surface and incorporated into the soil by plough annually to the original plots where the grain and straw were harvested (Han et al., 2006). Intensive tillage as a common soil management practice was used in all treatments. In each year, seeds of the crop were planted on the top of ridges with a conventional planter in the early of May. The second ridge was carried out at about half a month after planting. No rhizobial inoculation history was recorded in this experimental site.

ACCEPTED MANUSCRIPT TABLE I Application rates (Kg ha-1 yr-1) of different fertilizer treatments for a 3-year wheat-maize-soybean rotation cropping system since 1985. Treatmenta)

1985-1996

1997-2006

2007-2013

N

P

K

N

P

K

N

P

K

CK

0

0

0

0

0

0

0

0

0

ROM

0

0

0

0

0

0

0

0

0

N

107.2

0

0

90.0

0

0

90.0

0

0

N+ROM

107.2

0

0

90.0

0

0

90.0

0

0

NP

107.2

18.6

0

90.0

20

0

90.0

20

0

NP+ROM

107.2

18.6

0

90.0

20

0

90.0

20

0

NPK

107.2

18.6

187.5

90.0

20

60

90.0

20

60

NPK+ROM

107.2

18.6

187.5

90.0

20

60

90.0

20

60

CK

0

0

0

0

0

0

0

0

0

ROM

0

0

0

0

0

0

0

0

0

N

107.2

0

0

107.2

0

0

107.2

0

0

N+ROM

107.2

0

0

107.2

0

0

107.2

0

0

NP

107.2

18.6

0

107.2

20

0

107.2

20

0

NP+ROM

107.2

18.6

0

107.2

20

0

107.2

20

0

NPK

107.2

18.6

187.5

107.2

20

60

107.2

20

60

NPK+ROM

107.2

18.6

187.5

107.2

20

60

107.2

20

60

CK

0

0

0

0

0

0

0

0

0

ROM

0

0

0

0

0

0

0

0

0

N

0

0

0

0

0

0

17.9

0

0

N+ROM

0

0

0

0

0

0

17.9

0

0

NP

0

18.6

0

0

20

0

17.9

20

0

0

18.6

0

0

20

0

17.9

20

0

0

18.6

187.5

0

20

60

17.9

20

60

NPK

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NP+ROM

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Soybean phase

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Maize phase

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Wheat phase

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NPK+ROM 0 18.6 187.5 0 20 60 17.9 20 60 a) , ROM, with recycled organic manure at dose of all the straw and 80% of the grains produced in the plot; N+ROM, with N plus ROM; NP+ROM, with NP plus ROM; NPK+ROM, with NPK plus ROM. All treatments were laid out in 1985 and are still being applied.

Sampling All the samples used in the present study were obtained in 2013, when the soybean was cropped. For rhizobial isolation, roots of five soybean plants were randomly excavated from each plot and were washed immediately with tap water to eliminate attached soils at the full flower stage of soybean. Two or three healthy and intact nodules randomly sampled from each plant were put into plastic tubes filled with silica gel and were transported to laboratory. For isolation of rhizobia, the shriveled dry nodules were rehydrated overnight at 4 °C with sterile water and were surface sterilized with 3% (w/v) NaClO; then the surface-sterilized nodules were crushed and inoculated by cross-striking on the yeast mannitol agar (YMA) medium as described by Vincent (1970). After incubated 7 to 14 days at 28 C, the obtained rhizobial colonies were purified by repeatedly streaking on the same medium until all the colonies of the isolate presented the same morphology. Pure cultures were maintained on YMA slants at 4 °C for short-term storage

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or in YM broth supplied with 20 % (w/v) glycerol at -80 °C for long-term storage. In addition, the soybean yield and biomass production were recorded for each plot. For analysis of physicochemical features, soil samples were collected from surface layers (0--20 cm soil depth) of each plot in July 2013 at the full flower stage of soybean, with a soil drill cleaned with alcohol (95 %) and flamed. Five random subsamples in a plot were pooled together to generate a composite soil sample. Then 24 composite soil samples were separately placed in sterile plastic bags, and were transported to the laboratory immediately. In the laboratory, the composite samples were air dried at room temperature and passed through 2-mm sieve before they were used for estimation of soil organic carbon (SOC), pH, available N (AN), available P (AP), and available K (AK). The organic carbon content of the soils was determined by VarioEL CHN elemental analyzer (Heraeus Elementar Vario EL, Hanau, Germany). Because the soils were free of carbonates, the total C content was equivalent to SOC content. The soil pH was determined in 1:2.5 soil: water (w/v) suspension. Soil available N was analyzed through quantifying alkali-hydrolysable N in the Conway diffusion unit with Devarda’s alloy in the outer chamber and boric acid-indicator solution in the inner chamber (Shen et al., 2004). Available P was extracted from the sample with 0.5 M NaHCO3 and the AP content was determined colorimetrically (Olsen, 1954). Available potassium was extracted with ammonium acetate, and then determined by a flame photometry (Lu, 1999).

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PCR amplication and sequencing analysis of housekeeping and symbiotic genes

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Genomic DNA was extracted from each of the nodule isolates and the reference strains for Bradyrhizobium species using the guanidinium thiocyanate chloride (GuTC) method (Terefework et al., 2001). Amplification of recA genes using the obtained genomic DNA as template was carried out by employing the primers recA41F and recA640R and the protocol of Vinuesa et al. (2005). The obtained PCR products were then sequenced directly using primer recA41F with the method of Hurek et al. (1997). All the acquired recA sequences in this study were used to screen the phylogenetic relationships between the isolates and reference strains with the software Clustal W (Higgins et al., 1994). The isolates with identical recA sequences were designated as a single genotype (Yan et al., 2014). According to the recA typing results of all the isolates, six representative isolates were selected for the subsequent multilocus sequence analysis (MLSA). The genes glnII, atpD, dnaK, gyrB and rpoB were amplified by PCR from genomic DNA of the representative isolates and some reference strains with primer pairs glnII/glnII689R, atpD255F/atpD782R, TsdnaK3/TsdnaK2, gyrB343F/gyrB1043R, and rpoB454F/rpoB1364R, respectively (Stepkowski et al., 2003; Vinuesa et al., 2005; Vinuesa et al., 2008; Rivas et al., 2009). Fragment of nodulation gene nodC and nitrogen fixation gene nifH were also amplified with primer pairs nodCF540/nodCR1160 and nifHF/nifHR, respectively, using the protocol of Sarita et al. (2005) and Laguerre et al. (2001). The PCR products for each of the mentioned genes were sequenced directly by using the corresponding forward primers, same as the recA sequencing. The acquired sequences from the representative isolates N10, CK3, NPK+ROM11, NPK+ROM8, N+ROM18, and ROM2 were deposited in the GenBank database under the accession numbers KT965763 through KT9657668 for atpD, KT965769 through KT965774 for dnaK, KT965781 through KT965786 for gyrB, KT965775 through KT965780 for glnII, KT965805 through KT965810 for rpoB, KT965799 through KT965804 for recA, KT965793 through KT965798 for nodC and KT965787 through KT965792 for nifH. All sequences acquired in this study and homologous sequences obtained from the GenBank database by BLAST were aligned using the Clustal W software. Phylogenetic trees were reconstructed for each gene and for the combined housekeeping genes using the neighbor-joining method (Saitou and Nei, 1987) with the Kimura's two-parameter model and were bootstrapped with 1000 replications using MEGA 5.0.5 software (Tamura et al., 2011). The threshold value of 97.7% sequence similarity in the MLSA (recA, glnII, atpD, dnaK, gyrB, and rpoB) was used for genospecies separation (Yan et al., 2014). Statistical analysis Redundancy analysis (RDA) (Rao, 1964), the canonical version of principal component analysis (PCA), was used to examine the multiple relationships between soil factors (OC, AN, AP, AK and soil pH) and genospecies of soybean rhizobia in the 8 treatments. Community data and soil data (Table II) were preanalyzed using WCanoImp and then

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were analyzed with Detrended Correspondence Analysis (DCA) using CANOCO software 5.0 (Microcomputer Power, Ithaca, NY) (Šmilauer and Lepš, 2014). In the detrended correspondence analysis (DCA), the models of species response to environmental variables and the length of the gradient (first axis) was 0.433, so the linear models are suitable. After further model tests, RDA was proved to be the best method. Rhizobial diversity, genospecies richness, and evenness in different treatments were estimated by three popular alpha ecological indexes (Hill et al., 2003): the Shannon-Wiener (H') index calculating genospecies richness in a community; Simpson index (D) and the Pielou index (J), showing the genospecies dominance and evenness, respectively, in a community. These indexes of biodiversity were implemented in the Vegan package (version 2.0-10) and were calculated using the R statistical language (version 3.1.0) (R Core Team, 2014). Numbers of the genospecies and isolates from the eight treatments were counted and a stacked bar plot was drawn using the barplot function integrated in the R statistical language (R Core Team, 2014). Furthermore, the soybean production, organic carbon (SOC) content, pH, available N (AN), available P (AP), and available K (AK) of soils in the eight treatments were statistically by compared with one-way ANOVA. RESULTS

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Soil properties and soybean production

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The analyses of soil properties (Table II) revealed that the pH was significantly decreased (pH 6.02--6.21) in all the treatments in comparison with CK (pH 6.37), and that this decrease was significantly less in the ROM-involving treatments than that in the corresponding treatments of N, NP and NPK. Soil OC and AN were significantly increased (P < 0.05) in all the fertilization treatments comparing CK; while all the ROM-involving treatments presented greater OC and AN contents than the corresponding treatments with chemical fertilizers alone. The addition of P significantly increased the soil AP contents; while no difference was found in the content of AK among all the treatments (Table II). In the production analysis, the grain production and shoot biomass were significantly increased by fertilizer application, in which the NPK+ROM treatment presented the highest grain and biomass productions, and its grain production was significantly greater than those in the other treatments (Table II).

ACCEPTED MANUSCRIPT TABLE II The soybean production, chemical properties of soils and rhizobial composition in different treatments sampled in 2013 Productionb) -1

Original soil

Biomass

pH

(g plant ) (n=15)

-

f)

g)

AP

AK

(g kg-1)

(mg kg-1)

6.34a

31.3d

234bc

25.8b

191 a

-1

(kg ha ) (n=3)

AN

Rhizobial genospecies composition [number (%)] d) B. sp. I

B. jap

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Treatmentsa)

OCc)

B. dia

B. ott

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Grain

1501± 46 d

23.4±1.3 d

6.37a

30.5e

166f

19.2c

180a

19 (79.17)

3 (12.50)

0

2 (8.33)

0.652

ROM

1799± 82 c

36.9±6.2 c

6.21b

31.4d

220c

19.0c

187a

24 (80.00)

4 (13.34)

1 (3.33)

1 (3.33)

0.674

N

1682± 41 c

32.1±1.4 bc

6.02e

31.0d

182e

17.6d

181a

20 (86.95)

0

2 (8.70)

1 (4.35)

0.470

N+ROM

1833± 42 c

37.5±2.5 bc

6.12cd

33.7c

226c

18.7c

191a

22 (73.33)

3 (10.00)

1 (3.33)

4 (13.34)

0.840

NP

1873±141 c

44.5±5.2 ab

6.07de

32.2cd

200d

27.9b

187a

21(84.00)

0

3(12.00)

1( 4.00)

0.530

NP+ROM

2039± 83 b

46.4±3.8 ab

6.16c

34.5b

223c

29.9ab

190a

21(72.41)

3(10.34)

1(3.45)

4(13.79)

0.858

NPK

2104±118 b

48.8±6.5 ab

6.09d

34.3b

244b

30.1ab

190a

19 (82.60)

0

2 (8.70)

2 ( 8.70)

0.583

NPK+ROM

2469±100 a

50.1±7.8 a

6.17c

35.4a

252a

33.0a

192a

19 (65.52)

4 (13.79)

2 (6.90)

4 (13.79)

1.008

Total

-

-

-

-

-

-

-

165 (77.46)

17 (7.98)

12 (5.63)

19 (8.92)

-

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a)

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CK

CK, without fertilizer application; ROM, with recycled organic manure; N, with nitrogen fertilizer; N+ROM, with N plus ROM; NPK, with N and P and K fertilizer; NPK+ROM, with NPK plus ROM. All treatments were laid out in 1985 and are still being continued.

Data from the physical mature stage: production refers to seeds and biomass is for shoots.

c)

OC, organic carbon; AN, available nitrogen; AP, available phosphorus; AK, Available potassium. Different letters in the same column indicate a significant difference at P < 0.05 among treatments for each parameter.

d)

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d

b)

Genospecies was identified by multilocus sequence analysis (MLSA) including genes recA, atpD, glnII, gyrB, rpoB and dnaK. B. jap=B. japonicum; B. dia=B. diazoefficiens; B. ott=B.

ottawaense. H', Shannon-Weiner’s index.

f)

Mean ± standard deviation (n = 3).

g)

Values followed by the same letter(s) in the same column are not significantly different at P < 0.05.

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e)

ACCEPTED MANUSCRIPT Isolation and phylogenetic analyses of root nodule bacteria

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In total, 213 bacterial isolates were obtained from soybean root nodules of the 8 treatments: 24 from CK, 23 from N, 25 from NP, 23 from NPK, 30 from ROM, 30 from N+ROM, 29 from NP+ROM and 29 from NPK+ROM. All isolates were Gram-negative rods with slow growth rate (colonies of ≤ 1 mm in diameter in 7 days) and alkaline reaction in YMA medium. In recA gene sequence analysis, six genotypes were detected in all the isolates and they were grouped into 4 genospecies within the genus Bradyrhizobium (Supplementary Fig. S1), exhibiting highest sequence similarities 97.1% with B. japonicum USDA 6T, 99.0-99.8% with B. japonicum USDA 6T, 99.8-100% with B. ottawaense LMG26739T and 97.8% with B. diazoefficiens USDA 110T, respectively (Supplementary Fig. S1 and Table SI). The phylogenetic trees constructed with the separate housekeeping genes glnII, atpD, dnaK, gyrB, and rpoB (Supplementary Fig. S2-S6) showed topology similar to that of the recA, and were similar to the MLSA tree constructed with the combined sequences of the six genes (Fig. 1). The group containing 165 isolates represented by N10 was designed as Bradyrhizobium sp. I, which shared 97.6% MLSA similarity with B. japonicum USDA 6T. Nineteen isolates represented by ROM2 and N+ROM18 were designed as B. ottawaense based upon their 99.0-99.3% of similarities with B. ottawaense LMG26739T. Isolates CK3 and NPK+ROM11 representing 17 isolates were identified as B. japonicum since they shared 98.3% and 98.8% similarities with B. japonicum USDA 6T (Supplementary Table SI). Moreover, 12 isolates represented by NPK+ROM8 were identified as B. diazoefficiens, because they presented 99.4% similarity with B. diazoefficiens USDA 110T.

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Fig. 1 Phylogenetic tree of MLSA based on concatenated sequences of recA (418 nucleotides [nt]), glnII (505 nt), gyrB (590 nt), rpoB (761 nt), dnaK (223 nt) and atpD (419 nt). GenBank accession numbers in boldface were newly determined as a result of this study. The tree was constructed with Neighbor-Joining method. Bootstrap confidence levels of 50% are indicated at the

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internodes. The bar indicates 1% nucleotide divergence.

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Distribution of rhizobial genospecies in treatments

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As shown in Table II, the isolates of Bradyrhizobium sp. I and B. ottawaense were found in all treatments. The former was the most dominant group with relative abundance of 65.5--86.9%, while B. ottawaense varied between 3.3% and 13.8%. B. diazoefficiens was recovered from all the fertilization treatments, but not the CK, at a proportion between 3.3% and 12.0%. B. japonicum was disappeared in the three treatments with chemical fertilizers alone (N, NP and NPK) and presented at the proportion of 10.0% to 13.8% in CK and in other treatments with ROM application. Phylogeny of the symbiotic genes in the isolates Different from the phylogenies for the housekeeping genes, the six isolates representing different genotypes in the four genospecies formed two group in the phylogenetic tree constructed with the combined nodC and nifH sequences (Fig. 2), showing 92.5%-100% sequence similarities to those of the soybean nodulating reference strains B. japonicum USDA 6T, B. diazoefficiens USDA 110T, B. daqingense CCBAU 15774T, B. ottawaense LMG26739T and B. huanghuaihaiense CCBAU 23303T. The topologies of nodC and nifH phylogenetic trees (Supplementary Fig. S7 and S8) were very similar, except for isolate NPK+ROM8, which had a nifH gene different from the other isolates (Fig. S8).

ACCEPTED MANUSCRIPT Fig. 2 Phylogenetic relationships of the soybean rhizobia isolated from different fertilization treatments based on the combined sequence of nodC and nifH. The tree was constructed with Neighbor-joining methods. Bootstrap values greater than 50% were marked at the node of branch. The scale bar presents 2% nucleotide substitution.

Diversity index and RDA analysis

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The data in Table II demonstrated that the diversity index of rhizobia was decreased by application of the chemical fertilizers (N, NP, and NPK treatments), in which the NPK treatment presented the highest diversity index. The rhizobial diversity in all the ROM involved treatments was significantly increased in comparison with the corresponding chemical fertilizer treatments. The ROM treatment increased H' diversity index by 3.4% over CK; N+ROM increased 78.6% over the N treatment; NP+ROM increased 62.0% over the NP treatment, and NPK+ROM increased 73.0% over the NPK treatment. In RDA analysis (Fig. 3), all the analyzed soil factors (pH, OC, AP, AN and AK) presented correlation with the distribution of genospecies or community structure of rhizobia. According to the lengths of the arrows and the angles among them (Fig. 3), OC, pH and AP showed strong positive correlation with the distribution of B. japonicum and B. ottawaense; and strong negative correlation with the distribution of Bradyrhizobium sp. I and B. diazoefficiens. Contents of AK and AN had slight effects on the distribution of soybean rhizobia. It seems that higher pH (6.37) inhibited B. diazoefficiens, while lower pH (< 6.09) might eliminate B. japonicum from the soils (Fig. 3 and Table II). For B. ottawaense, OC content had stronger effect than the other factors (Fig. 3) and it was more abundant in treatments N+ROM, NP+ROM and NPK+ROM which had greater OC (Table I).

Fig. 3 Biplot of the RDA on the 4 genospecies of rhizobia and their soil factors from soil samples in different fertilizer by

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CANOCO. AP, available phosphorus; AK, available potassium; OC, organic carbon. CK, unfertilized control; ROM, recycled organic manure; N, nitrogen fertilizer; N+ROM, application of N fertilizers plus recycled organic manure; NP, nitrogen and phosphate fertilizer; NP+ROM, application of NP fertilizers plus recycled organic manure; NPK, nitrogen, phosphorus and

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potassium; NPK+ROM, application of NPK fertilizers plus recycled organic manure. All treatments were laid out in 1985 and are still being continued. The longer the arrow is, the greater the influence it has; the smaller the angle is between two arrows, the closer relationship they have.

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DISCUSSION

In the present study, the highest grain and biomass production, the highest contents of OC, AN, AP, and AK in soil, the highest rhizobial diversity (H´) and stable pH values, especially the significant increase in grain production, OC, AN and H´ values in the NPK+ROM treatment in comparison with all the other treatments (Table II) demonstrated that the fertilization strategy of NPK+ROM was an excellent management for maintaining the high soybean production and soil fertility in the Chinese Mollisol (Wang et al., 2015). During the 29 years of experiment, the NPK+ROM treatment increased 31.6% of the OC content comparing with the original soil in 1985, or 2.6-16.1% comparing with the other treatments in 2013. Since the high OC content is an essential characteristic for the Mollisol, the NPK+ROM fertilization strategy presents a great value for recovering and improving the Chinese Mollisol from degradation. The H´ value of 1.008 and OC content of 35.4 g kg-1 in the NPK+ROM treatment were rather similar to the remediation effects by recovered grass land (H´=1.325, OC = 35.5 g kg-1) in the same field (Yan et al., 2014). These results confirmed the previous observations (Han et al., 2006) that allocation of organic manure could recover the fertility of Mollisol and have instructive importance for the agricultural and ecological management to maintain the Chinese Mollisol ecosystem. In addition, the correlation among the high rhizobial diversity, high productivity

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and the fertilization of NPK+ROM (Table II) indicated the possibility to use rhizobial diversity as an indicator of productivity for the Chinese Mollisol. The results of the present study indicated that the long term fertilization strategy has direct effects on the soil features, such as pH and nutrient levels (Table II), which in turn affected the soil microbial communities represented by rhizobia (Fig. 3). The greater rhizobial diversity in ROM-involved treatments might be explained by that the high OC content (> 31.4 g kg-1) of ROM involved treatments acted as a source of C and provided a favorable environment (reduced the impression of N fertilizer and stable/slight acid pH values) for the bradyrhizobia (O'Hara, 2001; Zengeni et al., 2006; Grossman et al., 2011). Similar to the finding from other long term experiments (Pernes-Debuyser and Tessier, 2004; He et al., 2007), a significant decrease in soil pH was observed in the treatments N，NP, and NPK after 29 years of fertilizer applications in the present study. Generally, acidity does not only affect the growth, activity and persistence of rhizobia, but also has a major impact on their diversity (Fig. 3) (Yang et al., 2001; Andrade et al., 2002; Giongo et al., 2008). The positive correlation among the soil OC and AP contents, pH values and the distribution of soybean rhizobia in our study were similar to previous observations (Giongo et al., 2008; Zhang et al., 2011). In this study, the dominance of Bradyrhizobium sp. I and the definition of B. japonicum, B. diazoefficiens and B. ottawaense as the minor groups revealed a unique community structure of soybean rhizobia in the black soil (Table II and Fig. 1). The presence of B. diazoefficiens and absence of a sister species of B. japonicum differed this community structure from that previous reported in the same field with soil management and cropping system distinct from those involved in the present study (Yan et al., 2014). The absence of B. elkanii, B. liaoningense and Ensifer fredii differed the community in Mollisol from those in other soil types, such as the acid soils (Man et al., 2008) and the alkaline-saline soils (Han et al., 2009; Zhang et al., 2011) in other regions of China. These differences could been related to the soil pH, salinity and AP contents (Han et al., 2009; Zhang et al., 2011; Li et al., 2012). Despite the differences detected in the community structure of rhizobia among the different soil types, variations were also detected among the different treatments in the present study (Table II), which demonstrated the effects of long-term fertilization strategy on diversity of rhizobia. In this case, the variation in soil pH, OC and AP caused by the fertilization were found to be the most important determinants for the soybean rhizobial communities (Fig. 3) in the tested soil, which were similar to the previous observations (Giongo et al., 2008; Zhang et al., 2011). A new finding in the present study was the effects of ROM application on B. japonicum populations. B. japonicum was absent in the treatments N, NP and NPK, but presented in CK and the ROM-involved treatments, demonstrating the sensitivity of this species to the nitrogen fertilizer and the elimination of this impression by application of organic manure. Meanwhile, the abundance of Bradyrhizobium sp. I in all the treatments evidenced that it is a genospecies resistant to the presence of N fertilizer. The application of N fertilizer significantly decreased the rhizobial diversity compared with the CK treatment (Table II), which is similar to the previous reports of Caballero-Mellado and Martinez-Romero (1999), Palmer and Young (2000) and Herrmann et al. (2014); but different from those of Lindstrom et al. (2010), Bizarro et al. (2011) and Zahran (1999) who described the positive effect or no effect of N-fertilizer on rhizobial diversity. In addition, Bizarro et al. (2011) stated that soil under mineral fertilization had higher bradyrhizobial diversity when compared to that in soil under organic fertilization. These discrepancies may be explained by the differences in climatic factors, soil types, fertility (including OC content) and soil textures in these studies (Abaidoo et al., 2007; Herrmann et al., 2014). This estimation was confirmed by the fact that both the lowest rhizobial diversity index and the lowest soil pH were found in the N treatment; meanwhile pH was the strongest determinant for B. japonicum and B. diazoefficiens (Fig. 3), and even a small increase in pH (6.21 to 6.37) could eliminate B. diazoefficiens from the nodulation (Table I). Furthermore, the higher rhizobial diversity was companied by higher OC, pH, and AP contents in the ROM received treatments comparing with the treatments without ROM. Generally, the phylogenetic relationships of the symbiotic genes could be used to estimate the host range of the rhizobia (Laguerre et al., 2001), and comparison with the phylogentic relationships of housekeeping genes could give some idea for the evolution and origin of symbiotic genes (Man et al., 2008). Similar to the results in some previous studies, the nodC and nifH gene phylogenies revealed the possible lateral transfer of symbiotic genes

ACCEPTED MANUSCRIPT among the Bradyrhizobium genospecies since the strains in Bradyrhizobium sp. I, B. ottawaense and B. japonicum etc. shared very similar nodC and nifH genes (Fig. 2). In addition, the distinct relationships of strain B. diazoefficiens NPK+ROM8 in the phylogenetic trees of nodC and nifH evidenced a novel symbiotic gene lineage in the soybean nodulating rhizobia. The difference of this strain from all the other soybean nodulation rhizobia implied that the symbiotic genes in this strain might come from a distinct origin. This distinct lineage further revealed the promiscuous property of soybean in nodulation with diverse rhizobial species harboring different types of symbiotic genes. CONCLUSIONS

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Twenty-nine years application of ROM-combined with chemical fertilizer maintained or increased the fertility of the Chinese Mollisol, which is coupled with the high grain/biomass production and high diversity of soybean rhizobia. The fertilization strategies regulated the rhizobial diversity and community structure by their effects on soil pH and OC content. Novel Bradyrhizobium genospecies and novel symbiotic genotypes were maintained in the tested Mollisol. The sensitive feature of B. japonicum to N-fertilizer and of B. diazoefficiens to soil acidification, as well as the reducing of N-impression on the diversity of Bradyrhizobium by organic manure were recorded as new findings. ACKNOWLEDGEMENTS

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This work was supported by the National Natural Science Foundation of China (No. 41371296, 41671299, 31370108). ETW was supported by the projects SIP 20140124 and 20150597 authorized by Instituto Politecnico Nacional, Mexico. WFC was supported by the “Twelfth Five-Year” National Science and Technology Project in Rural Areas (No. 2011BAD11B03-03).

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ACCEPTED MANUSCRIPT B. japonicum USDA 6T (NC_017249)

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B. japonicum NPK+ROM11 (KT965777, KT965765, KT965801, KT965771, KT965783, KT965807)

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B. japonicum CK3 (KT965776, KT965764, KT965800, KT965770, KT965782, KT965806) Bradyrhizobium sp I N10 (KT965775, KT965763, KT965799, KT965769, KT965781, KT965805)

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B. betae LMG 21987T (AB353733, FM253129, AB353734, FM253303, AB353735, FM253260) B. canariense BTA-1T (AY386765, AY386739, AY591553, AY923047, FM253220, FM253263) 74 B.cytisi CTAW11T (GU001594, GU001613, GU001575, GU001575, JN186292, JN186288) 73 B. rifense CTAW71T (GU001604, GU001617, GU001585, JQ945187, KC569466, KC569468)

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B. diazoefficiens USDA 110T (NC_004463) B. diazoefficiens NPK+ROM8 (KT965778, KT965766, KT965802, KT965772, KT965784, KT965808)

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B. ottawaense ROM2 (KT965780, KT965768, KT965804, KT965774, KT965786, KT965810) 59

B. ottawaense N+ROM18 (KT965779, KT965767, KT965803, KT965773, KT965785, KT965809)

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B. huanghuaihaiense CCBAU 23303T

(HQ231639, HQ231682, HQ231595, JX437665, JX437672, JX437679)

B. arachidis CCBAU 051107T (HM107251, HM107217, HM107233, JX437668, JX437675, JX437682 ) 62

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B. ottawaense LMG26739T (HQ587750, HQ455212, HQ587287, JF308816, HQ873179, HQ587518 )

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B. yuanmingense CCBAU 10071T (AY386780, AY386760, AY591566, FM253312, FM253226, FM253269)

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B. daqingense CCBAU 15774T (HQ231301, HQ231289, HQ231270, JX437662, JX437669, JX437676) 58

B. liaoningense USDA 3622T (AY494803, AY493450, AY494833, FM253309, FM253223, EF190181)

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B. iriomotense LMG 24129T (AB300995, AB300994, AB300996, JF308944, HQ873308, HQ587646) B. jicamae PAC 68T (FJ428204, FJ428211, HM047133, JF308945, HQ873309, HQ587647)

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B. lablabi CCBAU 23086T (GU433498, GU433473, GU433522, JX437663, JX437670, JX437677)

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B. pachyrhizi PAC 48T (FJ428201, FJ428208, FJ428208, JF308946, HQ873310, HQ587648)

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B. elkanii USDA 76T (AY599117, AY386758, AY591568, AY328392, AB070584, EF190188))

Fig 1

ACCEPTED MANUSCRIPT B. ottawaense N+ROM18 (KT965797; KT965791) B. ottawaense ROM2 (KT965798; KT965792) B. ottawaense LMG26739T (HQ587980, JN186287) B. japonicum NPK+ROM11 (KT965795; KT965789) B. japonicum CK3 (KT965794; KT965788) B. huanghuaihaiense CCBAU 23303T (HQ231507, HQ231551) 56 B.japonicum USDA 6T (NC_017249) 100

B. diazoefficiens USDA 110T (NC_004463) B. daqingense CCBAU 15768T (HQ231326, HQ664976)

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Bradyrhizobium sp I N10 (KT965793; KT965787) 98 B. diazoefficiens NPK+ROM8 (KT965796; KT965790) 81

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B. arachidis CCBAU 051107T (HM107267, HM107283) B. pachyrhizi LMG 24246T (HM047128, HM047124) 74

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B. elkannii USDA 76 (AB354631, AB079619)

B. liaoningense LMG 18230T (GU263466, EU818925)

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B. jicamae LMG 24556T (HM047129, HM047127)

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B. lablabi CCBAU 23086T (GU433565, GU433546)

B. canariense BTA-1T (AJ560653, EU818926)

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B. cytisi CTAW11T (EU597844, GU001618)

100

B. rifense CTAW71T (EU597853, GU001627)

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B. yuanmingense CCBAU 10071T (AB354633, EU818927) 94