www.nture.com/scientificreports Received: 19 December 2017 Accepted: 6 July 2018 Published: xx xx xxxx OPEN Yield effect of pplying erthworm cstings produced during the oilseed rpe-growing seson in riceoilseed rpe cropping fields to rice Min Hung 1,2, Chunrong Zho 1, Yingbin Zou 1 & Normn Uphoff 2 In-field erthworm density cn be incresed by plnting oilseed rpe during the non-rice growing seson s compred to mintining the rice-growing fields in fllow. This study ws conducted to determine the effect on rice yield of erthworm cstings produced during the oilseed rpe-growing seson in riceoilseed rpe cropping fields nd to identify the criticl fctors tht contribute to the yield effect. Field microplot experiments were conducted in 2016 nd 2017. In 2016, rice cultivr ws grown under fctoril combintion of bsence (EC 0 : 0 kg m 2 ) nd presence of erthworm cstings (EC 1 : 17 kg m 2 ) with three N ppliction rtes (9, 12 nd 15 g m 2 ). In 2017, nine rice cultivrs were grown under EC 0 nd EC 1 with the moderte N rte s ws used in 2016. Results showed tht ppliction of erthworm cstings produced during the oilseed rpe-growing seson in rice-oilseed rpe cropping fields hd significnt positive yield effect on rice. This ws ttributed to incresed pnicle m 2 nd totl boveground biomss while spikelets pnicle 1, spikelet filling percentge, grin weight, nd hrvest index were not ffected. Our study indirectly provides new evidence tht oilseed rpe is n excellent previous crop for cerels. Rice is mjor stple food for lmost 50% of the world s popultion, nd rice fields ccount for more thn 12% of the globl croplnd re 1. Of these rice fields, nerly 90% re locted in Asi (World Rice Sttistics dtbse, vilble t http://ricestt.irri.org:8080/wrs, hereinfter referred to s WRS dtbse). In order to produce enough food for rpidly growing popultions, griculturl lnd use in Asi hs become very intense 2. In the pst 50 yers, the intensifiction of rice-bsed cropping systems hs helped ensure production of sufficient rice nd other food crops 3. However, the continuous intensive prctice of rice-bsed cropping systems such s rice-whet cropping system prcticed for severl decdes hs led to declines in productivity nd rised concerns bout sustinbility 4. Chin is one of the mjor rice-producing countries in the world with n re of bout 30 million hectres, ccounting for pproximtely 20% of the world s rice field re (WRS dtbse). Rice-bsed cropping systems re diverse in Chin due to differences in gro-climtic zones 5. Among those cropping systems, rice-whet nd rice-oilseed rpe re two long-estblished mjor ones 6. In contrst to the concerns bout sustinbility of continuous rice-whet cropping system s mentioned bove, our study suggests tht long-term rice-oilseed rpe cropping system cn increse soil fertility nd consequently reduce the dependence on externl nitrogen (N) inputs nd dverse impcts on the environment 7. This supports the viewpoint tht oilseed rpe is n excellent nd sustinble previous crop for cerels 8 10. Such viewpoint lso cn be supported by our recent investigtions which showed tht in-field erthworm density during the non-rice growing seson ws doubled by plnting oilseed rpe s compred to fllow in long-term, no-tillge rice-bsed fields 11. Although the erthworms re likely to migrte wy from fields during the erly stge of rice-growing when rice fields re flooded (Fig. 1A), their cstings will remin in the fields (Fig. 1B). These fcts indicte tht growing oilseed rpe s previous crop for rice cn enhnce erthworm ctivity nd hence my provide similr benefits s the option of pplying the erthworm cstings s fertilizer, especilly in regions tht re kept on fllow during the non-rice growing seson. This roused our interest to investigte 1 Southern Regionl Collbortive Innovtion Center for Grin nd Oil Crops (CICGO), Hunn Agriculturl University, Chngsh, 410128, Chin. 2 Interntionl Progrms-College of Agriculture nd Life Sciences (IP-CALS), Cornell University, Ithc, 14853, USA. Min Hung nd Chunrong Zho contributed eqully to this work. Correspondence nd requests for mterils should be ddressed to M.H. (emil: mhung@hunu.edu.cn) 1
www.nture.com/scientificreports/ Figure 1. Erthworms migrted wy from the rice field fter flooding (A), erthworm cstings produced in the oilseed rpe-growing seson (B), nd erthworm cstings collected fter hrvesting the oilseed rpe (C). These photos were tken from rice-oilseed rpe cropping fields locted in Nnxin, Hunn Province, Chin in 2016. the effect of the erthworm cstings produced during the oilseed rpe-growing seson in rice-oilseed rpe cropping fields on grin yield in rice. As consequence, we conducted pot experiment nd found tht grin yield ws incresed in rice cultivr with ppliction of the erthworm cstings collected from oilseed rpe fields 12. However, this finding could be subject to the constrints of pot cultivtion nd cultivr specificity. Therefore, more experimenttion should be done under field conditions with vrious rice cultivrs to obtin more concrete results. 2
www.nture.com/scientificreports/ Erthworm csting tretment N rte/cultivr EC 0 EC 1 Men 2016 N 1 846 (110) 1165 (116) 1006 N 2 884 (18) 1178 (73) 1031 N 3 932 (64) 1160 (41) 1046 Men 887 1168 Anlysis of vrince Erthworm csting tretment (EC) ** N rte (N) ns EC N ns 2017 Guihefeng 752 (63) 1002 (55) 877c Hunghuzhn 933 (54) 1113 (82) 1023b Lingyoupeijiu 967 (78) 1176 (102) 1072b Longlingyou 97 1157 (59) 1289 (84) 1223 Shenlingyou 5814 1028 (28) 1315 (43) 1172 Xinglingyou 396 1084 (43) 1306 (97) 1195 Y-lingyou 1 1035 (39) 1323 (74) 1179 Y-lingyou 2 1142 (119) 1293 (21) 1218 Zhunlingyou 608 1159 (42) 1273 (61) 1216 Men 1029 1232 Anlysis of vrince EC ** Cultivr (C) ** EC C ns Tble 1. Grin yields (g m 2 ) from rice cultivr Lingyoupeijiu grown under bsence (EC 0 : 0 kg m 2 ) nd presence of erthworm cstings (EC 1 : 17 kg m 2 ) with three N rtes (N 1 : 9 g m 2 ; N 2 : 12 g m 2 ; nd N 3 : 15 g m 2 ) in 2016, nd from nine rice cultivrs grown under EC 0 nd EC 1 with one N rte (N 2 ) in 2017. Vlues in prenthesis re the stndrd devitions. Mens of cultivrs with the sme letters re not significntly different t the 0.05 probbility level (LSD test). ** represents significnce t the 0.01 probbility level. ns denotes nonsignificnce t the 0.05 probbility level. Rice yield is determined by sink size (spikelets per unit lnd re), spikelet filling percentge, nd grin weight. Sink size is considered s the primry determinnt of the rice yield, nd it cn be incresed either by incresing the number of pnicles or pnicle size (spikelets per pnicle), or both 13. Becuse strong compenstion mechnism exists between the two yield components, concurrent increse in them is not esy to chieve 14,15. Incresing pnicle size is common pproch for the rice breeders to enhnce the sink size nd consequently to improve rice yield potentil 16. However, there hs contrdictory sttement tht more pnicle number should be emphsized to chieve higher grin yield in the super hybrid rice with lrge pnicle size 17. In nother pproch, grin yield of rice is function of totl boveground biomss nd hrvest index. It is generlly ccepted tht chieving greter rice yields depends on incresing totl boveground biomss, becuse there is little scope to chieve further increses in the hrvest index under fvorble conditions 18 20. The hrvest index of modern high-yielding rice is round 50% 21. However, in recent yers there hve been reports tht high grin yield cn chieved in rice with high hrvest index 22,23. In our present study, grin yield nd yield ttributes in rice were compred between with nd without pplictions of erthworm cstings tht were produced during the oilseed rpe-growing seson in rice-oilseed rpe cropping fields in two-yer field microplot experiments. Our objectives were to (1) determine the effect on rice yield of erthworm cstings produced during the oilseed rpe-growing seson in rice-oilseed rpe cropping fields, nd (2) identify the criticl fctors tht contributed to this yield effect. Results In 2016, grin yield ws significntly ffected by erthworm csting tretment, but not by N rte nd the interction between erthworm csting tretment nd N rte (Tble 1). In 2017, erthworm csting tretment nd cultivr hd significnt effect on grin yield, while the interction effect on grin yield between erthworm csting tretment nd cultivr ws not significnt. Therefore, only the mens of erthworm csting tretments in 2016 nd the mens of erthworm csting tretments nd cultivrs in 2017 were presented in subsequent tbles for evluting effect on yield ttributes, mking interprettion esier. EC 1 hd, respectively, 32% nd 20% higher grin yield thn EC 0 in 2016 nd 2017 (Tble 1). In 2017, Longlingyou 97 produced the highest grin yield, lthough this ws not significntly higher thn from Shenlingyou 5814, Xinglingyou 396, Y-lingyou 1, Y-lingyou 2 nd Zhunlingyou 608, but it ws 14 20% higher thn the yields from Lingyoupeijiu nd Hunghuzhn, nd 39% higher thn tht from Guihefeng. 3
www.nture.com/scientificreports/ Erthworm csting Spikelets tretment/cultivr Pnicles m 2 pnicle 1 2016 Spikelets m 2 ( 10 3 ) Spikelet filling (%) Grin weight (mg) EC 0 192b 251 48.2b 70.1 22.8 EC 1 233 262 61.0 71.4 23.1 2017 EC 0 247b 219 53.1b 79.8 21.3 EC 1 280 222 61.5 80.7 21.8 Guihefeng 198e 293 57.8b 79.7bc 16.6e Hunghuzhn 296b 203c 60.2b 82.2b 17.9e Lingyoupeijiu 269bc 208c 55.9b 71.2d 23.4b Longlingyou 97 298 207c 61.6b 77.2c 22.7bc Shenlingyou 5814 263cd 212c 55.3bc 87.7 21.0cd Xinglingyou 396 292b 201c 58.8b 79.2bc 22.6bc Y-lingyou 1 280bc 195c 54.6bc 82.4b 22.5bc Y-lingyou 2 239d 267b 63.8 79.8bc 20.7d Zhunlingyou 608 239d 200c 47.6c 83.1b 26.5 Tble 2. Yield components in rice s ffected by erthworm cstings in 2016, nd by erthworm cstings nd cultivrs in 2017. Within column for erthworm csting tretments in ech yer nd for cultivrs in 2017, dt with the sme letters re not significntly different t the 0.05 probbility level (LSD test). EC 0 nd EC 1 represent bsence (0 kg m 2 ) nd presence of erthworm cstings (17 kg m 2 ), respectively. Dt re the mens cross three N rtes in 2016. Pnicles m 2 under EC 1 were 21% nd 13% higher thn those under EC 0 in 2016 nd 2017, respectively (Tble 2). There ws no significnt difference in spikelets pnicle 1 between EC 1 nd EC 0 in either 2016 nd 2017. EC 1 hd higher spikelets m 2 thn EC 0, by 27% in 2016 nd by 16% in 2017. The differences in spikelet filling percentge nd in grin weight were insignificnt between EC 1 nd EC 0 in both yers. In 2017, Longlingyou 97 hd the highest pnicles m 2, nd similr vlues were observed in Hunghuzhn, Xinglingyou 396 nd Y-lingyou 1 (Tble 2). The lowest pnicles m 2 ws recorded in Guihefeng. This vriety lso hd the highest spikelets pnicle 1, followed by Y-lingyou 2 nd the other cultivrs, which were not significntly different from one nother. Y-lingyou 2 hd the highest spikelets m 2, but this ws not significntly different from the spikelets m 2 for Guihefeng, Hunghuzhn, Longlingyou 97 nd Xinglingyou 396, wheres Zhunlingyou 608 hd the lowest spikelets m 2. Spikelet filling percentge ws highest in Shenlingyou 5814 nd lowest in Lingyoupeijiu. Zhunlingyou 608 hd the highest grin weight, while Guihefeng nd Hunghuzhn hd the lowest grin weight. EC 1 produced 27% higher totl boveground biomss thn EC 0 in 2016 nd 19% more in 2017 (Tble 3). There ws no significnt in hrvest index between EC 1 nd EC 0 in either yer. In 2017, Y-lingyou 2 produced the highest totl boveground biomss, but this ws not significntly different from the biomss in Longlingyou 97, Shenlingyou 5814, Y-lingyou 1, nd Zhunlingyou 608, while Guihefeng hd the lowest totl boveground biomss. Xinglingyou 396 hd the highest hrvest index, but similr vlues were recorded in Hunghuzhn, Longlingyou 97, Y-lingyou 1 nd Zhunlingyou 608. The lowest hrvest index ws observed in Guihefeng. Non-fertilizer N uptke ws 32% higher under EC 1 thn EC 0 (Fig. 2A). There ws no significnt difference in non-fertilizer N uptke mong N 1, N 2 nd N 3 (Fig. 2B). Fertilizer N uptke ws not significntly different between EC 1 nd EC 0 (Fig. 2C), while it ws slightly but significntly incresed with n incresing rte of N (Fig. 2D). EC 1 hd 27% higher totl uptke of N thn did EC 0 (Fig. 2E). The difference in totl N uptke ws, however, insignificnt mong the three N rtes (Fig. 2F). Discussion Our results showed tht ppliction of erthworm cstings produced during the oilseed rpe-growing seson in the rice-oilseed rpe fields hd significnt positive yield effect on rice. This finding is in greement with our observtion in pot experiment 12. These works support the previously-expressed viewpoints tht oilseed rpe is n excellent previous crop for cerels 8 10 nd tht erthworm presence in groecosystems cn led to increse in crop yield 24. Anlysis of yield components indicted tht the yield increse effect of the ppliction of erthworm cstings ws minly ttributed to n enhncement of sink size tht resulted from incresed pnicle number. This finding is not in greement with the predominnt stndpoint mong rice breeders tht incresing pnicle size is the most promising pproch to enhncing sink size nd consequently improving rice yield potentil 16. More interestingly, the incresed pnicle number induced by the ppliction of erthworm cstings did not cuse significnt decrese in pnicle size in this study. This result is inconsistent with previous studies, such s Ying et l. 14 nd Hung et l. 15, which reported tht there ws compenstion between the two yield components. Moreover, in the present study, the enhnced sink size ws chieved not t the expense of spikelet filling percentge nd grin weight in rice tht hs been grown with erthworm cstings pplied. These results demonstrted tht comptible reltionships mong yield components were estblished in rice with the ppliction of erthworm cstings. In this regrd, it is suggested tht incresing biomss production is fesible wy to decouple the 4
www.nture.com/scientificreports/ Erthworm csting tretment/cultivr 2016 Totl boveground biomss (g m 2 ) Hrvest index (%) EC 0 1669b 45.9 EC 1 2097 47.9 2017 EC 0 1656b 53.4 EC 1 1970 53.7 Guihefeng 1478e 51.1e Hunghuzhn 1641d 53.6bc Lingyoupeijiu 1799c 51.3de Longlingyou 97 1897b 55.6b Shenlingyou 5814 1889bc 53.4bcd Xinglingyou 396 1844bc 55.7 Y-lingyou 1 1879bc 53.9bc Y-lingyou 2 1972 53.0cde Zhunlingyou 608 1920b 54.5bc Tble 3. Totl boveground biomss nd hrvest index in rice s ffected by erthworm cstings in 2016, nd by erthworm cstings nd cultivrs in 2017. Within column for erthworm csting tretments in ech yer nd for cultivrs in 2017, dt with the sme letters re not significntly different t the 0.05 probbility level (LSD test). EC 0 nd EC 1 represent bsence (0 kg m 2 ) nd presence of erthworm cstings (17 kg m 2 ), respectively. Dt re the mens cross three N rtes in 2016. compenstions mong yield components in cerels including rice 25,26. In this study, higher totl boveground biomss ws chieved in rice pplied with erthworm cstings, which could be responsible for the comptible reltionships mong yield components in rice with the ppliction of erthworm cstings. On the other hnd, the results of totl boveground biomss nd hrvest index reveled tht there ws positive yield effect from the ppliction of erthworm cstings which ws minly driven by incresed totl boveground biomss rther thn hrvest index. This is not surprising becuse it hs been well-documented tht there is little scope to further increse in hrvest index under fvorble conditions 18 20. However, perhps interestingly, the ppliction of erthworm significntly did not decrese nd even slightly incresed hrvest index. This is different from the effect of ppliction of chemicl fertilizer N on rice, in which hrvest index is generlly decresed 27. Hrvest index is determined by the remobiliztion of stored reserves into the growing grin nd the trnsient photosynthesis during grin formtion 28. An increse in the former is usully chieved ccompnied with erly senescence nd shortened grin-filling durtion, which cn be induced by unfvorble conditions such s wter stress 29. However, this must not be the cse in the present study, becuse we observed tht the ppliction of erthworm cstings mde the rice leves greener during the grin-filling period. Therefore, the slightly incresed hrvest index might be relted to the trnsient photosynthesis during grin formtion in rice pplied with erthworm cstings in this study. This highlights tht further investigtions re needed to determine the effect of ppliction of erthworm cstings on photosynthetic chrcteristics during the ripening period in rice. Decresing N rte from 150 to 90 kg h 1 did not result in significnt yield reduction in the hybrid rice cultivr Lingyoupeijiu in 2016. Hung et l. 30 determined N response of this cultivr over wide rnge of N rtes (60 410 kg h 1 ). Their results showed tht Lingyoupeijiu required minimum totl N rte of 120 150 kg h 1 to produce mximum grin yield. These suggest tht the hybrid rice Lingyoupeijiu does not necessrily need more N fertilizer to produce high grin yield. Consistent with this, Hung et l. 31 hve observed tht higher grin yield in hybrid rice is minly driven from higher grin yield without N fertilizer rther thn increses in grin yield with N fertilizer. This suggests tht greter ppliction of N fertilizer is not needed to benefit from hybrid production nd tht improving nd mintining soil fertility should be the focus for sustining hybrid rice production. This cn be further supported by this study s results in 2016 tht the totl N uptke minly depended on non-fertilizer N uptke in the hybrid rice cultivr Lingyoupeijiu. The non-fertilizer nd fertilizer N uptke ccounted for 87% nd 13% of the totl N uptke, respectively (Fig. 2). In ddition, the positive effect of ppliction of erthworm cstings on pnicle number, totl boveground biomss, nd grin yield in 2016 lso could be explined by n increse in non-fertilizer N uptke. The incresed non-fertilizer N uptke under ppliction of erthworm cstings ws prtilly due to tht the erthworm cstings contined certin mount of vilble N. Significnt cultivr differences in grin yield were detected in 2017. Inbred cultivrs generlly produced lower grin yields thn did hybrid cultivrs. The lower grin yields of inbred cultivrs were minly ttributed to lower grin weight nd to lower totl boveground biomss. When comprison ws mde mong the hybrid cultivrs, the lowest grin yield ws recorded in Lingyoupeijiu, which ws relesed in 1999 (Tble 1). The yield difference ws smll mong the other six hybrid cultivrs, which were relesed during 2008 to 2016 (Tble 1). It seems tht the breeding effort did not contribute much to incresed rice yield in the pst decde. Also interestingly, the six high-yielding hybrid cultivrs could be divided into four groups ccording to their yield component performnce: (1) Longlingyou 97, Xinglingyou 396 nd Y-lingyou 1 re chrcterized by more pnicle number, (2) Y-lingyou 2 is distinguished for its lrge pnicle size, (3) Shenlingyou 5814 hs higher spikelet filling 5
www.nture.com/scientificreports/ 25 20 15 A b B 10 5 0 N uptke (g m 2 ) 25 20 15 10 5 C D c b 0 25 20 15 E b F 10 5 0 EC 0 EC 1 Tretment N 1 N 2 N 3 Figure 2. Uptke of non-fertilizer N (A,B), fertilizer N (C,D), nd totl N (E,F) in boveground biomss in rice cultivr Lingyoupeijiu s ffected by erthworm cstings (EC 0 : 0 g m 2 ; EC 1 : 17 g m 2 ) nd N rtes (N 1 : 9 g m 2 ; N 2 : 12 g m 2 ; N 3 : 15 g m 2 ) in 2016. The interctive effect between erthworm csting tretment nd N rte on these prmeters were not significnt. Error brs represent SE (n = 9 for ech erthworm csting tretment, nd 6 for ech N rte). Within ech grph, columns with the sme letters re not significntly different t the 0.05 probbility level (LSD test). percentge, nd (4) Zhunlingyou 608 is notble for its lrge grin size. This suggests tht there re multiple strtegies or pthwys for developing high-yielding hybrid rice cultivrs. Conclusions Appliction of erthworm cstings produced during the oilseed rpe-growing seson in rice-oilseed rpe cropping fields hd significnt positive yield effect on rice. This ws ttributed to incresed pnicle m 2 nd totl boveground biomss while spikelets pnicle 1, spikelet filling percentge, grin weight, nd hrvest index were not ffected. Methods Field microplot experiments were conducted in rice field t the reserch frm of Hunn Agriculturl University (28 11 N, 113 04 E) in Chngsh, Hunn Province, Chin in 2016 nd 2017. The soil of the rice field ws tidl cly (Fluvisol, FAO txonomy) with the following properties: ph = 5.75, orgnic mtter = 34.2 g kg 1, vilble N = 81.6 mg kg 1, vilble P = 34.4 mg kg 1, nd vilble K = 56.7 mg kg 1. The soil test ws bsed on smples tken from the 0 20 cm lyer before the experiment ws begun in 2016. 6
www.nture.com/scientificreports/ In 2016, eighteen microplots were constructed by inserting bottomless PVC boxes (40 cm long 40 cm wide 30 cm deep) into the soil to depth of 20 cm with collr of 10 cm boveground. A hybrid rice cultivr Lingyoupeijiu ws grown fctoril combintion of bsence (EC 0 : 0 kg m 2 ) nd presence of erthworm cstings (EC 1 : 17 kg m 2 ) with three N ppliction rtes (N 1 : 9 g m 2 ; N 2 : 12 g m 2 ; nd N 3 : 15 g m 2 ). The erthworm csting mount of EC 1 ws bsed on n estimte obtined by multiplying dily production rte of erthworm cstings (78 g m 2 d 1 ) by the durtion of n oilseed rpe-growing seson (218 d). The dily production rte of erthworm cstings ws obtined on the first dy fter hrvesting the oilseed rpe from 10 rndomly selected 1-m 2 plots in rice-oilseed rpe cropping field locted in Nnxin (29 21 N, 112 25 E), Hunn Province, Chin in 2015. The N rtes were chosen ccording to the locl recommended N rte (150 kg h 1 ) for rice production in the study region. The tretments were rrnged in completely rndomized block design with three replictions. In 2017, seventy-two microplots were constructed using the sme procedures s described bove. Nine rice cultivrs, including two inbred cultivrs (Guihefeng nd Hunghuzhn) nd seven hybrid cultivrs (Lingyoupeijiu, Longlingyou 97, Shenlingyou 5814, Xinglingyou 396, Y-lingyou 1, Y-lingyou 2 nd Zhunlingyou 608), were grown under EC 0 nd EC 1 with N 2. The N rte ws chosen ccording to the results in 2016, when grin yield ws not significntly different mong the three N rtes (Tble 1). The tretments were lid out in split-plot design with erthworm csting tretments s the min plots nd cultivrs s subplots. The experiment ws replicted four times. All the cultivrs used in this study re ones tht hve been widely grown by rice frmers in southern Chin. The erthworm cstings used in the experiment were collected from rice-oilseed rpe cropping fields locted in Nnxin fter hrvesting the oilseed rpe in 2016 (Fig. 1C). The site hs moist subtropicl monsoon climte with n nnul verge temperture of 16.6 C, n nnul verge rinfll of 1238 mm, nd n nnul verge sunshine durtion of 1776 h. The soil in the fields is purple clcreous cly (Fluvisol, FAO txonomy). The dominnt erthworm species in the field is Pheretim guillelmi. The erthworm cstings hd the following properties: ph = 7.89, orgnic mtter = 61.4 g kg 1, vilble N = 128 mg kg 1, vilble P = 44.2 mg kg 1, nd vilble K = 254 mg kg 1. The N fertilizer used in 2016 ws 15 N-lbeled ure (5.18% isotopic bundnce, provided by Shnghi Institute of Chemicl Industry, Chin), nd unlbeled ure in 2017. Pre-geminted seeds were sown on seedbed on 10 My. Seedlings were trnsplnted on 5 June. Trnsplnting ws done with four hills per microplot nd one seedling per hill. Erthworm cstings were pplied t 1 dy before trnsplnting. N fertilizer ws split-pplied with 50% s bsl (1 dy before trnsplnting), 30% t erly tillering (7 dys fter trnsplnting), nd 20% t pnicle initition. Superphosphte (4.8 g P 2 O 5 m 2 ) ws pplied s bsl fertilizer. Potssium chloride (8.4 g K 2 O m 2 ) ws split eqully t bsl nd pnicle initition. A floodwter depth of bout 5 cm ws mintined in the microplots until 7 dys before mturity, when the microplots were drined. Insects, disese, nd weeds were controlled by using pproved pesticides to void yield loss. Plnts were smpled for ech microplot t mturity in both yers. Pnicle number ws counted in ech hill to determine pnicles m 2. Plnts were seprted into strw (including rchis) nd spikelets by hnd threshing. Filled spikelets were seprted from unfilled spikelets by submerging them in tp wter. Dry weights of strw nd filled nd unfilled spikelets were determined fter over-drying t 70 C to constnt weight. Three subsmples of 30 g of spikelets nd ll unfilled spikelets were tken to count the number of spikelets. Totl boveground biomss ws the totl dry mtter of strw nd of filled nd unfilled spikelets. Spikelets pnicle 1, spikelets m 2 (pnicles m 2 spikelets pnicle 1 ), spikelet filling percentge (100 filled spikelet number/totl spikelet number), grin weight, nd hrvest index (100 filled spikelet weight/totl boveground biomss) were clculted. Grin yield ws djusted to moisture content of 0.14 g H 2 O g 1. In 2016, the dried plnt smples were ground into fine powder for determining their N content (VAP50 Kjeldhl meter, Gerhrdt, Königswinter, Germny) nd 15 N bundnce (Delt V Advntge isotope mss spectrometer, Thermo Fisher, Wlthm, MA, USA). Totl N uptke, nd uptke of fertilizer nd non-fertilizer N in boveground biomss were clculted ccording to Hung et l. 32. Dt were nlyzed by nlysis of vrince with the use of Sttistix 8.0 softwre (Tllhssee, FL, USA). In 2016, the sttisticl model included repliction, erthworm csting tretment, N rte, nd the interction between erthworm csting tretment nd N rte. In 2017, the sttisticl model included repliction, erthworm csting tretment, cultivr, nd the interction between erthworm csting tretment nd cultivr. Mens were compred bsed on the lest significnt difference test (LSD). The 0.05 probbility level ws used to test for sttisticl significnce. Dt vilbility. All dt generted or nlysed during this study re included in the rticle. References 1. Zhng, G. et l. Mpping pddy rice plnting res through time series nlysis of MODIS lnd surfce temperture nd vegettion index dt. ISPRS J. Photogrm. Remote Sens. 106, 157 171 (2015). 2. Bronson, K. F. et l. Soil crbon dynmics in different cropping systems in principl ecoregions of Asi in Mngement of crbon sequestrtion in soil (eds Ll, R., Kimble, J. M., Follett, R. F. & Stewrt, B. A.) 35 57 (CRC Press, 1998). 3. Buresh, R. J., Lrzo, W. M., Lureles, E. V., Smson, M. I. & Pmpolino, M. F. Sustinble soil mngement in lowlnd rice ecosystems. 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Field Crops Res. 105, 70 80 (2008). 31. Hung, M. et l. Higher yields of hybrid rice do not depend on nitrogen fertiliztion under moderte to high soil fertility conditions. Rice 10, 43, https://doi.org/10.1186/s12284-017-0182-1 (2017). 32. Hung, M., Yng, L., Qin, H., Jing, L. & Zou, Y. Fertilizer nitrogen uptke by rice incresed by biochr ppliction. Biol. Fertil. Soils 50, 997 1000 (2014). Acknowledgements This work ws supported by the Ntionl Key R&D Progrm of Chin (2017YFD0301503) nd the Ermrked Fund of Chin Agriculture Reserch System (CARS-01). We would like to thnk Fngbo Co, Xiobing Xie, Jin Chen, Shunglü Shn, Wei Go, Zhibin Li, Yumei Wng nd Hengdong Zhng for joining the experiments. Author Contributions M.H. nd Y.Z. conceived the experiments. C.Z. performed the experiments. M.H. nd C.Z. nlyzed the dt nd wrote the mnuscript. N.U. revised the mnuscript. All uthors hve red nd pproved the finl mnuscript. Additionl Informtion Competing Interests: The uthors declre no competing interests. 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