Stoichiometric Characteristics And Soil Bacterial Diversity Of Robinia Pseudoacacia Sprout Forests

Robinia pseudoacacia（black locust）is a nitrogen-fixing species native to North America with root germination as its main mode of reproduction.The black locust seedlings are connected with the mother tree via root which can supply nutrients for the early growth and development.However,the seedlings usually have poor quality,with the second-generation black locust having difference in growth rate compared with the third-generation black locust forest.The root system plays an important role in plant growth and its interaction with soil may affect the nutrient cycling.In addition,soil microbes also play an important role in transferring materials.Therefore,studying the soil microbes of black locust may improve our understanding on nutrient cycling.Plants absorb nutrients through roots for self-growth and organic-matter synthesis.The stoichiometric characteristics of plants can reflect their health.We first used molecular markers to find clones from the second-and the third-generation black locust forests.These clones were used to study the stoichiometric characteristics of black locust.We aim to address if black locust has poor quality in the second-and the third-generation forest and if it does,what the role in which soil microbes played.Our main findings are as follows:（1）Identification of black locust clonesThe fact that the third-generation black locust forest has more clones than that of the second-generation black locust forest indicates the quality decline of the former.The density of three generations of locusts was 2752 plants/hm²,which was significantly larger than that of the second-generation germination forests of 1372 plants/hm².（2）Comparison of soil microbial communityThere was no significant difference in soil microbes between the second-and the third-generation black locust forest in the Chao1 index,ACE index,Shanon index and Simpson index（P>0.05）,Such as:Chao1（SR:2594,TR:2560）ACE index（SR:2670,TR:2597）and Shannon index（SR:9.41,TR:9.39）.This indicates that the diversity,richness and uniformity of soil bacteria did not decrease significantly with the increase of the number of germinal algebras.There was no significant difference in the rhizosphere bacterial diversity between the second and third generations of the secondary forests and the second and third generations of the non-rhizosphere（P>0.05）.The dominant genus of bacteria of the second and third generations of the black locust was identical with the main bacterial genera being Acidothermus,Rhizomicrobium,Gemmatimonas,Bradyrhizobium,Bryobacter,Haliangium,Variibacter,Acidibacter,Crossiella,c.Solibacter,Jatrophihabitans,Sphingomonas and Ralstonia.The third-generation black locust forest has a higher relative abundance of Gemmatimonas and Bradyrhizobium than that of the second-generation black locust forest.However,the soil N content of the third-generation black locust forest was not higher than that of the second-generation black locust forest,indicating the decrease in nitrogen-fixing capacity of the former.（3）Soil stoichiometricThere was no significant difference in soil carbon（C）,nitrogen（N）,phosphorus（P）,ammonium nitrogen（NH₄⁺）,nitrate nitrogen（NO₃^-）,available phosphorus（AP）,carbon to nitrogen ratio（C/N）,carbon/phosphorus ratio（C/P）and nitrogen/phosphorus ratio（N/P）between the second-and third-generation of black locust forests（P>0.05）,The contents of C,N,P,NH4+,NO3-and AP in the second and third generations of sprouting forest were 50.19,4.52,0.25,0.06,0.096,0.014（mg/g）and 47.23,4.34,0.24,0.063,0.128,0.016,respectively.However,the third-generation black locust forest has higher coefficient of variation,suggesting the uneven distribution of soil nutrients.The N/P ratio of the second-and third-generation forest in soil is over 20,which is much higher than the average N/P ratio of3.9 across the country,indicating the limited soil P.（4）Soil bacterial diversity and soil stoichiometry Pearson correlation resultsThere was no significant correlation between soil stoichiometry and stoichiometry ratio and soil bacteria Chao1 index and ACE index（P>0.05）.There was a significant positive correlation between soil P content and Shannon index of bacterial community.There was a positive feedback relationship between soil P content and soil bacterial community uniformity.In addition,there was a significant positive correlation between soil pH（3.59-4.35）and soil bacteria Shannon index and Simpson index（P<0.05）.Soil pH affected the uniformity of soil bacterial community.（5）Plant stoichiometricThere was no significant difference in C,N,P,C/N,C/P and N/P between the second-and the third-generation of Robinia pseudoacacia leaves（P>0.05）,The second and third generation sprouting forest roots C/N and C/P were 145.86,80.10 and 265.71,110.18,respectively.This is possibly due to the“dynamic balance”mechanism inherent in plant nutrient elements.Seedlings of the third-generation black locust have a higher ratio of C/N and C/P（P<0.05）than that of the second-generation black locus.This can reflect to some extent that the roots of the three generations of sprouting forests have many tillers and increased fine roots,which leads to the serious spread of the three generations of sprouting forest roots.The increase of the seedlings is also the cause of the decline of multiple generations of hedgehog.Pearson analysis showed that the C in soil had a positive correlation with N in roots,N/P in roots and C/N in branch and the N in soil had a significant positive correlation with N in root,N/P and soil NO₃^-versus root N/P（P<0.05）in second-generation of black locust forest.There was no significant correlation between the soil stoichiometry in the third-generation of black locust forest and the stoichiometric values of the tissues and organs（P>0.05）,but there was significant correlations in the second-generation of black locust forest.This indicated that the second-generation of black locust forest adapt to the changes in soil nutrients quickly.Therefore,the correlation between soil stoichiometry and plant stoichiometry may become weaker with generations,which may harm the development of the emerging black locust forest.In summary,our results showed that soil bacterial,soil stoichiometric and plant stoichiometric did not well explain the quality deterioration of the black locust forest as there was no significant difference in these aspects between the second-and the third-generation black locust forest.However,the experiment also found that there are differences between them.Since the number of germinating algebras is too small,the difference is not obvious.As the number of germinating algebras increases,the difference may gradually appear.More research is needed to better understand the causes of the quality deterioration of the black locust forest.