Root stoichiometry explains wheat endophytes and their link with crop production after four decades of fertilization.

Fertilization can impact root endophytic microbiomes and food production. However, the impacts of decades of continued fertilization on root microbiomes, and their link with ongoing crop production, remain poorly understood. Here, we used a four decade-long fertilization experiment, including contrasting types of organic and inorganic fertilization, to investigate the effects of long-term fertilization on multi-kingdom root endophytic microbiomes, including keystone species (modules within microbial networks), and their indirect associations with the production of wheat, which is one of the most important crops worldwide. We found that long-term inorganic (nitrogen, phosphorus, potassium (NPK)) and organic (NPK with straw (NPKS) and NPK with cow manure (NPKM)) fertilization had significant impacts on the community composition of endophytic arbuscular mycorrhizal fungi (AMF), bacteria, and non-mycorrhizal fungi. In addition, compared with NPK fertilization, NPKS and NPKM amendments significantly decreased the microbial network complexity, which was associated with changes in the root iron content. Finally, we identified an important subset of keystone root endophyte species within the microbial network (Module #2), which was positively correlated with wheat yield, and affected by changes in root carbon to phosphorus ratio. This study provides evidence that long-term fertilization can affect keystone root endophytic species in the root microbiome, with implications for food security in an over-fertilized world.

Phosphorus and Zinc Are Strongly Associated with Belowground Fungal Communities in Wheat Field under Long-Term Fertilization.

Belowground fungi are closely related to crop growth, and agricultural fertilization is widely known to affect soil fungal communities. Yet it remains unclear whether fungal communities in differing belowground habitats-root endosphere, rhizosphere soil, and bulk soil-respond differently to long-term fertilization. Here we investigated the variation in fungal communities of root endosphere, rhizosphere soil, and bulk soil under 35 years of fertilization in wheat fields. Specifically, the fertilization regimes were applied as five treatments: soils receiving NPK fertilizer, NPK and cow manure (NPK+CM), NPK and pig manure (NPK+PM), NPK and wheat straw (NPK+WS), and no fertilizer (Control). Long-term fertilization significantly impacted fungal community composition in all three habitats, and these effects were stronger in the rhizosphere and bulk soils than root endosphere. Mantel test results showed that fungal community composition was significantly correlated with phosphorus and zinc contents. Further, fungal alpha diversity was lowest in the NPK+PM treatment and was negatively correlated with both phosphorus and zinc contents. Moreover, NPK+PM treatment had the lowest complexity of fungal co-occurrence network, and in general network complexity was significantly negatively correlated with the zinc and phosphorus contents. Taken together, these results suggest that long-term fertilization can impact fungal communities not only in soils but in root endosphere, and this is strongly associated with the contents of phosphorus and zinc there, a finding important for guiding fertilization management practices and supporting sustainable agriculture. IMPORTANCE Fungi, an essential component in nutrient cycling and plant growth, are highly sensitive to fertilization. However, there are limited studies on fungi in root endosphere under long-term fertilization management. Our research extended the study on the endophytic fungal community of crop roots under agricultural management and found that its responses were similar to the communities in soil habitats. In addition, the type of organic materials was reported as the main driver affecting soil fungal community under long-term fertilization. Our research further revealed that the underlying mechanism of affecting the fungal communities in the soils and roots was the differences in phosphorus and zinc contents caused by the application of different organic materials. Therefore, our results highlight that except for phosphorus, zinc content of the organic materials should be considered in long-term organic fertilization systems.

Differential Responses of Arbuscular Mycorrhizal Fungal Communities to Long-Term Fertilization in the Wheat Rhizosphere and Root Endosphere.

Arbuscular mycorrhizal fungi (AMF) provide essential nutrients to crops and are critically impacted by fertilization in agricultural ecosystems. Understanding shifts in AMF communities in and around crop roots under different fertilization regimes can provide important lessons for improving agricultural production and sustainability. Here, we compared the responses of AMF communities in the rhizosphere (RS) and root endosphere (ES) of wheat (Triticum aestivum) to different fertilization treatments, nonfertilization (control), mineral fertilization only (NPK), mineral fertilization plus wheat straw (NPKS), and mineral fertilization plus cow manure (NPKM). We employed high-throughput amplicon sequencing and investigated the diversity, community composition, and network structure of AMF communities to assess their responses to fertilization. Our results elucidated that AMF communities in the RS and ES respond differently to fertilization schemes. Long-term NPK application decreased the RS AMF alpha diversity significantly, whereas additional organic amendments (straw or manure) had no effect. In contrast, NPK fertilization increased the ES AMF alpha diversity significantly, while additional organic amendments decreased it significantly. The effect of different fertilization schemes on AMF network complexity in the RS and ES were similar to their effects on alpha diversity. Changes to AMF communities in the RS and ES correlated mainly with the pH and phosphorus level of the rhizosphere soil under long-term inorganic and organic fertilization regimes. We suggest that the AMF community in the roots should be given more consideration when studying the effects of fertilization regimes on AMF in agroecosystems. IMPORTANCE Arbuscular mycorrhizal fungi are an integral component of rhizospheres, bridging the soil and plant systems and are highly sensitive to fertilization. However, surprisingly little is known about how the response differs between the roots and the surrounding soil. Decreasing arbuscular mycorrhizal fungal diversity under fertilization has been reported, implying a potential reduction in the mutualism between plants and arbuscular mycorrhizal fungi. However, we found opposing responses to long-term fertilization managements of arbuscular mycorrhizal fungi in the wheat roots and rhizosphere soil. These results suggested that changes in the arbuscular mycorrhizal fungal community in soils do not reflect those in the roots, highlighting that the root arbuscular mycorrhizal fungal community is pertinent to understand arbuscular mycorrhizal fungi and their crop hosts' responses to anthropogenic influences.

Divergent responses of bacterial activity, structure, and co-occurrence patterns to long-term unbalanced fertilization without nitrogen, phosphorus, or potassium in a cultivated vertisol.

Unbalanced fertilization lacking nitrogen (N), phosphorus (P), or potassium (K) is a worldwide phenomenon; however, whether they affect bacterial community composition and intraspecific interactions in a similar pattern and how they affect bacterial activity are not systematically compared. Soils under different kinds of unbalanced fertilization in a 21-year field experiment were collected to investigate the variation in dehydrogenase activity (DHA), bacterial community diversity, structure, composition, and possible interactions. Compared to the balanced fertilization of NPK, the DHA from unbalanced fertilization of NP, PK, and NK was 8.70, 11.59, and 14.17% lower, respectively, and from the unfertilized treatment (Nil) was 13.41% lower; however, the Shannon index from NP, PK, and Nil was 4.48-7.21% higher and from NK was 3.95% lower. Based on principal coordinate analyses (PCoA), bacterial community structure was separated by N application or not along PCo1 and was further separated by P application or not along PCo2, indicating a more influence by N deficiency. Moreover, the structure was mainly determined by soil pH, soil organic carbon (SOC), and total phosphorus (TP). The network complexity using co-occurrence analysis followed the order NP > NPK > PK > NK > Nil, indicating a more influence by P deficiency on intraspecific interactions. Structural equation modeling (SEM) revealed that the reduced DHA in NP was mainly regulated by the decreased SOC and increased Shannon index, in PK by the decreased SOC and increased Shannon index and pH, and in NK by the decreased SOC and TP and increased PCo2. The significantly lower abundance of Bacteroidetes and Chitinophagaceae in NK may also contribute to the reduced DHA. Our results imply that N deficiency had the greatest impact on bacterial community structure and composition, P deficiency had the greatest impact on network construction and bacterial activity, and K deficiency has minimal effect. Our results also suggest that main factors regulating the variation in soil functions may vary among different nutrient deficiencies.

Carbon sequestration efficiency of organic amendments in a long-term experiment on a vertisol in Huang-Huai-Hai Plain, China.

Soil organic carbon (SOC) sequestration is important for improving soil fertility of cropland and for the mitigation of greenhouse gas emissions to the atmosphere. The efficiency of SOC sequestration depends on the quantity and quality of the organic matter, soil type, and climate. Little is known about the SOC sequestration efficiency of organic amendments in Vertisols. Thus, we conducted the research based on 29 years (1982-2011) of long-term fertilization experiment with a no fertilizer control and five fertilization regimes: CK (control, no fertilizer), NPK (mineral NPK fertilizers alone), NPK+1/2W (mineral NPK fertilizers combined with half the amount of wheat straw), NPK+W (mineral NPK fertilizers combined with full the amount of wheat straw), NPK+PM (mineral NPK fertilizers combined with pig manure) and NPK+CM (mineral NPK fertilizers combined cattle manure). Total mean annual C inputs were 0.45, 1.55, 2.66, 3.71, 4.68 and 6.56 ton/ha/yr for CK, NPK, NPKW1/2, NPKW, NPKPM and NPKCM, respectively. Mean SOC sequestration rate was 0.20 ton/ha/yr in the NPK treatment, and 0.39, 0.50, 0.51 and 0.97 ton/ha/yr in the NPKW1/2, NPKW, NPKPM, and NPKCM treatments, respectively. A linear relationship was observed between annual C input and SOC sequestration rate (SOCsequestration rate  = 0.16 Cinput -0.10, R = 0.95, P<0.01), suggesting a C sequestration efficiency of 16%. The Vertisol required an annual C input of 0.63 ton/ha/yr to maintain the initial SOC level. Moreover, the C sequestration efficiencies of wheat straw, pig manure and cattle manure were 17%, 11% and 17%, respectively. The results indicate that the Vertisol has a large potential to sequester SOC with a high efficiency, and applying cattle manure or wheat straw is a recommendable SOC sequestration practice in Vertisols.