The significance of the linear model was tested with type III analysis of variance with Kenward-Roger’s method with function anova.OTU data from the amplicon sequencing was normalized using the geometric mean of pairwise ratios method. We performed permutational multivariate analysis of variance using distance matrices with function adonis from vegan 2.5–5 to test the effect of farming system and crop type , and the effect of manure addition, including plants grown in 2018, on microbial community composition. We also conducted 2-D nonmetric multidimensional scaling with stable solution from random starts, axis scaling and species scores with function meta MDS from vegan using the Bray-Curtis dissimilarity index and plotted the NMDS with fitted environmental variables from function envfit in vegan. Fungal and bacterial OTUs indicative for specific rotations were obtained by differential abundance analysis which identified significant groups. Due to the PERMANOVA results we also did additional differential abundance analyses to obtain OTUs indicative for rotation plots that have either received manure or not. The results are presented as paired comparisons between the farming systems for forage and cereal crop rotations, and plots with manure or not, and for the spring and autumn data separately.Bacterial richness, assessed by OTU reads, was significantly higher in the organic rotations compared to the conventional rotations in the autumn data . Furthermore, fungal richness was higher in the organic systems in the autumn data but only significantly for the OMilk rotation. In the spring data bacterial and fungal richness did not differ between farming systems. Neither did bacterial and archaeal 16S rRNA gene copy numbers differ between farming systems in either the spring or autumn data,plastic pots 30 liters whereas fungal ITS copy numbers were higher in the OCer rotation compared to the CCer rotation in the autumn data . The NMDS ordination showed minor differences in bacterial community composition between the farming systems for both crop rotations.

Differences in bacterial community composition, however, were more distinct in the autumn data and higher basal respiration, microbial biomass C and N, as well as extractable C and N variables fitted best with the CMilk rotation. Whereas changes in the fungal community composition between farming systems were more pronounced for both crop rotations and between the seasons . The fungal community composition in the organic rotations were more similar than between the conventional rotations. In the springdata, higher basal respiration rates and microbial biomass C and N variables fitted best with the OMilk rotation. In autumn data, microbial C and N, and extractable C fitted best with the OMilk rotation, and soil pH and basal respiration rates with the OCer rotation, and extractable N with the CMilk rotation. The first PERMANOVA indicated that both farming system and crop type significantly affected the variation in microbial community composition both in the spring and autumn data. However, crop type did not explain the variation in the bacterial community composition in the spring data . Farming system explained 10 and 14% of the variation in bacterial community composition in the spring and autumn data, respectively . Crop type explained 10% of the variation in the autumn bacterial data. In turn, the farming system explained 11 and 14% of the variation in fungal community composition in the spring and autumn data, respectively. Crop type explained even more, 21 and 36% of the variation, in the spring and autumn data, respectively. We also tested the effect of manure in an additional PERMANOVA test, which showed that in addition to farming system manure did not affect the variation in bacterial community composition . However, manure explained additional 14% and 11% of the variation in fungal community composition in the spring and autumn data, respectively. In addition, the impact of manure could not be separated from the impact of crop plant which was grown in all manure fertilized rotation plots in 2018. In the ITS2 based fungal data we could identify on average from 9 to 15 Glomeromycotan AMF OTUs in the spring, and from 12 to 25 in the autumn. In the spring data, the OMilk rotation had the lowest OTU number and the OCer rotation the highest . In the autumn data, the CMilk rotation had the lowest and the OCer rotation the highest AMF OTU number.

The majority of AMF OTUs were shared between the farming systems. PERMANOVA showed that season explained the most variation in the AMF community composition, while crop type and farming system also had an effect. There were large differences in the relative abundances of AMF families between the spring and autumn data of the organic systems . Claroideoglomeraceae was the most abundant family, especially in the autumn data, whereas Acaulosporaceae and Pacisporaceae were not present at all in the CMilk rotation. Archaeosporaceae were abundant in the organic systems and almost absent from the conventional systems. It also seems that Pacisporaceae, while not abundant, is characteristic to the cereal rotations, regardless of the season. The 18S rDNA-based AMF data from composite samples obtained in the autumn identified 38 OTUs with unique genbank accession numbers . Moreover, two Scutellospora species were obtained from the Gigasporaceae family that were not seen in the ITS2 data . Paraglomeraceae, Claroideoglomeracea and Diversisporaceae were the most abundant AMF families based on the 18S data. Organic systems had higher AMF richness; AMF OTU numbers varied from 22 in soil with conventional systems to 35 in soil from OMilk. One out of two Acaulospora sp. and four out of seven Glomus sp. OTUs were only present in the organic systems, with two of them only in the OMilk rotation. Altogether, 19 OTUs were shared between all four rotations. Glomus species were abundant in the OMilk rotation, and similarly to the ITS2 based data, Pacisporaceae was not present in the CMilk rotation, but we could find one Acaulosporaceae OTU present in all rotations, contrary to the ITS2 data. Cereal systems were characteristically associated with Archaeospora trappei, Archaeospora sp, Glomus mosseae, and Pacispora sp. . The more frequent tillage in the conventional system for cereal crops explains partly the lower microbial activity rates and biomasses observed in spring and autumn, a phenomenon which has been observed also by others. Furthermore, the conventional cereal system had bare soil over the winter and is the only treatment without manure addition in the rotation. Thus, the absence of continuous plant cover may have further reduced the microbial biomass C in the spring compared to the respective organic system as earlier reported.

Moreover, the second cut of grass and clover ley was left on the soil surface as a green manure in the organic system for cereal rotation and this may have led to the higher microbial activity and biomass in the following autumn. The lack of chemical agents may also have been reflected in the results since earlier studies have shown negative effects of agrochemicals on soil microbial communities.Higher springtime microbial activity and biomass in the conventional forage crop rotation compared to that of organic cannot be easily explained by the differences in the rotation types as the rotations include various management practices. However, there was 30–40% higher soil P concentration in the conventional system for both cereal and forage crop rotations compared to the organic ones. Indeed, lower water soluble and inorganic P amounts have been reported from organic systems compared to systems receiving synthetic fertilization in a long-term field experiment. Possibly the springtime bacterial community in the conventional forage crop rotation gained competitive advantage from the higher availability of P, since P has been reported to limit bacterial growth in agricultural soils. Later in autumn, the summertime amendments of cow manure would equalize the P availability, and differences between organic and conventional systems for forage crop rotation are no longer detected. The slightly higher autumn pH in the organic system for cereal crop rotation may result from microbial decomposition of fresh plant residues with high N content and mineralization of ammonium which temporarily is known to increase pH. Furthermore, it has been reported that long-term application of manure maintains the soil pH,round plastic pots but inorganic fertilizer decreased it. Consequently, since bacterial growth is known to increase multi-fold with increasing pH, this may explain the increased microbial biomass in the organic system for cereal crop rotation. Alternatively, the increased fungal abundance in the organic system for cereal crop rotation in the autumn indicated that fungi could also be responsible for the higher respiration activity and sequestration of C into their biomass.

Moreover, the organic system for cereal crop rotation had timothy and clover ley as the main crop plant in the sampling year instead of barley as in the respective conventional system. Indeed, higher microbial biomass in production systems including ley grasses have been detected compared to single crop systems only. Our results showed that the farming system induced a clear shift in microbial community composition and that the overall impact of the farming system was about the same magnitude for both bacterial and fungal community composition. Our results are comparable to previous findings that about 10% of variation in microbial communities was explained by the farming practices of conventional and organic systems. Yet, crop type affected fungal community composition in particular, especially in the autumn. A simple explanation would be that changing cultivated plants from barley in the year 2017 to ley in the sampling year 2018 induced a shift in fungal community composition. Furthermore, it is likely that the summertime amendments of synthetic fertilization in the conventional systems have also contributed to the lowered bacterial and fungal richness in the autumn, since the quality of fertilizer is known to impact largely on microbial communities. Thus, the differences in crop rotation, tillage and fertilization practices may all have contributed to the differences in the microbial community but with the current experimental layout we are not able to determine which practices have the strongest impact. Nevertheless, comparisons within the cereal rotations were valuable for indicating the long-term impacts of manure addition and overwintering as bare soil, while comparing the forage crop rotations it was possible to assess the other effects of organic practises beyond manure addition and undersown ley. Moreover, the differences in AMF richness between the farming systems were only moderate and non-existing under the cereal crop rotation. A higher diversity of Acaulospora species, typical to organic systems was also supported by our study. We did not observe Clareideoglomus species to be characteristic to organic systems, instead finding the opposite, which contradicts previous results. Thus, our study supports previous observations which concluded that mycorrhizal diversity is not influenced by the farming system but rather cultivation practices and conditions, and that finding a universal AMF indicator for farming systems is not feasible. Results suggest that the cow manure applied in the forage crop rotation under both the conventional and the organic systems over the years has shaped the bacterial and fungal communities more than any other farming system specific practice, since only a few representatives were typical of either farming system.

This also highlights the commonness of pathogenic fungi in the fields cultivated for fodder and fertilized with manure irrespective of the farming system. On the contrary, AMF richness in the forage crop rotation varied clearly due to the farming system; for instance, Glomus species were indicative in the organic system, while Pacispora sp. was totally missing from the conventional system. Since plants acquire P directly and through their symbiotic AMF, the 40% higher levels of P in the conventional forage crop rotation compared to the respective organic rotation may partly explain the lower AMF richness. However, bacterial representatives were less diverse in the conventional farming system of the cereal rotation compared to the respective organic system. Representative taxa typical of the conventional cereal crop rotation in autumn were affiliated to decomposer and plant-growth promoting and cellulose decomposing bacterial taxa. In contrast, both the spring and autumn data obtained from the organic system for cereal crop rotation revealed a variety of specific taxa with diverse functional roles benefiting soil health. Most of these taxa were also linked to manure fertilization, and included for instance, plant growth-promoting rhizobacteria that include species capable of N-fixation, P solubilization, phytohormone production, and repression of soil-borne plant pathogens, and genera with anti-fungal and antibiotic capability. Furthermore, the spring and autumn data of the organic system for cereal crop rotation contained also season specific taxa, which shared bacterial representatives with similar functional roles.