Initial sample processing can influence the soil microbial metabarcoding surveys, revealed by Leucocalocybe mongolica fairy ring ecosystem

In this study, we aimed to investigate the influence of soil preservation approaches, especially cryopreservation and high temperature-drying on the sequencing quality of its microbial community and the background microbial diversity information of fairy ring soil from Leucocalocybe mongolica. Through DNA metabarcoding surveys based on 16S rDNA and ITS barcodes, we observed that the bacterial abundance was notably changed when the soil samples were exposed in room temperature for 4 hours, whereas the fungal composition was not significantly changed. Moreover, the soil samples preserved their major microbial structures even after high temperature-drying for 12 hours, whereas their microbial diversity was influenced. Overall, a total of 9283 and 1871 OTUs were obtained from soil bacteria and fungi, respectively, and we observed that Chthoniobacteraceae and Tricholomataceae were the dominant bacterial and fungal families in the fairy ring soil, respectively. Our study reveals the impact of soil processing methods on the microbial community compositions and contributes to the understanding of fairy ring ecology.


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Soil biodiversity is mostly attributed to its rich contents of microorganisms, but 32 currently most of these microbial taxa remain undescribed, which provides the 33 importance for cultivation and edaphology studies [1]. Sequencing technology is 34 considered as the most effective approach on studying soil microbial diversity, but the 35 sequencing results were differential and usually influenced by different soil processing 36 methods [2]. Moreover, the preservation process of soil samples has been reported to 37 affect microbial DNA extraction, thus directly determining the sequencing quality[3-38 7]. The internal microbial diversity in soil, as a living biotic environment, after 39 sampling can gradually or drastically change from its original habitat over time. In this 40 case, cryopreservation is one of the common methods applied for soil processing,  Since the soil storage temperature can influence the quality of high-throughput 49 sequencing [6,12], there is possibility that cryopreservation for the first few hours after 50 sampling might also have such impact, though extremely low temperature and fast 51 preservation are commonly the best ways to ensure the authenticity of soil samples. As 52 a matter of fact, similar sequencing results were observed previously when comparing 53 the soil samples preserved in liquid nitrogen (<-80°C) and in cooler (-20°C) [11]. 54 However, no study has directly assessed the influence of immediate cryopreservation, 55 which is usually needed for sampling at remote locations. On the other hand, efficient 56 and radical method, with time-saving and high safety properties, in preserving soil 57 samples other than ethanol processing [3,13], air-drying [8], and freeze-drying[7] is 58 scarce. Drying at high temperature is a promising approach for long-term preservation, 59 which is widespread applied in the biodiversity surveys for microbial DNA of the 60 preserved specimen [14]. 61 The fairy ring is a unique mycological growth pattern usually shown in grasslands 62 and formed by certain fungi living in the soil of special grassland habitat, which has the 63 ecological values in promoting the yield of crops [15]. Leucocalocybe mongolica, as 64 one of the representative fairy ring fungi with unique taxonomic status (belongs to 65 monotypic genus), is an incomplete domestication of rare wild mushroom, which is 66 edible, possesses medicinal values, and only lives in fairy soil [16]. Therefore, it is of 67 great significance to investigate fairy ring soil with L. mongolica and its microbial 68 compositions, through which we could advance the studies of both fairy rings and L. 69 mongolica domestication.

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As soil serves as an unstable environment [1,2], large-scale and multi-point 71 sampling from different geographical environments is crucial for studying the 72 ecological diversity of fairy rings in the soil. Therefore, soil preservation methods, such 73 as high temperature-drying approach, are critical to be evaluated for their influences on 74 detecting the main microbial diversity structures of the soil samples. In addition, the 75 microbial diversity of fairy ring soils could provide important reference values for the 76 subsequent large-scale investigation, due to limited numbers of studies and inconsistent 77 sequencing results [17,18]. 78 Accordantly, we aimed to discuss the following 3 issues in this study: (i) whether 79 the sequencing results could be affected by immediate in situ soil cryopreservation after 80 sampling, (ii) whether the microbial structure in the soil sample could be preserved after 81 high temperature-drying, and (iii) to obtain the microbial diversity information and 82 major species distribution patterns of bacteria and fungi in a fairy ring soil of L. 83 mongolica. We used the same fairy ring soil sample and divided it by 3 groups, with 6 84 biological replicates for each group. The sub-samples were processed with 85 cryopreservation in situ, with initial 4 h exposure at room temperature, and with further 86 12 h drying at high temperature. (S1 Table) 16S rDNA and ITS barcodes for bacteria 87 and fungi analyses and DNA metabarcoding surveys for all the soil samples were 88 further performed. Finally, we discussed the 3 issues mentioned above based on the 89 analyses of microbial community compositions and diversity.    (821/1871) fungal OTUs were shared among the 3 groups of soil samples (Fig 1b). The indicated the near saturation of the metabarcoding sequencing (Fig 1c). Generally, we 182 found that the OTUs number of bacteria in the fairy ring soil was significantly higher 183 than that of fungi.   However, based on the heat maps (Fig 2ef), when species were compared 218 separately across all soil samples, we observed differences in the microbial abundance Pyrinomonadaceae was found in the DI group (Fig 2e), indicating that the difference in  Fig 2e), and less 251 differences in fungal abundances were observed between LN and DI groups (Fig 2f). Kruskal-Wallis test of the top 5 families of bacteria (g) and fungi (h), in which the x-263 axis represents the relative abundance and the y-axis represents the family.

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Tricholomataceae was excluded from the results of fungi.

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Alpha diversity analysis. 266 We further sought to better understand the changes of both bacterial and fungal 267 diversity in the soil samples introduced by in situ cryopreservation. As shown in Fig 3   268 and Table 2, a total of 6 alpha diversity indices were estimated, including Sobs Goods_coverage values all exceeded 0.99 (Table 2). The soil samples in the DRY group 282 also had the lowest Sobs value (P-value = 0.01) but the highest Shannon value (P-value 283 = 0.002), indicating that the high temperature-drying process could reduce the richness 284 of fungal species but increase their diversity (Fig 3b and Table 2). the DI group, while the DRY group clustered alone (Fig 4b). Similar results were found 303 in the NMDS analysis (Fig 4d). The P-value of Anosim was less than 0.001 (Fig 4f), 304 which indicates that the diversity differences in soil fungi among groups were greater 305 than those in soil bacteria. Overall, soil bacterial diversity in LN and DI groups formed 306 2 complete clusters, and compared with the DRY group, they contained less intra-group 307 difference; for fungi, soil bacterial diversity in LN and DI groups could not be 308 significantly distinguished by OTU genetic distance, but they were clustered separately 309 from the DRY group. which the x-axis represents the group ID, the y-axis represents the relative difference 318 grade, the R value represents the difference level, and the P value represents the 319 significance of the difference.

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In this study, we performed community compositional analysis as well as alpha Pirellulaceae, and differences were also found within the biological replicates (Fig 2eg).

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Secondly, we detected that the soil bacteria in the DI group diverged significantly from 335 those in the LN group through UPGMA clustering analysis (Fig 4a), with all biological 336 replicates from both groups clustered into their own branches. Finally, the analyses of 337 NMDS (Fig 4c) and Anosim (Fig 4e) (Fig 2bdfh, 3b, and 4bd), which is consistent with the studies by Cui[5]. In 346 addition, the Ace index in bacteria showed that the LN group contained less intra-group 347 difference within the biological replications than that of the DI group (Fig 3a). Since 348 the Ace index could cover the microbial species with low abundance (OTUs number 349 below 10 but more than 1)[23], this result indicates that the low abundance-species in 350 the soil samples were well preserved by in situ cryopreservation. Furthermore, our 351 study is related to a recent study by Delavaux[11], which also supported that low although the drying process reduced microbial alpha diversity (Fig 3) and leads to less 370 repeatability in the analyses of soil bacteria and fungi (Fig 2ef and 4ae). Overall, the 371 approach of high temperature-drying is only limited in preserving the major microbial 372 community composition of the soil samples, whereas the method could not ensure high-373 precision of the sequencing results. In addition, this approach is also not suitable for 374 ensuring high repeatability within biological replicates and preserving microbial 375 species with low abundances. Therefore, in situ cryopreservation is a more ideal 376 processing method for high-throughput sequencing study of soil microbial ecosystem.

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The basic information of microbial composition and diversity in soil of fairy ring  Chthoniobacteraceae and Tricholomataceae, respectively, whose abundances were 394 much higher than the following families (Gemmataceae and Geoglossaceae,  (Fig 2b), from which we speculated that the dominance was attributed to the 404 mycelium of L. mongolica during its fruiting body formation. In this study, we assessed the importance of in situ cryopreservation and high