Microbial community response to cover cropping varied with time after termination

This study evaluates cover crop (CC) effects on microbial community structure in a winter wheat–sorghum–fallow rotation with pea, oat, and canola; mixtures of pea and oat; pea and canola; pea, oat, and canola; and six species mixture (SSM) of pea, oat, canola, hairy vetch, forage radish, and barley as CCs, and fallow as treatments. Soil microbial community structure was analyzed at CC termination (phase I), 36 days (phase II), and a year (phase III) after termination using an ester‐linked fatty acid methyl ester analysis. Total microbial biomass (TMB) under oats was significantly greater than under canola (by 47%) in phase I (p ≤ 0.05). The TMB was >48% under pea, pea + canola, and SSM, and arbuscular mycorrhizal fungi was 70%–93% more under pea, canola, and their mixtures than fallow in phase II. While microbial abundance varied with CCs at and after 36 days post‐termination, these effects did not persist for a year. Long fallow period after cropping or cover cropping appears detrimental to microbial community proliferation.

also reported that grass CC significantly increases soil organic matter in long-term (∼30 years) continuous no-till rotations on silty clay loam soils.
Incorporating legumes and grasses in CC mixtures can bring additional benefits critical to water-limited regions through nitrogen fixation, improved water uptake, and nutrient cycling through symbiotic associations with microbes such as rhizobia or arbuscular mycorrhizal fungi (AMF) (Lehman et al., 2012).Additionally, CC mixtures suppress weeds by rapidly producing canopy cover and allelochemicals (Koehler-Cole et al., 2020), control wind and water erosion, and prevent nutrient leaching.While several studies (Blanco-Canqui et al., 2015;Ogunleye et al., 2023) have reported cover crop diversity influence on physicochemical properties of the soil, there is a lack of information on the temporal dynamics of soil microbial communities following CC termination in arid and semiarid environments.Therefore, the primary objective of this study was to assess microbial community size and composition in response to diverse CCs and their mixtures in a winter wheat (Triticum aestivum L.)-sorghum (Sorghum bicolor L. Moench.)-fallowrotation under limited irrigation management in the water-limited environment of the U.S. Southern High Plains region.

MATERIALS AND METHODS
This study (2019)(2020)  Soil samples were collected from 0-to 15-cm depth from each plot at all phases of crop rotation in the first week of June

Core Ideas
• Cover crops improved microbial community abundance compared to fallow.• Microbial community responses varied among cover crops and time after termination.• The AMF abundance was 70%−93% greater under pea, canola, and their mixtures than fallow.
2019 and 2020.The sampling time represented three different stages of the field after CC termination: at spring CC termination (phase I), 36 days after fall CC termination (phase II), and a year after spring CC termination during active winter wheat growth (phase III).Soil microbial community structure was characterized using an ester-linked fatty acid methyl ester analysis (Schutter & Dick, 2000).The effect of CC, sampling year, and their interaction on soil microbial groups were analyzed by the CC termination phase using a generalized linear mixed model procedure in SAS (version 9.4).The normality of residuals and equality of variance was tested using the Shapiro-Wilk and Levene tests, respectively.The analysis considered treatment and year as fixed and replication as a random factor in the model.Unless otherwise stated, means were separated using Tukey's test at p ≤ 0.05.Principal component analysis using covariance matrix structure was performed in PAST 4.11.

RESULTS
Microbial community responses differed among CC treatments and sampling year, but there was no significant interaction between treatment and sampling year in all phases (Table 1).At CC termination (phase I), CC treatments had a significant effect on total microbial community (TMB) size, AMF, Gram-positive bacteria (Gram+), Gram-negative bacteria (Gram−), actinomycetes (ACTM), total bacteria (TB), and total fungi (TF) as observed in their markers.TMB was significantly greater under oats (47%) than under canola but was not different from other treatments.The AMF under oats, a mixture of pea + oat + canola, and SSM behaved similarly and were about 67%-86% more than under canola and fallow.The Gram+ and Gram− bacteria were also significantly greater (44% and 28% greater, respectively) under oats than canola.All other treatments except pea + oat + canola remained intermediate of oats and canola for Gram+ bacteria.The ACTM and TB followed a similar trend as Gram+ bacteria, where oats and pea + oat + canola had the greatest and canola had the lowest abundance.However, only oats had 54% greater TF than canola; other treatments ranged    Abbreviations: ACTM, actinomycetes; AMF, arbuscular mycorrhizal fungi; F/B, fungi to bacteria ratio; Gram+, Gram-positive bacteria; Gram−, Gram-negative bacteria; G+/G−, Gram-positive to Gram-negative bacteria ratio; SAP-F, saprophytic fungi; SSM, six species mixture of pea + oats + canola + hairy vetch + forage brassica + barley; TB, total bacteria; TF, total fungi; TMB, total microbial biomass.
between oats and canola.The FAME markers for protozoa and saprophytic fungi (SAP-F) were in the range of 0.5-1.6 and 39.5-59.0nmol g −1 soil, and Gram+/Gram− and fungal to bacterial (F/B) ratio were in the range of 4.4-4.9 and 1.0-1.1,respectively (Table 1).Averaged across treatments, all microbial groups except protozoa and Gram+/Gram− were significantly greater in 2019 than in 2020.The abundance of TMB, AMF, Gram+, Gram−, ACTM, TB, and TF varied by CC treatments at 36 days after CC termination (phase II) (Table 1).Pea, canola, pea + canola, and SSM exhibited greater soil microbial abundance than fallow.Averaged across sampling years for pea, pea + canola, and SSM, which were not different from one another, resulted in 48%-50% greater TMB than under fallow.The FAME marker for AMF was similar in soils under pea, canola, and pea + canola but was 70%-93% greater than under fallow.Gram+ bacteria was 44% greater under pea + canola than under fallow.Microbial including Gram−, ACTM, and TB, were 42%, 32%, and 39% greater under pea than under fallow, respectively.However, fungal abundance was 64%-71% greater under pea + canola and SSM than fallow.Averaged across treatments, all microbial groups except TMB and F/B ratio were significantly greater in 2020 than in 2019.The legacy effect of CC was observed only in Gram+ bacteria in phase III.Pea + oat had significantly higher (by 29%) Gram+ bacteria than those of monoculture canola.All other treatments were intermediate of pea + oat and canola and were not different from one another (Table 1).
Principal component analysis (PCA) revealed distinct communities under fallow than under CCs and a distinct community under pea and mixtures with pea than the rest of the CC treatments (Figure 1).Independent of the sampling year and CC termination phase, PC1 explained most of the variance (93.5%) and was principally related to SAP-F, while PC2 explained 3.8% of the variance and was mostly associated with Gram+ bacteria (Figure 1).Biplot revealed that all microbial groups except protozoa had a positive loading along PC1.Along PC2, most microbial groups (Gram+, AMF, ACTM, and Gram−) had a positive loading, whereas SAP-F had a negative loading.The loading on protozoa along both PC1 and PC2 was close to zero.It was observed that peas and mixtures containing peas favored Gram+ bacteria and AMF, while SAP-F was more associated with the oat and pea + oat combination.Fallow and canola treatment had inverse loading with most of the microbial groups.

DISCUSSION
An increase in organic residue inputs often supports high soil microbial abundance (R. Ghimire et al., 2014;Acosta-Martinez et al., 2023), and the microbial substrate availability and quality affect the community structure (Mbuthia et al., 2015;Nautiyal et al., 2010).At the time of CC termination in our study, the higher TMB under oats, compared to canola, was likely attributed to the greater addition of biomass C. Immediately after CC termination, decomposing CC residues serve as a readily available C source, fueling microbial growth (Lehman et al., 2012).In a concurrent study conducted at the same site, oats as CC exhibited significantly greater average aboveground biomass production, greater C:N ratios, and more soil organic carbon and total N accumulation than canola (Thapa et al., 2022).Additionally, grass CCs, such as oats, often generate dense root systems and more root biomass, providing a C-rich substrate that stimulates microbial activity (Amsili & Kaye, 2021), which can result in a surge in microbial biomass and shifts in community structure.Correspondingly, CCs with dense rooting systems, like oats, increase the abundance of AMF (Kabir & Koide, 2002;Lehman et al., 2012).Gram+ bacteria can efficiently utilize fresh organic residues with higher C:N ratios and flourish in response to the newly available resources, while Gram− bacteria respond quickly to easily decomposable substrates (Carpenter-Boggs et al., 2003;Fierer et al., 2003).The rhizosphere of living canola roots releases an anti-fungal fumigant-like compound (2-phenylethyl isothiocyanate), which might also affect bacterial and eukaryotic communities (Rumberger & Marschner, 2003).
Typically, plant materials characterized by low C:N and lignin:cellulose ratios favor early mineralization and rapid recycling of soil nutrients (B.Ghimire et al., 2017), promoting soil microbial growth and activity (Bartelt-Ryser et al., 2005).The lower C:N ratio of pea and canola compared to oats may have favored fast decomposition, providing a C source for diverse microbial groups within a month of CC termination (Thapa et al., 2022).Based on the results obtained by Thapa et al. (2022) on soil properties such as potentially mineralizable C and N, C:N ratio, etc., in previous research involving the same treatments, CCs such as pea, canola, and brassicas, either alone or in combination, that is, pea + canola and SSM, resulted in greater particulate carbon and maintained belowground food chains for various microbial groups (Table 1; phase II).A mixture of high biomass and high C:N ratio CC species, like oats, with low C:N ratio compared to species, such as peas, have slowly and gradually facilitated nutrient mineralization, making it accessible to microorganisms, specifically Gram+ bacteria, over time.It appears that stronger cell walls and more advanced osmoregulatory functions in Gram+ bacteria than Gram− result in Gram+ bacteria responding slowly to management changes, soil water content, and substrate availability (Fierer et al., 2003;R. Ghimire et al., 2014).Several studies have reported that a mixture of diverse CC species could boost diverse microbial communities by providing a variety of substrates and altering habitat conditions over time (Carrera et al., 2007).While some studies have reported a positive influence of CC diversity F I G U R E 1 Relationships among microbial groups at the system scale through principal component analysis and their loading score.ACTM, actinomycetes; AMF, arbuscular mycorrhizal fungi; Gram+, Gram-positive bacteria; Gram−, Gram-negative bacteria; SAP-F, saprophytic fungi.(Daryanto et al., 2018;King & Hofmockel, 2017), our study evaluating how CC diversity impacts microbial functionality and evenness compared to fallow showed no significant difference in the diversity and evenness indexes, or microbial functions measured by enzyme activities (data not presented).While cover cropping appears to help microbial community proliferation compared to leaving land fallow, the impact did not last for a year after CC termination for most of the microbial groups.Continuous cover is needed to have a lasting impact on soil microbial communities.

CONCLUSION
The legacy effect of CC in influencing microbial abundance and functionality up to or beyond a year after termination is not a common concept.In our study, this concept was only observed in the pea + oat mixture, which only influenced Gram+ bacteria.This study also observed that the lack of CC residue inputs under fallow resulted in lower microbial biomass, indicating that cover cropping, whether monoculture or in mixtures, provides a smarter option compared to fallow.More research will reveal the complex interaction of microbial community structure and functionality following the termination of diverse CCs in arid and semiarid environments.However, cover cropping encourages microbial community proliferation compared to leaving land fallow, and continuous cover may help maintain their high abundance.

C O N F L I C T O F I N T E R E S T S T A T E M E N T
The authors declare no conflicts of interest.

T A B L E 1
Microbial groups by cover crop (CC) treatment and sampling year in each crop rotation phase.
Data curation; formal analysis; writing-original draft.Vesh R. Thapa: Data curation; investigation; writing-original draft.Deb R. Aryal: Data curation; writing-original draft.Rajan Ghimire: Conceptualization; data curation; funding acquisition; investigation; resources; software; supervision; validation; writingoriginal draft.Veronica Acosta-Martinez: Formal analysis; investigation; methodology; writing-review and editing.A C K N O W L E D G M E N T SThis research was funded by project No. 2016-68007-25066 and 2022-67019-36106 of the USDA National Institute for Food and Agriculture's Agriculture and Food Research Initiative.The authors would like to thank the ASC Clovis staff for their help in maintaining the field experiment and soil sampling.

year after spring CC termination/during active winter wheat growth (phase III)
Note:Means within columns followed by the same letter in superscripts are not statistically different among CC treatments (p < 0.05).