Abstract

   Continuous-cropping (CC) obstacles are among the primary factors
   limiting the development of global agriculture. Although most plants
   are negatively affected by barriers that develop with CC, they may also
   overcome such obstacles by altering the soil biological and chemical
   environment to favor plant growth. In this study, we investigated the
   mechanism by which plants recruit beneficial microorganisms using root
   exudates to alleviate obstacles in a 10-year CC potato system. On day
   20 after potato emergence, soil microorganisms in the CC system
   promoted an increase in adventitious root (AR) numbers by increasing
   the indole-3-acetic acid (IAA) content of the rhizosphere soil.
   Analysis of rhizosphere bacterial communities using 16S rRNA sequencing
   revealed that CC alters community structure, increasing the abundance
   of Pantoea sp. MCC16. Irrigation with root exudates from CC potato
   significantly increased AR numbers and Pantoea sp. MCC16 abundance.
   Through untargeted metabolomic analysis, we identified nobiletin as key
   metabolite that promotes Pantoea sp. MCC16 colonization in the
   rhizosphere. Furthermore, application of either nobiletin or Pantoea
   sp. MCC16 significantly improved the yield of CC potatoes. These
   findings demonstrate that CC plants can actively secrete the unique
   metabolite nobiletin to recruit Pantoea sp. MCC16, a high IAA producer,
   to help plants recover functional traits and mitigate CC obstacles.

   Key words: potato, continuous cropping, Pantoea sp. MCC16, nobiletin,
   adventitious root numbers
     __________________________________________________________________

   Potato exhibits autonomous adaptation to continuous-cropping stress.
   This study provides evidence that continuously cropped potato secretes
   nobiletin to recruit Pantoea sp. MCC16, which increases rhizosphere IAA
   levels and stimulates adventitious root proliferation during the
   seedling stage, thereby alleviating the decline in root biomass and
   yield caused by self-toxic stress.

Introduction

   Although the traditional practice of continuous cropping (CC) can meet
   short-term yield demands, the prolonged use of CC engenders a complex
   of persistent challenges, collectively termed CC obstacles. CC
   obstacles significantly impair crop productivity and sustainability,
   thereby hindering the long-term development of modern agriculture
   ([45]Li et al., 2021; [46]Ma et al., 2023a).

   Geographic constraints, arable-land limitations, economic demands, and
   intensified modern farming practices reduce species diversity in
   continuously cropped areas. Combined with excessive fertilizer
   application aimed at maximizing crop yields, these factors disrupt
   ecosystem self-regulation, exacerbating CC obstacles. It is challenging
   to completely eliminate such obstacles when implementing a CC system.
   However, artificial means can be used to mitigate the suppression of
   plant growth in CC systems. The main obstacles to CC are usually caused
   by soil-borne diseases and autotoxicity. Approaches to prevent and
   control soil-borne diseases in CC systems have evolved from extensive
   management aimed at eliminating all soil microorganisms, to more
   refined management focusing on individual pathogenic bacteria, and
   finally to management of the entire microbial community, which involves
   making overall adjustments to soil structure ([47]Wang et al., 2024a).
   The introduction of microorganisms with specific functions has
   historically been the primary technique used to regulate soil microbial
   community structure. However, soil colonization is challenging because
   of factors such as poor environmental adaptability, competition with
   native flora, and host immune responses. Therefore, an effective
   strategy to prevent and control soil-borne diseases under CC conditions
   is the direct soil application of prebiotics required by beneficial
   bacteria to increase their competitiveness or the application of
   functional strains that can inhibit the main active substances of
   pathogenic bacteria ([48]Chung et al., 2016; [49]Wen et al., 2023). In
   addition, ecosystem function can be effectively optimized by leveraging
   the “home-field advantage” of native beneficial microorganisms
   ([50]Jiang et al., 2023).

   Autotoxicity, mainly mediated by phenolic acids produced by the plants
   themselves, remains a difficult problem. Small amounts of phenolic
   acids can temporarily improve plant tolerance and reduce oxidative
   stress ([51]Martinez et al., 2016; [52]Sun et al., 2024). However,
   long-term CC leads to the accumulation of large amounts of phenolic
   acids in the soil, which disrupt root membrane structures and hinder
   the development of root morphology ([53]Ni et al., 2021). Autotoxicity
   is most commonly observed during the seedling stage, although the
   specific phenolic acids responsible for autotoxicity vary among
   different plants ([54]Wang et al., 2022). Therefore, to alleviate
   autotoxicity, in addition to developing new varieties that are
   resistant to CC obstacles, a common practice is to screen for
   beneficial bacteria that can degrade specific phenolic acids on a large
   scale and then apply them to CC soil in a targeted manner ([55]Chen et
   al., 2023). However, with this method, challenges remain in terms of
   colonization and efficacy. Artificial stimulation of root system
   establishment during the seedling stage is another approach to
   alleviate autotoxicity, and it has been shown to increase yield by
   approximately 20% ([56]Ma et al., 2023b). With this method, negative
   plant responses to external stress weaken, gradually reducing the
   release of phenolic acids into the soil.

   Although CC leads to an imbalance in overall ecosystem function, plants
   can still promote their own growth by adjusting surrounding
   microhabitats in a “cry-for-help” strategy that is common in
   plant–microbe systems ([57]Rolfe et al., 2019). Plants actively secrete
   metabolites that attract beneficial bacteria to accumulate in the
   rhizosphere. The specific metabolites serve as signaling or energy
   substances for the bacteria, with specific bacteria selectively
   recruited during certain periods and their functions maintained. Under
   long-term stress conditions, nonresistant plant varieties tend to be
   more effective at stimulating soil microbial communities to suppress
   pathogens and alleviate environmental stresses—a phenomenon indicative
   of the development of resistant soil—compared with their resistant
   counterparts, although the extent of this effect varies depending on
   the specific environmental conditions and the particular plant–microbe
   interactions involved ([58]Raaijmakers and Mazzola, 2016). For example,
   susceptible varieties of cucumber secrete more organic acids to recruit
   Comamonadaceae, which inhibit Fusarium oxysporum f.sp. cucumerinum
   (Foc) ([59]Wen et al., 2020). Similarly, after infection by Pseudomonas
   syringae pv tomato (Pst), the subsequent generation of Arabidopsis
   secretes more long-chain organic acids and amino acids to regulate the
   soil bacterial community and increase plant resistance ([60]Yuan et
   al., 2018). Some of the obstacles posed by CC can be alleviated or even
   eliminated with time, usually more than 10 years ([61]Zhang and Chen,
   2014), suggesting that plants develop strategies to cope with CC
   obstacles over the long term. Such strategies are closely linked to the
   “root exudate–soil microbial” system. Therefore, harnessing the
   restoration capabilities of the ecosystem itself may be the most
   effective approach for mitigation of CC obstacles.

   Potatoes, a top-four food crop, are known for their nutrient-rich and
   stress-resistant properties and are widely grown in CC systems.
   However, the inherent intolerance of Solanaceae plants to CC makes the
   CC of potato a persistent industry challenge ([62]Chen et al., 2023),
   and potato seedling growth is typically significantly inhibited in CC
   systems. In the absence of noticeable pest and disease symptoms, the
   accumulation of autotoxic substances in soil is thought to be a primary
   factor underlying this phenomenon ([63]Guo et al., 2016; [64]Lyu et
   al., 2022; [65]Xiang et al., 2022). In previous research, we discovered
   that the autotoxic substance vanillin, present in CC systems, initially
   led to significantly lower adventitious root (AR) numbers than those of
   normal plants ([66]Ma et al., 2023b). However, AR numbers increased
   significantly later in the seedling stage. We hypothesized that
   long-term CC stimulates plants to autonomously recruit beneficial
   microorganisms, thereby increasing AR numbers. Changes in potato root
   exudates are considered to drive increases in beneficial plant
   microorganisms, with exudates influenced by autotoxic substances in CC
   soil. To test this hypothesis, we performed experiments to (1) evaluate
   the establishment of a microbiome that promotes increased AR numbers in
   a CC system, (2) analyze root exudates of CC potatoes to identify
   signaling compounds for the recruitment of beneficial bacterial taxa,
   and (3) examine changes in plant-growth-promoting rhizosphere bacteria
   after exposure to root exudates from CC potatoes and examine the effect
   of using plant-growth-promoting bacteria and growth-promoting
   metabolites to alleviate CC obstacles ([67]Supplemental Figure 1).

   The results of this study shows that AR numbers increased significantly
   at a later seedling stage under vanillin stress in CC conditions. The
   increase in AR numbers was due to significant enrichment of Pantoea sp.
   MCC16 in the rhizosphere soil. By increasing the indole-3-acetic acid
   (IAA) content of rhizosphere soil, the bacterium increased the content
   of root IAA, which in turn promoted increases in AR. In addition, we
   found that enrichment of Pantoea sp. MCC16 was due to the recruitment
   ability of nobiletin, which was secreted by CC potato roots under
   autotoxic conditions.

Results

Autotoxicity inhibits potato plant growth under continuous cropping

   CC significantly inhibited potato seedling growth ([68]Figure 1, ① in
   [69]Supplemental Figure 1). At the seedling stage, AR numbers increased
   significantly at 10–20 days after emergence (DAE) under noncontinuous
   cropping (NCC) conditions but at 20–30 DAE under CC conditions. The
   maximum IAA content occurred at 10 DAE in NCC potato roots but at 20
   DAE in CC potato roots ([70]Figure 1A). Although plant height, shoot
   diameter, and leaf and root dry weights of CC potato increased
   significantly at 20–30 DAE, these values were always lower than those
   of NCC potato, and potato yield was significantly lower (−24.43%) under
   CC conditions than under NCC conditions ([71]Supplemental Figure 2;
   [72]Figure 1C). In rhizosphere soil at the seedling stage, the vanillin
   content of CC soil was significantly higher than that of NCC soil on
   all sample dates ([73]Figure 1B). According to RT–qPCR analysis, CC
   significantly inhibited expression of the root IAA-synthesis genes
   StYUCCA5 and StTAR2 throughout the seedling stage, whereas expression
   of the root IAA-response genes StARF3 and StIAA4 increased
   significantly at 10–20 DAE ([74]Figure 1D). These results indicated
   that CC significantly inhibited the growth of potato seedlings but that
   substances in CC soil promoted an increase in root IAA content at 20
   DAE.

Figure 1.

   [75]Figure 1
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   Inhibition of potato plant growth under continuous cropping.

   (A) Effects of continuous cropping (CC) on the number of potato ARs and
   the root indole-3-acetic acid (IAA) content.

   (B) Effects of CC on soil vanillin content.

   (C) Effects of CC on potato yield.

   (D) Effects of CC on the expression of IAA-synthesis (StYUCCA5, StTAR2)
   and IAA-response (StARF3, StIAA4) genes. NCC, noncontinuous cropping;
   DAE-X, X days after potato emergence. Data are shown as the mean ± SEM
   (n = 3). Different letters indicate significant differences (Tukey’s
   HSD test, p < 0.05).

Soil microorganisms promotes potato plant growth under continuous cropping

   To determine whether microorganisms in CC soil could promote plant
   growth, we set up a soil sterilization test. In CC soil, the presence
   of microorganisms promoted potato plant growth ([77]Supplemental Figure
   3A). Soil vanillin content was significantly higher in CC soil than in
   NCC soil, regardless of whether the CC soil had been sterilized,
   indicating the development of autotoxicity in CC soil ([78]Supplemental
   Figure 3D). Soil sterilization had no significant effect on AR numbers
   at any seedling stage in NCC soil, and AR numbers increased
   significantly at 10–20 DAE ([79]Figure 2A). In addition, IAA content
   did not differ significantly between sterilized and unsterilized NCC
   soil during the seedling stage, and root IAA content was highest at 10
   DAE ([80]Supplemental Figures 3B and 3C).

Figure 2.

   [81]Figure 2
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   Rhizosphere microorganisms promote potato plant growth in continuously
   cropped soil 20 days after emergence.

   (A) Effects of soil sterilization on the AR number and yield of potato.
   NCC, unsterilized noncontinuous-cropping soil; NCCS, sterilized
   noncontinuous-cropping soil; CC, unsterilized continuous-cropping soil;
   CCS, sterilized continuous cropping soil; DAE-X, X days after potato
   emergence.

   (B) Effects of soil sterilization on the expression of IAA-synthesis
   (StYUCCA5, StTAR2) and IAA-response (StARF3, StIAA4) genes in potato
   roots.

   (C) Effects of microflora transfer on AR numbers and potato yield.
   NCC-X, rhizosphere microorganisms on day X after potato emergence in
   noncontinuous cropping soils; CC-X, rhizosphere microorganisms on day X
   after potato emergence in CC soils.

   Data are shown as the mean ± SEM (n = 3). Different letters indicate
   significant differences (Tukey’s HSD test, p < 0.05).

   In CC soil, AR numbers in sterilized soil did not increase
   significantly during the seedling stage, but in unsterilized soil,
   numbers increased significantly at 20–30 DAE ([83]Figure 2A). In CC
   rhizosphere soil, IAA content increased significantly at 20 DAE
   ([84]Supplemental Figure 3C). Notably, expression of the root
   IAA-synthesis genes StYUCCA5 and StTAR2 decreased significantly,
   whereas expression of the root IAA-response genes StARF3 and StIAA4
   increased significantly ([85]Figure 2B). In addition, although potato
   yield was significantly lower in CC soil than in NCC soil (regardless
   of sterilization), potato yield was significantly lower in sterilized
   continuously cropped soil (CCS) than in in unsterilized CC soil
   ([86]Figure 2A). These results indicated that soil microorganisms
   caused the increase in AR numbers under CC conditions (with
   autotoxicity) (② in [87]Supplemental Figure 1).

   To further explore the stage at which microorganisms in CC soil played
   the main role in increasing AR numbers, we collected rhizosphere flora
   of potato seedlings in CC and NCC soil and transferred them to
   sterilized CC soil. There was no significant difference in the effect
   of bacterial transfer on soil vanillin content, and vanillin stress was
   present in each treatment ([88]Supplemental Figure 3G). Soil
   microorganisms from NCC soil did not significantly affect the IAA
   content of rhizosphere soil or potato roots, whereas soil
   microorganisms from CC soil significantly increased IAA content in both
   soil and roots. Regarding the increases in IAA content in rhizosphere
   soil and roots, the most significant effect of CC rhizosphere
   microorganisms occurred at 20 DAE ([89]Supplemental Figure 3E and 3F).

   In addition, microorganisms from the rhizosphere of NCC potato
   seedlings produced no significant increases in AR numbers or potato
   yield. However, microorganisms from CC soil at 20 DAE significantly
   increased AR numbers throughout the seedling stage, with the first
   significant difference detected at 10 DAE ([90]Figure 2C). Therefore,
   microorganisms from CC soil at 20 DAE significantly increased the yield
   of CC potatoes ([91]Figure 2C, ③ in [92]Supplemental Figure 1). On the
   basis of these results, we hypothesized that microorganisms in CC soil
   could significantly increase potato yield under CC vanillin stress,
   with the main functions of rhizosphere microorganisms apparent at 20
   DAE of CC potatoes.

Pantoea sp. MCC16 promotes potato growth under continuous cropping

   After confirming that rhizosphere soil microorganisms at 20 DAE had a
   significant growth-promoting effect on CC potatoes, we analyzed the
   bacterial community structure of CC and NCC soil by performing
   sequencing for absolute quantification of 16S rRNA at 10 and 20 DAE.

   The difference in rhizosphere bacterial community structure between CC
   and NCC was more significant at 20 DAE than at 10 DAE ([93]Figure 3A).
   Bacterial community diversity was significantly higher in CC soil than
   in NCC soil, especially at 20 DAE, whereas there was little difference
   in community richness ([94]Supplemental Figure 4A and 4B). The absolute
   abundance of the top 10 bacterial phyla was highest in CC soil at 20
   DAE (CC20), especially for Proteobacteria and Bacteroidetes ([95]Figure
   3B). To identify which bacteria in CC20 caused significant differences
   compared with the other treatments, we identified the bacteria that
   were present in CC20 but not in CC10, NCC10, or NCC20. Eleven bacteria
   were screened in total, and Rudaea (3ASV), Pantoea (1ASV), and
   Melioribacter (2ASV) were present only in the CC20 treatment; Rudaea
   and Pantoea are members of Proteobacteria ([96]Figure 3C).

Figure 3.

   [97]Figure 3
   [98]Open in a new tab

   Pantoea sp. MCC16 promotes plant growth in continuous cropping soil.

   (A) Principal coordinate analysis of differences in rhizosphere soil
   community structure under continuous cropping (CC) and noncontinuous
   cropping (NCC) conditions on days 10 and 20 after emergence. NCC-X,
   rhizosphere microorganisms on day X after potato emergence in NCC
   soils; CC-X, rhizosphere microorganisms on day X after potato emergence
   in CC soils.

   (B) Absolute abundance of the top 10 bacterial phyla in CC10, CC20,
   NCC10, and NCC20.

   (C) Bacterial genera absent (11) in CC10, NCC10, or NCC20 and unique
   genera (3) in CC20.

   (D) Strains with an indole-3-acetic acid (IAA) production capacity >20
   μg ml^−1 in CC and NCC rhizosphere soils. I-X, 10 days after potato
   emergence (DAE); M-X, 20 DAE; F-X, 30 DAE.

   (E) Evolutionary tree of bacterial species ICC5 and MCC16. The red
   plate contains E. coli identification medium.

   (F) Effect of irrigation with a solution of Pantoea sp. MCC16 on the
   yield of CC potato.

   Data are shown as the mean ± SEM (n = 3). Different letters indicate
   significant differences (Tukey’s HSD test, p < 0.05).

   Because previous experiments confirmed that the bacteria in CC20
   rhizosphere soil could increase soil IAA content, we isolated and
   cultured rhizosphere soil bacteria in the seedling stage under CC and
   NCC. Seven IAA-producing strains (IAA-producing ability >20 μg ml^−1)
   were isolated: three strains in CC and four strains in NCC. The IAA
   production capacity of MCC16 in CC was as high as 77.022 μg ml^−1,
   which was 125.5% higher than that of FNCC5, the strain with the highest
   IAA production capacity in NCC ([99]Figure 3D). The seven strains were
   then identified by analyzing their 16S rDNA sequences. Five of the
   strains were in Firmicutes, whereas the other two were in
   Proteobacteria (ICC5, MCC16), and the abundance of Proteobacteria in
   CC20 increased significantly ([100]Figure 3B and 3D). Strain ICC5 was
   identified as Escherichia coli, and strain MCC16 showed 100% homology
   to Pantoea sp. SAS9 ([101]Figure 3E). Absolute quantification of 16S
   rRNA sequencing results revealed that only one taxon, ASV7733, was in
   the genus Pantoea. This taxon was only found at 20 DAE, and strain
   MCC16 was isolated from potatoes at 20 DAE, indicating that ASV7733 was
   the MCC16 strain ([102]Figure 3E; [103]Supplemental Figure 4C, ④ in
   [104]Supplemental Figure 1).

   When determining whether Pantoea sp. MCC16 could promote plant growth
   in CC potato, we found that the IAA content in rhizosphere soil and
   roots of CC potato increased significantly after bacterial suspension
   treatment, leading to increases in AR numbers and potato yield.
   Notably, expression of the IAA-response genes StARF3 and StIAA4 was
   significantly enhanced in the roots after Pantoea sp. MCC16 treatment,
   whereas expression of the IAA-synthesis genes StYUCCA5 and StTAR2
   showed no significant change ([105]Figure 3F; [106]Supplemental Figure
   4D and 4E). These results also confirmed that Pantoea sp. MCC16 was the
   main strain that promoted plant growth in CC soil (⑤ in
   [107]Supplemental Figure 1).

Potato root exudates under continuous cropping promote plant growth

   To determine why Pantoea sp. MCC16 appeared only in CC20, we collected
   root exudates of CC and NCC potatoes at 10 and 20 DAE and performed
   irrigation tests of root exudates against the background of
   unsterilized CC soil. Only the CC20 root exudates significantly
   increased AR numbers at the beginning of the potato seedling stage and
   maintained this enhancement effect over time. In addition, strain MCC16
   in rhizosphere soil appeared only under irrigation with CC20 root
   exudates at 10 DAE ([108]Figure 4A). These results indicated that CC20
   root exudates could directly promote Pantoea sp. MCC16 colonization of
   the potato rhizosphere (⑥ in [109]Supplemental Figure 1).

Figure 4.

   [110]Figure 4
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   Pantoea sp. MCC16 colonization was stimulated by root exudates on day
   20 after emergence of continuously cropped potato.

   (A) Effects of root exudates from CC and NCC potato on AR numbers and
   Pantoea sp. MCC16 abundance of CC potato. NCC-X, root exudates on day X
   after potato emergence in NCC soils; CC-X, root exudates on day X after
   potato emergence in CC soils; CFU, colony-forming unit.

   (B) Principal coordinate analysis of root exudates in CC and NCC.

   (C) Four methods used to screen for metabolites that were concentrated
   in the root exudates of CC20.

   (D) Correlation analysis of metabolites enriched in CC20 (29
   metabolites) and Pantoea sp. MCC16.

   Data are shown as the mean ± SEM (n = 3). Different letters indicate
   significant differences (Tukey’s HSD test, p < 0.05).

   To further explore which specific metabolites in CC20 could recruit
   Pantoea sp. MCC16, we performed untargeted metabolomics analysis of
   root exudates in CC and NCC. The components and contents of root
   exudates treated with CC and NCC differed significantly in both
   positive and negative modes ([112]Figure 4B). Using Kyoto Encyclopedia
   of Genes and Genomes (KEGG) pathway enrichment analysis (TOP10)
   ([113]Supplemental Figure 5; [114]Supplementary Table 3),
   principal-component analysis variable-importance analysis (TOP40)
   ([115]Supplemental Figure 6), random forest variable importance
   analysis (TOP40) ([116]Supplemental Figure 7), and significant
   difference analysis (TOP50) ([117]Supplemental Figure 8), we selected
   29 metabolites that increased significantly only in CC20 ([118]Figure
   4C, ⑦ in [119]Supplemental Figure 1). The absolute abundance of MCC16
   was significantly positively correlated with the relative contents of
   11 metabolites: N-isovalerylglycine, epimedin C, D-turanose, nobiletin,
   sauchinone, praeruptorin A, isomucronulatol-7-O-glucoside, methyl
   4-hydroxy-3-methoxycinnamate, 3-succinoylpyridine, thioctic acid, and
   kaempferol ([120]Figure 4D).

Nobiletin alleviates potato continuous cropping obstacles by recruiting
Pantoea sp. MCC16

   To determine which substances could recruit Pantoea sp. MCC16, we
   performed an in vitro test to assess the chemotaxis of Pantoea sp.
   MCC16 toward the 11 substances. Only nobiletin significantly stimulated
   chemotaxis in Pantoea sp. MCC16, and this chemotaxis reached its
   maximum value at 20 μmol l^−1 nobiletin ([121]Figure 5A). We next
   examined the effect of nobiletin on biofilm formation by Pantoea sp.
   MCC16 and found that biofilm formation was significantly promoted by 20
   μmol L^−1 nobiletin ([122]Figure 5B, ⑧ in [123]Supplemental Figure 1).
   To confirm this result, we set up an experiment with exogenous
   nobiletin irrigation in CC soil. In CCS, exogenous nobiletin had little
   effect on AR numbers. In unsterilized CC soil, however, nobiletin
   significantly promoted AR numbers, increasing them by 73.91% at DAE-10
   and 125.49% at DAE-20 compared with CCS ([124]Supplemental Figure 9).
   Finally, we set up an experiment with exogenous nobiletin irrigation in
   CC soil. Compared with CC or CCS control soil, nobiletin significantly
   increased the abundance of Pantoea sp. MCC16 in rhizosphere soil. These
   results suggested that nobiletin was a major functional substance
   secreted by CC potato roots that could recruit Pantoea sp. MCC16
   ([125]Figure 5C, ⑨ in [126]Supplemental Figure 1).

Figure 5.

   [127]Figure 5
   [128]Open in a new tab

   Validation of the ability of nobiletin to alleviate continuous cropping
   obstacles in potato.

   (A) Chemotactic analysis of 11 different concentrations of metabolites
   and Pantoea sp. MCC16.

   (B) Effect of different concentrations of nobiletin on the biofilm
   formation of Pantoea sp. MCC16.

   (C) Verification of the chemotaxis of Pantoea sp. MCC16 toward
   exogenous metabolites in sterilized CC soil. “Rhizosphere soil” denotes
   rhizosphere soil inoculated with CC20; “MCC16” denotes inoculation with
   Pantoea sp. MCC16.

   (D) Effects of exogenous nobiletin and Pantoea sp. MCC16 on the growth
   of CC potato. Different droplet properties represent different
   treatments. The blue droplet represents the control with sterile water.
   DAE, days after emergence.

   (E) Effects of exogenous nobiletin and Pantoea sp. MCC16 on the yield
   of CC potato.

   Data are shown as the mean ± SEM (n = 3). Different letters indicate
   significant differences (Tukey’s HSD test, p < 0.05).

   Finally, we performed a pot test with exogenous application of
   nobiletin and a Pantoea sp. MCC16 bacterial solution. Nobiletin and
   Pantoea sp. MCC16 significantly increased AR numbers at 10 and 20 DAE
   ([129]Figure 5D). In further studies, no significant difference was
   found in soil vanillin content under different treatments. However, the
   IAA content of potato rhizosphere soil and roots increased
   significantly ([130]Supplemental Figure 10). Exogenous application of
   nobiletin and Pantoea sp. MCC16 also significantly increased potato
   yield, although their modes of action differed. Nobiletin mainly
   increased potato quantity, whereas Pantoea sp. MCC16 mainly increased
   potato unit weight ([131]Figure 5E, ⑩ in [132]Supplemental Figure 1;
   [133]Supplemental Figure 12). This difference may have occurred because
   exogenous nobiletin changed the structure of the rhizosphere microbial
   community, thus changing microbial community function.

Discussion

   Agricultural ecosystems possess an inherent capacity for self-repair,
   which manifests as the alleviation of growth suppression caused by
   biotic or abiotic stresses when environmental conditions improve
   ([134]Ma et al., 2019; [135]Davis et al., 2023). However, the ability
   of agricultural ecosystems to overcome CC obstacles has not yet been
   investigated extensively. This failure may be due to plant–soil systems
   not reaching self-repair thresholds after an insufficient number of CC
   years ([136]Xuan et al., 2024). In addition, the initial self-repair of
   plant–soil systems cannot compensate for losses in the final economic
   yield, leading to the misconception that CC systems lack self-repair
   ability ([137]Wang et al., 2024a). In this study, even though the final
   yield of CC potato was significantly lower than that of NCC potato, the
   roots of CC potato actively recruited beneficial bacteria during the
   seedling stage to increase rhizosphere IAA content, thereby alleviating
   the decline in root biomass caused by self-toxic stress and further
   promoting the morphogenesis of CC potato ([138]Figure 6). Previous
   studies on CC obstacles have focused primarily on healthy plants that
   do not show CC obstacles in a small number of fields ([139]Song et al.,
   2021; [140]Sui et al., 2023). In this study, our findings suggest that
   the CC system leads to positive regulation by the majority of plants
   experiencing CC obstacles. Given the prevalence of these plants, the
   findings of this study may have significant implications for artificial
   regulation strategies aimed at enhancing plant adaptation to CC
   environments. In addition, the results revealed a new mechanism by
   which CC plants autonomously recruit plant-growth-promoting bacteria to
   alleviate CC obstacles. Thus, this study demonstrates that beneficial
   microorganisms can be obtained from rhizosphere soil with CC obstacles,
   providing a new way to overcome difficulties due to CC.

Figure 6.

   [141]Figure 6
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   Conceptual model of plant self-repair under continuous cropping
   obstacles.

   (1) When potatoes are replanted into continuous cropping (CC) potato
   soil with accumulated vanillin, seedlings suffer vanillin stress,
   resulting in inhibited IAA synthesis in the roots, lower adventitious
   root (AR) numbers, and suppressed aboveground growth. (2) Under CC
   vanillin stress, potato plants adopt autonomous adaptive strategies by
   secreting substantial amounts of nobiletin from roots, which acts as a
   signaling molecule to recruit Pantoea sp. MCC16 for rhizosphere
   enrichment. (3) In the CC potato rhizosphere, Pantoea sp. MCC16
   secretes abundant IAA (indole-3-acetic acid), increasing rhizosphere
   IAA levels and subsequently enhancing IAA accumulation in the roots.
   (4) Consequently, under vanillin stress, CC potato seedlings exhibit
   increased AR numbers, promoting aboveground plant growth and ultimately
   achieving mitigation of CC obstacles.

Self-repair in a continuous cropping system dose not directly reduce the
content of an autotoxic substance

   The accumulation of autotoxic substances is the main factor that causes
   autotoxicity during CC of plants. To date, most research on mitigating
   plant autotoxicity has focused on how to eliminate autotoxic substances
   ([143]Zhang et al., 2010; [144]Chen et al., 2011; [145]Liu et al.,
   2023). However, according to the results of this study, the ultimate
   goal of the measures taken by potato plants to deal with autotoxic
   stress was not to reduce the content of autotoxic substances but to
   compensate for the plant growth defects caused by autotoxic stress,
   mainly by increasing AR numbers to promote plant growth at the seedling
   stage ([146]Figure 1). This strategy may be a sustainable positive
   regulatory measure chosen by plants independently under long-term
   autotoxic stress to obtain greater stability. A recent study also
   demonstrated for the first time that continued allelopathic stress can
   promote plant microorganisms that have positive effects on plant
   performance ([147]Revillini et al., 2023). Research also indicates that
   nonresistant varieties that have been exposed to the same stress for an
   extended period are more likely to induce soil resistance to the
   stress, resulting in what is commonly referred to as resistant soil
   ([148]Raaijmakers and Mazzola, 2016). In addition, when a plant chooses
   to alleviate its own stress by reducing autotoxic substances in the
   soil environment, the plant’s continued input of those substances into
   the soil is not affected. Moreover, relying on microorganisms to
   degrade autotoxic substances over the long term will continue to
   consume the plant’s energy. By contrast, improving the plant’s own
   growth under autotoxic stress can adjust metabolism in the short term,
   thereby reducing the input of autotoxic substances into the soil and
   stably improving the soil environment under CC ([149]Xuan et al.,
   2024).

   Under vanillin stress during CC, a decrease in AR numbers due to
   vanillin inhibition of root IAA content is the main factor that causes
   reduced root biomass ([150]Ma et al., 2023b). However, under continued
   vanillin stress in the present study, the IAA content of potato roots
   increased, the expression of root IAA-responsive genes increased
   significantly, and the expression of root IAA-synthesis genes decreased
   significantly ([151]Figure 1D). The results indicated that the
   significant increase in IAA content in CC potato roots was closely
   associated with increased IAA content in the soil. [152]Li et al.
   (2019) observed a significant decrease in the expression of
   IAA-synthesis and -response genes in the roots of CC peanuts. In this
   study, this decrease may have been attributable to the relatively short
   duration of CC, during which the system did not reach the threshold at
   which plants initiate self-repair. Furthermore, although CC potatoes
   did exhibit self-repair, the increase in AR numbers was delayed by
   approximately 10 days compared with that of normal plants ([153]Figure
   1A; [154]Supplemental Figure 2A). This may have been because, after
   potato seedlings are stressed by vanillin, they require some time to
   respond to the recruitment of specific functional microorganisms.
   Previous studies have also shown that, in addition to the perception of
   stress signals, the initiation of in vivo signal transduction, and the
   activation of molecular network cascades of gene expression ([155]Yang
   et al., 2023), plant adaptation to environmental stress also requires
   host–microbe interaction signaling ([156]Rodriguez et al., 2019;
   [157]Song et al., 2021). Although insufficient AR numbers in the early
   stage may be the main reason why the yield of CC potatoes is
   significantly lower than that of normal plants ([158]Huang et al.,
   2017), AR numbers are expected to rise as the degree of self-repair
   gradually increases.

Change in the rhizosphere microbial community promotes root morphogenesis of
continuous cropping plants

   Rhizosphere microorganisms have vital roles in plant growth,
   participating in growth and development, resistance to stress, and
   other processes ([159]Berendsen et al., 2012; [160]Kwak et al., 2018;
   [161]Zhang et al., 2019). The roles of microorganisms in plant growth
   extend beyond the current growing season and also help future
   generations of plants to adjust to the growth environment
   ([162]Wilkinson, 1997; [163]Wilkinson and Sherratt, 2001; [164]Bakker
   et al., 2018). In plants that reproduce vegetatively, progeny also
   receive a considerable proportion of symbiotic bacteria and fungi from
   the parent plant ([165]Vannier et al., 2018). However, although host
   genetics can significantly influence the assembly of rhizosphere
   microbial communities, these effects are weaker than those of
   environmental factors ([166]Pérez-Jaramillo et al., 2017;
   [167]Thiergart et al., 2019). In addition, unlike other vegetatively
   propagated plants, the seed potatoes planted each year are virus free
   and propagated from tissue-culture seedlings. With this approach, it is
   not possible to pass the stress memory of potato CC obstacles to
   offspring. Thus, the most effective strategy for alleviating the CC
   obstacles encountered during potato cultivation is to develop soil
   resistance to CC through sustained planting practices. This process
   enables the soil to acquire resilience in the face of environmental
   changes, resulting in the formation of resistant soil.

   In current agricultural research, greater attention is given to the
   development of resistant soil formed through the persistent induction
   of a specific pathogen, in which the soil acquires a strong resistance
   to that pathogen. This acquisition proceeds from no resistance to
   significant resistance and is due primarily to significant enrichment
   of antagonistic bacteria with specific resistance to the pathogen in
   the soil ([168]Song et al., 2021). However, production problems caused
   by non-pathogenic factors can also lead to the formation of resistant
   soil, such as the enhancement of soil recovery capacity by long-term
   drought stress through changes in soil microbial community function
   ([169]Canarini et al., 2021). Less research has focused on the
   induction of resistant soil by long-term CC stress. In this study,
   rhizosphere microorganisms from CC potatoes increased AR numbers by
   increasing root IAA content ([170]Figure 2; [171]Supplemental Figure
   3). The presence of these microorganisms was likely the result of
   independent selection by potatoes under long-term CC conditions.
   Studies have shown that the microbiome selected by allelopathic
   substances can moderately reduce the plant growth inhibition caused by
   allelopathy ([172]Revillini et al., 2023). However, the selective
   effect of root signals on soil microorganisms during plant growth may
   be stronger than that of a single allelopathic substance ([173]Venturi
   and Keel, 2016). In addition, plants show dynamic changes in microbial
   recruitment during different growth stages ([174]Shi et al., 2015;
   [175]Cordovez et al., 2021; [176]Santoyo, 2022). In this study, the
   difference in microbial communities between CC and normal potato plants
   was much smaller at 10 DAE than at 20 DAE. At the same time, the
   absolute abundance of rhizosphere microorganisms in CC potato plants
   increased from 10 to 20 DAE. This result was the opposite of that
   observed for normal potato plants ([177]Figures 3A and 3B) and may
   reflect an effect of CC soil on the components and contents of potato
   root exudates, which in turn increased soil bacterial diversity
   ([178]Supplemental Figure 4A). However, in contrast to this study, most
   studies have found that CC significantly reduces bacterial diversity
   ([179]Tan et al., 2017; [180]Zhao et al., 2020), a result that may be
   related to the different backgrounds of the plant samples. The CC
   barriers to plant growth in the present study were caused by
   autotoxicity rather than disease, which reduced the negative
   interactions between microorganisms to a certain extent. In addition,
   owing to the dynamic changes in soil microorganisms, sampling time also
   has an important effect on the measurement results. We believe that the
   seedling stage, a critical period for the establishment of plant
   morphology, is more important than other periods.

   Plant–microbe interactions can be long term or short term
   ([181]Cordovez et al., 2021; [182]Orozco-Mosqueda and Santoyo, 2021;
   [183]Santoyo, 2022). Previous studies have suggested that short-term
   interactions with specific probiotic bacteria may contribute to plant
   health and productivity ([184]Berlanga-Clavero et al., 2020). In the
   present study, Pantoea sp. MCC16 appeared only at 20 DAE in CC potatoes
   ([185]Figure 3C), indicating that there was a short-term interaction
   between CC potato plants and Pantoea sp. MCC16. This may have been
   because the IAA required to establish the potato root system reached
   its peak at the seedling stage, whereas later growth does not require
   abundant IAA in the soil. However, change in the abundance of Pantoea
   sp. MCC16 is a dynamic process. Although the findings of this study
   suggest that Pantoea sp. MCC16 appears only in CC20, this does not mean
   that no Pantoea sp. MCC16 is present in CC10 and CC30 but rather that
   the detection of low-abundance strains is limited by sequencing
   technology and bacterial isolation and culture methods. In addition,
   beneficial microorganisms can improve plant growth by secreting plant
   hormones into the surrounding environment and also by affecting plant
   gene expression to cause physiological changes ([186]Angulo et al.,
   2022).

   Microorganisms in the environment typically alter plant IAA levels
   through two mechanisms: one is the synthesis of IAA by microorganisms
   for direct absorption and utilization by the plant, and the other is
   the induction of IAA production by the plant itself ([187]Etesami and
   Glick, 2024). Plant absorption of exogenous IAA can occur through
   diffusion or via transport proteins that facilitate its entry into the
   root system. The process by which microorganisms stimulate the
   autonomous production of IAA in plants often involves signaling
   pathways activated by metabolic byproducts or other compounds from the
   microorganisms. For instance, rhizobia AS5 and AS55 stimulate the
   expression of IAA12 and YUCCA2 in Arabidopsis, leading to an increase
   in endogenous IAA levels ([188]Sujkowska-Rybkowska et al., 2022).
   Lysinibacillus spp. can promote plant growth by producing IAA and
   influencing the plant’s own IAA production ([189]Pantoja-Guerra et al.,
   2023). Our previous research found that exogenous IAA can enhance the
   IAA level of potato roots under vanillin stress in a sterile matrix,
   demonstrating that the root can use exogenous IAA directly ([190]Ma et
   al., 2023b). In the present study, the rhizosphere microorganisms of CC
   potato did not affect the expression of auxin synthesis genes in potato
   roots under autotoxic stress but did significantly increase the
   expression of auxin-response genes ([191]Figure 2B), indicating that
   the increased AR numbers may have been due to direct utilization of IAA
   produced by MCC16 in the soil. Given that transport of IAA into and out
   of plant cells involves both intramembrane receptors (such as
   TIR1/AFBs) and extramembrane receptors (such as ABL and TMK) and that
   IAA does not directly enter plant cells from the outside, it will be
   necessary to further investigate the activity of both intramembrane and
   extramembrane receptors in order to characterize the mechanism by which
   Pantoea sp. MCC16 increases root IAA levels. In addition, although more
   IAA-producing bacteria were screened in the rhizosphere of normal
   potato plants, the IAA-producing ability of such bacteria in the
   rhizosphere of CC potatoes was stronger ([192]Figure 3D). Moreover,
   similar to previous studies ([193]Zhao et al., 2020), the IAA-producing
   bacteria screened in the rhizosphere of normal potato plants were all
   Firmicutes, whereas those in CC soil were all Proteobacteria
   ([194]Figure 3E; [195]Supplemental Figure 4C). This result may reflect
   the continuous accumulation of root exudates due to CC, which changed
   the soil environment and excluded those microorganisms for which the
   growth conditions were not suitable. Although many studies have
   reported the potential of Pantoea to promote plant growth
   ([196]Hernández-Forte et al., 2022; [197]Kumar et al., 2022), the genus
   is not common in CC systems, possibly because of differences in
   sampling times and CC duration.

Importance of root exudates in self-repair of the plant–soil system

   Changes in the composition of root exudates during plant growth lead to
   corresponding changes in the structure of the rhizosphere microbial
   community ([198]Shi et al., 2015; [199]Cordovez et al., 2021;
   [200]Santoyo, 2022). However, when plants are exposed to external
   environmental stress, the composition and content of root exudates
   often differ significantly from those observed under normal conditions
   during the same period ([201]Stringlis et al., 2018; [202]Gong et al.,
   2023). There has been extensive research on root secretions during the
   formation of resilient soil. For example, after infection by P.
   syringae pv. tomato for five consecutive generations, Arabidopsis
   thaliana secretes increased amounts of long-chain organic acids and
   amino acids to regulate the soil bacterial community and increase plant
   resistance ([203]Yuan et al., 2018). Wild soybean (Glycine soja) exudes
   key metabolites, such as purines, to recruit beneficial Pseudomonas
   strains against salt stress ([204]Zheng et al., 2024). In this study,
   at 20 DAE of CC potatoes, roots increased the secretion of nobiletin to
   recruit Pantoea sp. MCC16 and promote the establishment of seedling
   morphology ([205]Figures 4D and [206]5C). The secondary metabolite
   nobiletin is a member of the polymethoxyflavonoids, a subclass of
   flavonoids ([207]Kawai et al., 2007). Flavonoids, phenylpropanoids,
   nitrogenous compounds, and terpenoids are the main categories of
   root-specific metabolites and play crucial roles in regulating the
   assembly of specific microbial populations in the rhizosphere
   ([208]Singh et al., 2023; [209]Wang et al., 2024b), mainly serving as
   signaling substances or growth substrates in the microbial recruitment
   process ([210]He et al., 2022). In this study, through preliminary
   verification, we found that Pantoea sp. MCC16 could not use nobiletin
   as a substrate ([211]Supplemental Figure 11). Therefore, nobiletin may
   effectively recruit Pantoea sp. MCC16 because the bacterium contains
   flavonoid-sensing regulators. Plant-derived flavonoids can be sensed by
   the TetR regulator PhlH in P. fluorescens 2P24 and act as important
   signaling molecules, providing a sensing mechanism that facilitates the
   movement of microorganisms at the root–soil interface and increases
   their colonization on the root surface ([212]Yu et al., 2020). However,
   the proliferation of Pantoea sp. MCC16 requires nutrients whose
   availability may be significantly influenced by certain substances in
   the soil. It is currently unclear whether these substances are produced
   by the host plant’s root exudates or result from changes in soil
   microbial community structure, potentially leading to an increase in
   microorganisms whose secreted metabolites support the growth of Pantoea
   sp. MCC16 or to a decrease in microorganisms that inhibit its growth.

   The shikimic acid pathway is one of the main secondary metabolic
   pathways involved in plant responses to external stress, and IAA,
   nobiletin, and vanillin are all secondary metabolites produced through
   the shikimic acid pathway in potatoes. Both nobiletin and vanillin are
   produced by the phenylalanine metabolism pathway, whereas IAA is
   produced by the tryptophan metabolism pathway. Both pathways use
   chorismate as the initial precursor for synthesis ([213]Maeda and
   Dudareva, 2012). In this study, the IAA content of CC potatoes
   decreased, suggesting that the contents of phenylalanine-pathway
   products may have increased. In addition, vanillin is synthesized from
   ferulic acid ([214]Gallage and Møller, 2015), whereas nobiletin is a
   flavonoid. Synthesis of both ferulic acid and flavonoids relies on a
   common precursor, p-coumaric acid ([215]Mandal et al., 2010). We found
   that the nobiletin content of CC potatoes increased significantly,
   suggesting that vanillin synthesis in these plants may have decreased,
   consistent with previous results ([216]Ma et al., 2023b). This strategy
   not only promotes plant growth in the current season but also gradually
   reduces the amount of autotoxic substances released into the soil,
   thereby improving the soil environment in subsequent CC seasons.
   Because self-repair is initiated by promoting plant growth at the
   seedling stage under CC vanillin stress, this strategy also explains,
   at least in part, why CC potato plants choose to secrete nobiletin to
   recruit Pantoea sp. MCC16.

   Although the findings of this study demonstrate that potato plants
   adapt to CC obstacles under vanillin stress in the soil, this does not
   preclude the possibility that soil vanillin content may decrease as the
   adaptive process progresses. A recent study showed that tobacco adapts
   to CC obstacles primarily by reducing soil vanillin content ([217]Xuan
   et al., 2024), in contrast to the results of the present study. This
   discrepancy may be attributed to the shorter duration of CC in the
   current study or to variations in microbial community composition
   across different regions, which lead to distinct optimal strategies for
   different plants. Furthermore, the findings of this study indicate that
   Pantoea sp. MCC16 is a key functional strain that alleviates potato CC
   obstacles, nobiletin is the key root exudate that recruits Pantoea sp.
   MCC16, and 20 DAE is a critical period for adaptation to CC obstacles.
   However, factors such as region, climate, and cultivar may lead to
   differences in the primary functional strains that help potatoes adapt
   to CC obstacles, the main root exudates that recruit functional
   strains, and the critical timing of recruitment. Despite these
   differences, the underlying mechanism for alleviation of CC obstacles
   identified in this study remains broadly applicable.

   Although self-repair capabilities have been demonstrated in long-term
   CC systems, this ability cannot be relied upon to compensate for the
   consequences of inappropriate agricultural practices. In practical
   production, annual yield losses can severely impact laborers’ income
   and industrial development. To protect and enhance these vital
   agricultural ecosystems, sustainable agricultural management strategies
   must be implemented. The direct application of beneficial bacteria to
   the soil is currently the primary method used in agriculture to
   regulate plant growth. However, owing to unsuitable soil environments
   and ecological niches, as well as competition for resources with
   indigenous microorganisms, beneficial bacteria often fail to
   effectively colonize the rhizosphere ([218]Alabouvette et al., 2009;
   [219]Wang et al., 2019). The use of beneficial bacteria from the
   indigenous microbial community may therefore represent an effective
   solution. In this study, application of the beneficial bacterium
   Pantoea sp. MCC16 to CC soil significantly increased potato yields by
   51.09% compared with the control ([220]Figure 3F). Moreover, in
   large-scale field production, the direct large-scale application of
   bacterial inoculants may consume substantial resources. The enrichment
   of target bacterial strains in the soil at the appropriate time may
   thus provide a more efficient approach. In this study, Pantoea sp.
   MCC16 only became enriched in the rhizosphere 20 days after the
   emergence of CC potatoes, which was clearly disadvantageous for early
   plant development. Therefore, nobiletin was applied to the soil on the
   day that the potatoes emerged. Potato root biomass and aboveground
   growth were significantly higher in nobiletin-treated plants than in
   the controls at 10 DAE, ultimately leading to a 35.86% increase in
   potato yield ([221]Figure 5D and 5E; [222]Supplemental Figure 9).
   Although the direct application of nobiletin was less effective than
   the direct application of Pantoea sp. MCC16, the findings confirmed the
   feasibility of this approach. Therefore, manipulating the timing and
   composition of root exudates can effectively enhance the colonization
   of beneficial bacteria and lead to more sustainable and efficient
   agricultural practices, reducing reliance on synthetic fertilizers and
   pesticides. By identifying and promoting the growth of indigenous
   beneficial bacteria, farmers can enhance plant health and resilience,
   leading to higher yields and reduced environmental impacts. The use of
   natural compounds such as nobiletin to stimulate beneficial bacterial
   growth could also represent a cost-effective and environmentally
   friendly strategy for improving crop performance.

   In conclusion, this study revealed that potatoes mitigate CC obstacles
   by increasing root IAA content and AR numbers under vanillin stress in
   the soil. Potato roots counteract vanillin stress by secreting
   nobiletin, attracting the high-IAA producing bacterium Pantoea sp.
   MCC16, raising the levels of IAA in the rhizosphere, enhancing root IAA
   uptake, increasing AR numbers, and improving plant resilience, thereby
   alleviating CC challenges ([223]Figure 6).

Methods

CC field experiment and sample collection

   The potato CC positioning test was initiated in 2011 at the potato
   experimental station (35°6′41″ N, 103°59′4″ E, 2276 m above sea level)
   in Weiyuan County, Dingxi City, Gansu Province, China, with an annual
   average temperature of 6.8°C, an annual rainfall amount of 500 mm, and
   a frost-free period of 166 days. The soil type was black loam, which is
   loose, soft, and easy to cultivate. The physical and chemical
   properties of the soil in the cultivated layer (0–20 cm) of the
   abandoned land were as follows: total nitrogen 0.746 g kg^−1, total
   phosphorus 0.638 g kg^−1, alkaline nitrogen 42.02 mg kg^−1, available
   phosphorus 13.80 mg kg^−1, available potassium 184.99 mg kg^−1, organic
   matter 16.26 g kg^−1, and a pH of 8.46. The only variable in this test
   was the number of CC years. Each year, a new piece of land was
   introduced as a CC treatment for 1 year. By 2020, the plots had
   durations of CC ranging from 1 to 10 years. Owing to the short
   frost-free period in the area, potatoes are planted in early May and
   harvested in early October each year. The potato variety was Longshu
   no. 7 (pre-elite seed), which was provided by the Potato Research
   Institute of the Gansu Academy of Agricultural Sciences (Dingxi,
   China). Each CC period was represented by one plot, with an area of
   48.6 m^2 (9 × 5.4 m), row spacing of 60 cm, plant spacing of 30 cm, and
   planting density of 5.56 × 10^4 plants ha^−1. Fertilization occurred on
   the day of potato planting and included 150 kg ha^−1 nitrogen
   fertilizer (urea, 46% N; one-quarter of the nitrogen fertilizer was
   top-dressed during the budding stage in combination with soil
   cultivation), 120 kg ha^−1 phosphate fertilizer (diammonium phosphate,
   18% N and 46% P[2]O[5]), 75 kg ha^−1 potassium fertilizer (52% K[2]O),
   and 37.5 t ha^−1 of organic fertilizer (fresh sheep manure). Weeding
   was performed manually, and 25% leucine was applied during the budding
   stage to control aphids. Metalaxyl manganese zinc (58%) was used to
   control late blight, with four sprays performed based on field
   conditions.

   On the basis of previous research ([224]Ma et al., 2023b), 7 years was
   selected as the representative period for potato CC obstacles.
   Therefore, the CC treatment in this study refers to 7 years of CC,
   whereas the NCC treatment refers to 1 year of CC. Previous studies
   ([225]Ma et al., 2023b) also indicated that the autotoxicity effect of
   CC significantly inhibits AR numbers in potato seedlings during the
   mid-seedling stage. However, field experiment observations revealed no
   significant difference in AR numbers between CC and NCC during the late
   seedling stage. To further investigate the changes in AR number during
   the seedling stage, we collected CC and NCC potato plants from the
   field and analyzed plant morphology, root IAA content, root expression
   of genes related to IAA synthesis and response, soil vanillin content,
   and yield. Details of sample collection are provided in Supplementary
   Method 1.

Soil sterilization and microbial transfer experiments to identify soil
microbial functions in CC

CC soil sterilization test

   Four treatments were used: unsterilized CC soil, CCS, unsterilized NCC
   soil, and sterilized NCC soil. The test soil and seed potatoes (Longshu
   no. 7, pre-elite seed) were transported from Weiyuan Potato
   Experimental Station, Dingxi City, Gansu Province, China, to Sichuan
   Agricultural University (Chengdu, China) in late November 2021. For the
   sterilization treatment, we used two high-pressure steam sterilization
   events (121°C, 1.5 h) with an interval of 3 days ([226]Zheng et al.,
   2017). After the second sterilization, sterilized soil extract was
   isolated and cultured on Luria–Bertani (LB) solid medium to ensure that
   no bacteria could be isolated from the soil. The specific test
   procedure was as follows. First, 30 pots were set up for each treatment
   (300 mm in upper diameter × 210 mm in height). Each pot was filled with
   4 kg of soil and then planted with one grain of whole potato weighing
   approximately 50 g at a sowing depth of 10 cm. The seed potatoes were
   disinfected with 0.1% sodium hypochlorite solution for 20 min and then
   washed with sterile distilled water. When potato plants had emerged to
   approximately 1 cm, they were watered with 200 ml Hoagland nutrient
   solution once every 7 days. The application times of the Hoagland
   nutrient solution were increased appropriately according to the potato
   growth, and watering ceased after the tuber-formation stage. Throughout
   the potato growth process, the water supply was appropriate, pests and
   diseases were controlled, and weeding was performed manually. For
   details of sample collection, please see Supplementary Method 2.

Microbial flora transfer test

   The rhizosphere soils of CC and NCC plants in the CC potato field test
   were used as the source of microbial flora, and potatoes were planted
   in the field as described in the first experiment. First, field
   cultivation was performed in early May 2022, and a new plot was used
   for the NCC treatment (with specific cultivation measures such as those
   in the first experiment). Before potato planting, 400 kg of CC topsoil
   was collected and transported to Sichuan Agricultural University for
   sterilization treatment (as described above), then used as the basic
   soil for the subsequent experiment. At 10, 20, and 30 DAE, 10 CC and 10
   NCC plants were selected. Soil clods of approximately 20 cm around the
   stem base of the plants were loosened with a shovel. After the plants
   were gently pulled out, some of the soil was shaken off, but the roots
   remained wrapped in soil. The rhizosphere soil of different plants from
   the same treatment was then collected into the same aseptic bag with a
   sterile brush, mixed, and stored temporarily at 4°C.

   To ensure maximum microbial activity in the rhizosphere soils, pot
   tests were performed on the day after rhizosphere soil collection (10,
   20, and 30 DAE). Therefore, the experiment was divided into three
   periods with intervals of 10 days. The method for microflora transfer
   was based on [227]Jin et al. (2023). The specific method was as
   follows. CC and NCC rhizosphere soils were transferred to sterile soil
   at a 3% (g) inoculation rate, with a total of 4 kg soil per pot (300-mm
   upper diameter × 210-mm height). Potatoes were disinfected with 0.1%
   sodium hypochlorite solution for 20 min before planting and then washed
   with sterile distilled water. One seed potato was planted in each pot
   at a planting depth of 10 cm. Fifteen pots were set up for each
   treatment, and the pots were placed in a light culture room for
   cultivation (12-h light, 12-h dark). When the potato plants had emerged
   to approximately 1 cm, 200 ml Hoagland nutrient solution was applied
   once every 7 days. The application times of the Hoagland nutrient
   solution were increased appropriately according to potato growth, and
   watering ceased after the tuber-formation stage. During the potato
   growth process, the water supply was appropriate, pests and diseases
   were controlled, and weeding was performed manually. Potato plant
   samples were collected from each treatment at 10, 20, and 30 DAE, and
   the effects of rhizosphere flora on potato AR number, root IAA content,
   rhizosphere soil IAA content, soil vanillin content, and yield were
   determined. Potato yield was measured in five pots.

Profiling the potato rhizosphere microbiome

   16S rRNA absolute quantitative analysis of rhizosphere bacterial
   communities was performed at 10 and 20 DAE in CC (CC10, CC20) and NCC
   (NCC10, NCC20) soils to explore the structure of the rhizosphere soil
   microbial community during the period. For details of 16S rRNA
   sequencing, please see Supplementary Method 3.

Isolation of rhizosphere bacteria and screening of IAA-producing strains and
their effects on potato growth

   Rhizosphere bacteria were isolated and cultured at 10 (I), 20 (M), and
   30 (F) DAE in the CC and NCC treatments. The specific steps were as
   follows. In June 2022, plants in CC and NCC soils were retrieved from
   the field, and rhizosphere soil was collected. Owing to the difference
   in sampling times, the standard continuous-dilution culture method was
   used to isolate culturable bacteria immediately after retrieval. Soil
   suspensions with different concentrations (n = 5) were plated on solid
   LB medium and cultured overnight at 37°C. After colony formation,
   labels were made successively according to colony morphology, and
   bacteria were picked out. Purified single colonies were obtained by
   plate marking on the corresponding LB medium, and the lines of single
   colonies were redrawn twice to ensure colony purity. Finally, the
   strains were inoculated into LB broth medium and stored at −80°C.

   Salkowski’s reagent method ([228]Sarwar and Kremer, 1995) was used to
   examine the IAA production capacity of all isolated bacteria. Seven
   strains of bacteria with an IAA production capacity greater than 20 μg
   ml^−1 were identified, followed by sequence alignment
   ([229]Supplementary Table 2; [230]Supplemental Figure 4C). According to
   the identifications, the MCC16 strain and the unique strain ASV7733
   identified in CC20 rhizosphere soil were the same strain, and this
   strain also had the strongest IAA production capacity. Therefore, an
   irrigation test with MCC16 bacterial solution was designed to determine
   whether this strain could promote the growth of CC potatoes. The
   specific design of this experiment is described in Supplementary Method
   4.

Pot experiment to determine the effect of CC root exudates on potato growth
and Pantoea sp. MCC16

   Root exudates of CC and NCC potato were collected at 10 and 20 DAE and
   used to irrigate CC potato. The specific procedure is described in
   Supplementary Method 5.

Nontargeted metabolomics analysis and differential metabolite screening of
root exudates

   Nontargeted metabolomics analysis of root exudates from CC10, CC20,
   NCC10, and NCC20 was performed to identify which metabolites had major
   effects. Root exudates were collected, then freeze-dried in an LGJ-18S
   vacuum freeze dryer (Bonnin Instrument Technology, Wuhan, China) and
   stored at −80°C. The nontargeted metabolomics procedure is described in
   Supplementary Method 6. The purpose of the differential metabolite
   screening was to screen for substances that increased significantly in
   the root exudates of CC20 potato. Significance analysis (TOP50), random
   forest analysis (TOP40), KEGG pathway enrichment analysis (TOP10), and
   PCA (TOP40) were used to screen for substances that increased
   significantly in the root exudates of CC20 potato. The screened
   metabolic substances were significantly more abundant in CC20 than in
   CC10 and NCC20.

Effects of differential metabolites on chemotaxis and biofilm formation by
Pantoea sp. MCC16

   The procedures used to detect chemotaxis and biofilm formation in vivo
   are described in Supplementary Method 7. Supplementary Method 8
   describes the approach used to verify whether the target metabolite
   (nobiletin) increased AR numbers in CC potatoes through soil
   microorganisms. Chemotaxis and biofilm formation were then verified in
   the soil. First, sterilized CC soil was placed in pots (10 cm in upper
   diameter × 10 cm in height). Ten pots received injections of nobiletin,
   and 10 pots received injections of sterile water as a control. Each pot
   contained 400 g of soil, and 1 ml of Pantoea sp. MCC16 bacterial
   solution (1.0 × 10^8 colony-forming units [CFU] ml^−1) was injected
   vertically into the soil with a 1-ml syringe at four locations 4 cm
   from the center of the pot. A syringe was also used to vertically
   inject 10 ml of 20 μM nobiletin solution or sterile water as the
   control, and the pots were then placed at 37°C for constant temperature
   culture, during which the same amount of nobiletin solution was added
   every 3 days. The central soil column of the pots was collected with a
   hole punch (1-cm diameter) on the 10th day. Bacteria were isolated and
   cultured by the standard continuous-dilution culture method, and the
   number of Pantoea sp. MCC16 was counted.

Effects of exogenous application of Pantoea sp. MCC16 and nobiletin on the
growth of CC potato

   Using CC soil as the background, the following four treatments were
   established: sterile water, nobiletin solution (20 μM), Pantoea sp.
   MCC16 bacterial solution (1.0 × 10^8 CFU ml^−1), and nobiletin solution
   combined with Pantoea sp. MCC16 bacterial solution. Twenty pots were
   set up for each treatment, and each pot (300-mm upper diameter × 210-mm
   height) was filled with 4 kg of CC soil and planted with one seed
   potato. In the nobiletin treatment, potatoes were watered at 3, 6, and
   9 DAE. In the Pantoea sp. MCC16 bacterial solution treatment, pots were
   irrigated once with 350 ml on the day of potato emergence. At 10 and 20
   DAE, potato plants were collected to determine plant morphology, AR
   numbers, root IAA content, rhizosphere soil IAA content, and soil
   vanillin content. The yield of 10 plants per treatment was determined
   at harvest.

Statistical analyses

   Community alpha diversity indices (i.e., operational taxonomic unit
   [OTU] number, Shannon index, and abundance-based coverage estimator
   [ACE] index) were calculated using the vegan package in R ([231]Oksanen
   et al., 2019). Beta diversity was analyzed and visualized using
   Bray–Curtis dissimilarity-based principal coordinate analysis. To test
   the effects of CC on community dissimilarities (i.e., whether there
   were differences in beta diversity), a PERMANOVA test was performed
   using the Adonis function in the vegan 2.6-4 package (Helsinki
   University, Finland) for 9999 permutations ([232]Oksanen et al., 2019).
   Functional inference was performed using PICRUSt2.0 against the KEGG
   pathway database ([233]Douglas et al., 2020). The p values were
   corrected for multiple comparisons using the Benjamini–Hochberg false
   discovery rate method ([234]Lydersen, 2024). To identify the exudate
   compounds that made the greatest contributions to the classification,
   the random forest method and default parameters of the algorithm R
   implementation were used (R package RandomForest, ntree = 1000).
   Correlations were examined using Pearson correlation analysis.
   Enrichment analysis of metabolic pathways was performed using the
   online platform MetaboAnalyst
   ([235]http://www.metaboanalyst.ca/faces/home.xhtml). A phylogenetic
   tree of bacterial rhizosphere isolates was constructed using MEGA 6
   (Temple University, Philadelphia, PA, USA). Phylogenetic trees were
   annotated and visualized using Adobe Illustrator software (Adobe, San
   Jose, CA, USA).

   All data were tested for normality (Shapiro–Wilk test) and homogeneity
   of variance (Levene’s test). To analyze microbial abundance data, we
   applied logarithmic transformation. Comparisons between two groups were
   performed using Welch’s t-test, whereas comparisons among three or more
   groups were performed by one-way ANOVA followed by Tukey’s honestly
   significant difference (HSD) test. Statistical significance was
   determined at p < 0.05. [236]Figure 3B was plotted using the
   Bioinformatics online platform
   ([237]https://www.bioinformatics.com.cn/). [238]Figures 4D and [239]5A
   were plotted using the Chiplot online platform
   ([240]https://www.chiplot.online/). [241]Figure 6 was plotted using the
   Figdraw online platform
   ([242]https://www.figdraw.com/static/index.html#/). [243]Supplemental
   Figure 1 was prepared in PowerPoint (Microsoft Corporation, Redmond,
   WA, USA). Other figures were plotted using GraphPad Prism 9 (GraphPad
   Software, La Jolla, CA, USA).

Data and code availability

   Raw 16S rRNA sequencing data were deposited in the Genome Sequence
   Archive at the China National Genomics Data Center under accession
   number CRA015542 ([244]https://ngdc.cncb.ac.cn/gsa/), and metabolite
   profiling data were deposited in the EMBL-EBI metabolomics repository
   under the identifier MTBLS12358
   ([245]https://www.ebi.ac.uk/metabolights/search).

Funding

   This research was supported by the Natural Science Foundation of
   Sichuan Province (grant number 2022NSFSC0014), the Tackling Key
   Problems and Supporting Projects of Breeding in Sichuan Province (grant
   numbers 2021YFYZ0019 and 2021YFYZ0005), the Sichuan Potato Innovation
   Team (grant number sccxtd–2023–09), and the National Natural Science
   Foundation of China (grant number 32060443).

Acknowledgments