**1. Introduction**

Replant diseases, also known as consecutive monoculture problems or sick soil syndrome, are widespread in the production of different crops, especially in medicinal crops, fruit tree and vegetables, such as *Rehmannia glutinosa*, *Panax notoginseng*, apples, peach, strawberry, soybean etc. [1–6], which usually lead to disease aggravation, poor growth status, yield reduction and quality deterioration [7]. *Rehmannia glutinosa* Libosch, a perennial herbaceous plant, is one of the 50 traditional Chinese medicines with high value [8]. However, consecutive monoculture of *R. glutinosa* results in its abnormal growth and a significant decline in the yield and quality of tuberous roots. Moreover, it cannot be replanted on

the same land for 8–10 years [9]. The causes of problems associated with consecutive monoculture of *R. glutinosa* have become a research priority in China [10].

Allelochemicals, soil-borne diseases and soil quality deterioration are usually considered as primary factors involved in the formation of replant disease [11]. Some studies concluded that the effect of soil chemical properties is inconsistent with replant disease over time [12,13]. Furthermore, allelochemicals, such as phenolic acid and flavonoids, are quickly metabolized by soil microorganisms and shape the composition and diversity of microbial community in rhizosphere soils [14–19]. Recent studies have appointed that the healthy growth of plants is closely related to the balance of the rhizosphere microbes [20–22]. There is mounting evidence that the biotic factors mediated by rhizosphere allelochemicals is a causal agent of replant disease [10,23–25]. Previous studies have discovered that the pathogenic microorganisms in replanted *R. glutinosa* rhizosphere soil were selectively attracted by root exudates, and then colonized the root surface to proliferate, resulting in rhizosphere micro-ecology catastrophe, mainly in the transformation of rhizosphere microorganisms from "bacterial" to "fungal" types and a decline in rhizosphere microbial diversity [10,11,13,26,27]. The previous evidences have confirmed that the pathogenic *Fusarium oxysporum* f.sp. *R. glutinosa* (FO) was enriched and beneficial *Pseudomonas* spp. (PS) specifically decreased in rhizosphere soil of replanted *R. glutinosa* [28–30]. However, it remains largely unknown the mechanism of how replanted *R. glutinosa* responded to the changes in composition and diversity of microbial community in rhizosphere soil.

In natural environments, plants can regulate the rhizosphere micro-ecological status to preserve healthy growth by constantly renewing the composition and diversity of microbiome [31]. Plant immune response, containing recognition to pathogens and signal transduction, thus plays a key role in coordinating microbial communities in rhizospheres [32–34]. As we know, plant innate immunity co-evolving with pathogenic microbes have developed two strategies, including pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) [35,36]. PTI is generally effective against non-adapted pathogens in a phenomenon called non-host resistance, and the pattern recognition receptors (PRRs) of PTI systems can recognize conserved PAMP features of different species or genera. ETI is active against adapted pathogens, and the receptors of ETI systems specifically and robustly respond to pathogen effectors through nucleotide-binding-leucine-rich repeat (NB-LRR) domain-mediated perception [37]. NB-LRRs are now believed to include the majority of plant R proteins and recognize fast-evolving effectors [38,39]. Our previous studies found that pathogenic FO was specifically enriched in the rhizosphere soil of replanted *R. glutinosa* [28], and PTI of two plant innate immunity silently responded to replant disease [40], which suggested that PTI was not sufficient to cope with fast-evolving effectors. However, some NB-LRR receptors in ETI were upregulated [41]. It remains unclear why these upregulated NB-LRRs fail to prevent the death of replant *R. glutinosa*.

Plant hormones are essential regulators for triggering plant immune resistance to cope with various pathogens [42]. The salicylic acid (SA) and jasmonic acid-ethylene (JA-ET) are believed to form the hormonal backbone of plant immune responses to pathogens, with SA involved in resistance to biotrophic pathogens and JA–ET involved in responses to necrotrophic pathogens and chewing insects [37,43]. The antagonism between SA and JA-ET often occurs through the regulatory protein non-expressor of pathogenesis-related proteins 1 (NPR1), which mediates the SA-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR) [44]. Enhanced disease susceptibility 1 (EDS1) acts as a SA-pathway inducer and a JA-ET pathways repressor, and mitogen-activated protein kinase 4 (MPK4) acts as a negative regulator of systemic acquired resistance, which is controlled by the SA pathway [37]. In addition, abscisic acid (ABA) can increase susceptibility to pathogens to some extent, although it is mainly associated with responses to abiotic stresses [37,45]. Some studies have described that SA was involved in the defense against *Fusarium oxysporum* and associated with acteoside accumulation, one of important pharmacodynamic component [46,47]. Our recent study displayed that plant hormones involved in the formation mechanism of *R. glutinosa*

replant disease and are closely related to immune resistance [48]. However, a comprehensive survey on the relationship between plant hormones and replant disease in *R. glutinosa* is still unknown.

Given the interferences from complex field environment and vegetative propagation, such as various pathogens, variable light and temperature conditions, maternal resistance, size of vegetative mass, germination rate and site of shoot, pot experiment in controlled conditions with regenerated plantlets is thus an ideal research method. Here, the soils from the sites in which *R. glutinosa* had never been planted and had been consecutively planted *R. glutinosa* were proportionally mixed to control the stress levels of replant disease. The regenerated plantlets of *R. glutinosa* with feeble immunity were acclimatized and transplanted in a phytotron. The dynamic changes of pathogenic microbes in the rhizosphere soils were investigated. At same time, *NB-LRRs* expression, plant hormone contents and physiological index levels were examined in the roots of *R. glutinosa*. These research results will provide important insights for further revealing the mechanism of formation of replant disease.

#### **2. Results**
