Leaf litter from Cynanchum auriculatum Royle ex Wight leads to root rot outbreaks by Fusarium solani, hindering continuous cropping

Abstract Cynanchum auriculatum Royle ex Wight (CA) is experiencing challenges with continuous cropping obstacle (CCO) due to soil-borne fungal pathogens. The leaf litter from CA is regularly incorporated into the soil after root harvesting, but the impact of this practice on pathogen outbreaks remains uncertain. In this study, a fungal strain D1, identified as Fusarium solani, was isolated and confirmed as a potential factor in CCO. Both leave extract (LE) and root extract (RE) were found to inhibit seed germination and the activities of plant defense-related enzymes. The combinations of extracts and D1 exacerbated these negative effects. Beyond promoting the proliferation of D1 in soil, the extracts also enhanced the hypha weight, spore number, and spore germination rate of D1. Compared to RE, LE exhibited a greater degree of promotion in the activities of pathogenesis-related enzymes in D1. Additionally, caffeic acid and ferulic acid were identified as potential active compounds. LE, particularly in combination with D1, induced a shift in the composition of fungal communities rather than bacterial communities. These findings indicate that the water extract of leaf litter stimulated the growth and proliferation of fungal strain D1, thereby augmenting its pathogenicity toward CA and ultimately contributing to the CCO process.


Introduction
Continuous planting fr equentl y leads to the suppression of seed germination and plant growth, a phenomenon r eferr ed to as the continuous cropping obstacle (CCO).This obstacle is observed in numerous plant species, particularly in medicinal plants like Panax quinquefolius L. (Dong et al. 2016, Tan et al. 2017, Liao et al. 2018, Li et al. 2020, 2021, Zhang et al. 2020 ), Pogostemon cablin (Zeng et al. 2020 ), Angelica sinensis (Xinhui 2010 ), and Amomum villosum (Wang et al. 2022 ).Additionally, CCO is also evident in Cynanchum auriculatum Royle ex Wight ( CA ), commonly known as "Binhai Baishouwu," primarily found in Binhai county, Jiangsu Pro vince , China (Jiang et al. 2011 ).Several studies have demonstrated the significant medicinal properties of CA , such as its antitumor, imm unomodulatory, hepatopr otectiv e, anti-inflammatory, and antidepressant activities (Chen et al. 2019 ).Consequently, CA cultivation is extensiv el y pr acticed in China and Kor ea due to its substantial economic adv anta ges (Kim et al. 2018 ).Ne v ertheless, the limited expansion of CA planting areas can be attributed to the decrease in CA yields caused by CCO (Chen et al. 2022b ).
Various perspectives have been emplo y ed to investigate the mec hanisms underl ying CCO.Numer ous studies hav e r eported and widel y ac knowledged alter ations in micr obial structur e and diversity, as well as the degradation of soil properties resulting fr om the accum ulation of phenolic acids and other autotoxins (Dong et al. 2016, Tan et al. 2017, Bai et al. 2019, Li et al. 2020, Wang et al. 2020, 2022, Chen et al. 2022b ).Chen et al. ( 2020 ) additionally highlighted that different phases of strawberry cultivation exhibit distinct changes in CCO.Building upon these findings, se v er al str ategies hav e been pr oposed and successfull y implemented, including the addition of arbuscular mycorrhizal fungi and calcium (CUI et al. 2019 ), the utilization of microalgae (Feng et al. 2022 ), nov el bioor ganic fertilizer (Ling et al. 2014 ), and the adoption of ster eoscopic cultiv ation (Liao et al. 2018 ) and intercropping systems (Zeng et al. 2020 ).
To date, no r ele v ant r eports pertaining to CCO in CA have been identified.Based on our field investigation, it has been observed that root rot disease is highly prevalent during the continuous cropping of CA .We also observed that that the aboveground components of CA , particularly the lea ves , are left to fall to the ground without being harvested or utilized due to their unappealing taste to animals.Consequently, these components ar e subsequentl y incor por ated into the ploughed layers after the r oots hav e been harv ested.Although leaf litter has been shown to contribute more to soil organic carbon than fine roots in certain plantations (Cao et al. 2020 ) and serves as a pr otectiv e measur e against runoff and erosion (Li et al. 2014 ), this practice does not align with the r equir ements of medicinal plants.A study demonstrated that the leachate derived from the stems and leaves of commonly used medicinal materials exhibited noteworthy inhibitory effects on the release of carbon, nitrogen, and phosphorus during litter decomposition, as well as on the activities of all se v en types of soil enzymes (Yu-Peng et al. 2017 ).Given the substantial amount of leaf litter that accumulates in the soil during each planting season in CA , coupled with the presence of various allelopathic substances in the leaves of numerous medicinal plants (Basotra et al. 2005, Gupta 2016, Aniy a et al. 2020 ), w e propose the hypothesis that the inhibitory effects on CA growth ar e primaril y attributed to the deposition of leaf litter r ather than the r oots extr action.Pr e vious r esearc h has demonstr ated the potential inhibitory impact of leaf litter on the growth of plants.Furthermore, the primary association between leaf litter and the plant growth index is attributed to alterations in the composition and diversity of soil microbial communities (Chen et al. 2022b ), particularl y c har acterized by an incr ease in pathogenic bacteria and a decrease in beneficial microorganisms (Pang et al. 2021, Wei-Ye et al. 2022 ).Ho w e v er, it is important to note that these findings wer e deriv ed fr om high-thr oughput sequencing tec hniques, and ther e r emains a dearth of e vidence fr om pur e cultur e experiments.
To mec hanisticall y test our hypothesis that inhibitory effects on CA gr owth ar e primaril y attributed to the deposition of leaf litter rather than the roots extraction, we contacted inoculation experiments with an isolated a significant fungal pathogen.Subsequently, we conducted a comparative analysis of the distinct impacts of root and leaf extracts on various indices pertaining to plant growth, soil quality, and pathogen activity.The outcomes of this investigation offer a theoretical foundation for elucidating the mechanism of CCO and enhancing the field management of CA .

Isolation and identification of fungal pathogen causing root rot disease
On 22 May 2022, soil samples were obtained from the rhizosphere of CA plants afflicted with root rot disease in a planting base (117.71071E, 39.0032 N) with a cultivation history of at least 15 years.To collect the soil, CA roots were vigorously shaken to dislodge loosel y adher ed soil particles .T he dislodged soil was carefull y separ ated and collected using glo ved hands , and considered as rhizosphere soil.Subsequently, the soil sample was promptly transported to the laboratory at Yancheng Teachers University in a cooler box, ensuring a maximum time lapse of 12 h.The soil pr operties wer e assessed as follo ws: pH ( H2O ) w as determined to be 8.30 using a pr ecalibr ated glass electrode in a 1:2 soil/distilled water suspension (Mclean 2015 ).The total nitrogen content was measured to be 1.07 g/kg by using UV radiation digestion and subsequent oxidation with potassium persulfate in an alkaline medium following a 2-h extraction with a 0.01 M CaCl 2 (Houba et al. 2000 ).The organic matter content was found to be 1.5% using an acidified solution of ferrous ammonium sulfate method after a digestion of the soil sample with an acidified dic hr omate (Br emner 2018 ).To pr epar e the soil sample for further analysis, it was diluted with sterile distilled water (SDW) and subjected to vortexing for 3 min.The diluted sample was then spread onto a potato dextr ose a gar (PDA) medium supplemented with 10 mg/l rifampicin and 200 mg/l ampicillin.The plates were incubated at room temper atur e in the laboratory for a period of 5-7 da ys .Fungal colonies present on the plate were subjected to repeated streaking onto a new plate until pure cultures were achieved.
Subsequently, a total of seven distinct fungal strains exhibiting different morphologies were individually assessed for their pathogenicity.The fungi were individually cultured on PDA for a period of 3-4 days at a temper atur e of 28 • C, following which spor es wer e harv ested and pr eserv ed in SDW at 4 • C before utilization.Seedlings of CA plants, possessing three to four leaves and cultivated in quartz sand, were then tr ansferr ed to pots containing 2 kg of soil each.The soil utilized for the pot experiment was collected from the campus of Y ancheng T eac hers Univ ersity.Each of the seven fungal spore suspensions was inoculated into five pots, with the final concentration of 10 9 colony-forming units (CFU) per kg of soil, specificall y ar ound the r oot ar ea.Concurr entl y, a contr ol gr oup of soil without inoculation was emplo y ed.The disease se v erity index of CA leaves was assessed on a scale ranging from 0 to 4, where 0 = no disease symptom, 1 = 0.1%-5%, 2 = 5.1%-20%, 3 = 20.1%-40%, and 4 = 40.1%-100%(the percentage of diseased leaf area).The disease se v erity v alue was determined utilizing the subsequent formula: Disease index (%) = [ (the number of diseased leaves × disease severity index)/(4 × the number of leaves evaluated)] ×100 (Lee et al. 2006 ).Subsequent to the pathogenicity analysis and symptom observation, the fungal pathogen exhibiting the highest disease index was c hosen and subsequentl y r eisolated fr om the plant r oots.Subsequentl y, the r eisolated str ain was subjected to testing to ascertain its pathogenicity to w ar ds CA .
The genomic DNA of the chosen pathogenic fungus was extracted utilizing a Rapid Fungi Genomic DNA Isolation Kit (Sangon Biotech Co., Ltd., Shanghai, China) in accordance with the manufacturer's instructions .T he internal transcribed spacer gene ( ITS ) region of the ribosomal RNA, translation elongation factor 1-α gene ( TEF1 ), and the 6-7 region of RNA polymerase II gene ( RPB2 ) were amplified using the universal primers ITS1/ITS4 (forw ar d primer: 5 -TCCGTA GGTGAA CCTGCGG-3 , reverse primer: 5 -TCCTCC GCTT A TTGA T A TGC-3 ) (White et al. 1990 ), EF1-983F/EF1-1567R (forw ar d primer: GCYCCYGGHCAY-CGTGA YTTY A T, r e v erse primer: ACHGTRCCRA T ACCACCSA TC) (Rehner and Buckley 2005 ), and RPB2-b6F/RPB2-b7.1R(forw ar d primer: 5 -TGGGGY A TGGTNTGYCCYGC-3 , r e v erse primer: 5 -CCC ATRGCYTGYTTMCCC ATDGC-3 ) (Matheny 2005 ), respectively.The resulting PCR products were subsequently submitted to Sangon Biotech.Co., Ltd for sequencing, and the obtained sequences were deposited in the NCBI database with the accession numbers PP577661 ( ITS ), PP692189 (TEF1), and PP692190 (RPB2).Subsequentl y, phylogenetic tr ees wer e constructed using the DNA sequences through the employment of Mega X software and the neighbor-joining method (Kumar et al. 2018 ).The identification of the fungal str ain r esponsible for the r oot r ot disease in CA was conducted, resulting in the identification of Fusarium solani as the causativ e a gent.

Prepar a tions of leaf, root extracts, and fungal pathogen inoculum
Fallen y ello w leav es and matur e r oots wer e collected fr om the planting base and subjected to grinding and drying at a constant weight of 65 • C. A total of 30 g of dried leaves or roots were then immersed in 1 l of SDW at a temper atur e of 24 • C for a duration of 48 h, with periodic shaking e v ery 12 h.Following the 48-h incubation period, the liquid was subjected to centrifugation at a speed of 10 000 r/min for a duration of 20 min, and the resulting supernatant was collected.The extracts were concentrated 3-fold over an 18-h period using a vacuum freeze dryer, resulting in a concentration of 90 g/l as determined by the oven-drying method at 105 • C for 5-6 h.T hus , the water extracts of leave extracts (LE) and r oot extr acts (RE) wer e obtained separ atel y and stor ed at 4 • C for future use .T he extracts were divided into two portions, filtered through a 0.22-μm pore size membrane, and autoclaved.Fusarium solani D1 was cultured on PDA media at 28 • C for a duration of 3 da ys .Subsequentl y, spor es fr om the plates wer e collected using SDW and adjusted to a concentration of 10 8 CFU/ml.The spores were also stored at 4 • C for < 1 week.

Effects of LE, RE, and D1 on seed germination
A minimum of 360 seeds of C A (variety: Binwu No .1) was subjected to surface sterilization using a 50% bleach solution for 15 min, follo w ed b y three w ashes with SDW.The surface sterilized seeds were then stored at 4 • C overnight before utilization.Half the seeds were soaked in the spore suspension (10 8 CFU/ml) for 20 min at room temperature, and then air dried.Meanwhile, the leftover seeds were soaked in SDW under the same conditions.These two portions were regarded as inoculated and uninoculated seeds, r espectiv el y.LE and RE wer e diluted to a concentr ation of 30 g/l to create stock solutions.Six treatments were established, including the control (consisting of uninoculated seeds receiving 2 ml of SDW e v ery 2 days), D1 group (consisting of inoculated seeds receiving 2 ml of SDW e v ery 2 days), LE gr oup (consisting of uninoculated seeds receiving 2 ml of LE stock solution e v ery 2 days), LE + D1 group (consisting of inoculated seeds receiving 2 ml of LE stock solution every 2 days), RE group (consisting of uninoculated seeds receiving 2 ml of RE stock solution e v ery 2 days), and RE + D1 group (consisting of inoculated seeds receiving 2 ml of RE stoc k solution e v ery 2 days).A total of 20 seeds wer e placed on a sterile filter paper in a Petri dish containing various ad diti ves as indicated abo ve , and were subsequently subjected to germination at a temper atur e of 24 • C. Eac h tr eatment and contr ol wer e r eplicated thr ee times in the experiment.On the 10th day, the germination rate of each Petri dish was determined.Additionally, in order to assess the heat resistance of the active substances in the extracts , autocla ved and filtered extracts were compared through the germination experiment.
To validate the findings of the aforementioned experiment, a supplementary pot experiment was conducted.Each treatment included six replicates, with 15 seeds sown in a pot with 2 kg of campus soil.The treatments remained consistent with those pr e viousl y enumer ated.To incor por ate the extr acts, 667 ml of both LE and RE (90 g/l) wer e uniforml y added to 2 kg of soil, resulting in a final concentration of 30 g/kg of extracts in the soil.In the D1-containing treatments, the soil surrounding the seeds was supplemented with D1 spore suspensions at a final concentration of 10 9 CFU/kg.On the 15th day, the germination r ates wer e computed by considering the number of seedling emergence.

Effects of LE, RE, and D1 on photosynthetic pigment contents
In light of the susceptibility of certain plant indicators to environmental stress, the levels of Chlorophyll Chl a , Chl b , carotenoid (Car), and phenylalanine ammonia lyase (PAL) were assessed over a period of time.A subsequent pot experiment was conducted, following the aforementioned protocol.The only deviation was the pr earr angement of seedlings at the third true leaf stage, whic h wer e subsequentl y tr ansplanted into individual pots (one seedling per pot) for this particular experiment.A minimum of 12 pots were prepared for each treatment.The methodology for the introduction of extracts and D1 remained consistent with the pr e viousl y outlined pr ocedur e.At thr ee time points, specifically day 0 (within 30 min after additions), day 15, and day 50, samples were collected from three randomly selected pots for eac h tr eatment.Additionall y, soil samples wer e obtained fr om the same three pots.Soil sampling was conducted using a soil corer with an inner diameter of 3 cm, at a depth of 10 cm around the roots of each plant.Following the r emov al of root residues, the soil samples were divided into two portions.One portion was stored at 4 • C for enzymatic activity determination within 1 week, while the other portion was stored at −80 • C for molecular analysis.
For the assays of photosynthetic pigment contents, a updated pr ocedur e with new equations were adopted (Chazaux et al. 2022 )

Effects of LE, RE, and D1 on plant defense-related enzymes
In the second pot experiment, the determination of plant defenser elated enzymes, specificall y super oxide dism utase (SOD) and peroxidase (POD), was conducted (Datt 1999, Huang et al. 2010, Kong et al. 2014, Zameer et al. 2022 ).The pr epar ation of crude extracts of these antioxidant enzymes involved homogenization of frozen leaves in a buffer medium.Specifically, a 10-g sample was homogenized in a 100-mM sodium phosphate buffer (pH 7.0) containing 1 mM ascorbic acid and 0.5% (w/v) pol yvin ylpyrr olidone for a duration of 5 min at a temper atur e of 4 • C. The resulting homogenate was then filtered through three layers of gauze towel, and the filtrate was subsequently subjected to centrifugation at 5000 × g for a duration of 15 min, after which the supernatants were collected.The activity of PAL was assessed through the quantification of the absorbance of transcinnamic acid at a wavelength of 290 nm, as modified by González-Mendoza et al. ( 2018 ).A single unit (U) of this enzymatic activity was defined as the quantity that induces a 0.01 increase in absorbance per hour.The determination of SOD involved measuring the inhibition of nitroblue tetr azolium (NBT) photor eduction by the SOD enzyme (Kumar et al. 2012 ).One unit (U) of SOD activity was defined as the quantity of enzyme that causes a 50% inhibition of the photoc hemical r eduction of NBT.The determination of POD activity involv ed the measur ement of the incr ease in absor ption at 420 nm spectr ophotometricall y, whic h r esulted fr om the oxidation of 4methylcatechol by H 2 O 2 .The enzymatic activity was quantified as one U, defined as a 0.001 change in absorbance per minute (Onsa et al. 2004 ).

Effects of LE, RE, and D1 on soil enzymes
Invertase activity was assessed using the 3,5-dinitrosalicylic acid colorimetric method and expressed as the amount of released reducing sugars derived from a 10% sucrose solution per gram of soil per 24 h at 37 • C (Fr ankeber ger and Johanson 1983 ).Urease activity was determined through the colorimetric measurement of ammonium pr oduced fr om a 0.72-M ur ea solution and expr essed as the amount of NH 3 -N in 1 g of soil after 24 h (Kandeler and Gerber 1988 ).Due to the alkaline nature of the soil samples, only alkaline phosphatase activity was detected.This was ac hie v ed by utilizing 1 mM p -nitr ophen yl phosphate ( p NPP) as a c hr omogenic substrate, and the activity was quantified as mg p -nitrophenol ( p NP) per gram of soil per 24 h (Li et al. 2016a ).

Quantitati v e polymerase chain reaction analysis of D1
To extract the total microbial DNA from ∼0.5 g of soil, a Po w erSoil ® DNA Isolation Kit (MO BIO Laboratories Inc., CA, USA) was emplo y ed, and the extracted DN A w as assessed using agarose gel electr ophor esis.For the quantification of the abundance of strain D1, a primer pair FSGq1 (5 -GGCTGAA CTGGCAA CTTGGA-3 ) and FSGq2 (5 -C AAAGCTTC ATTC AATCCTAATAC AATC-3 ) (Li et al. 2008 ), together with a specific probe (5 -6FAM-TCTTCT AGGA TGGGCTGGT-MGBNFQ-3 ) (Li and Hartman 2003 ) targeting the minor groove-binding region were utilized in quantitativ e pol ymer ase c hain r eaction (qPCR) anal ysis.A volume of 1 μl of DN A w as added to a 25-μl qPCR reaction mixture using the OmniMix HS Beads (Cepheid, Sunn yv ale , C A).Each DNA sample was tested three times by qPCR.The reaction conditions were as follows: 95 • C for 120 s, then 45 cycles of 95 • C for 120 s and 60 • C for 30 s.A standard curv e of str ain D1 DN A w as pr epar ed in triplicate using the probe in qPCR.Specifically, a 10-ng/ μl solution of the DNA of strain D1 was diluted 10-fold serially to 10 −6 ng/ μl.It was calculated dir ectl y fr om the concentr ation of extr acting plasmid carrying target genes from soil samples according to the standard method.A volume of 1 μl DNA from each dilution was added to the OmniMix HS master mix and each dilution was tested three times .T he DNA concentr ations of str ain D1 and r esulting thr eshold cycle ( C t ) v alues wer e used to construct a standard curve.

Detections of phenolic acids in soil and plant tissues
On 10 September 2022, soil samples (0-20 cm) from a planting base (120.29523E, 34.20 406 N) were collected at different durations of CA : 0 years (0 a), 2 years (2 a), and 3 years (3 a).The quantification of phenolic compounds was conducted using highperformance liquid c hr omatogr a phy (HPLC).Leaf or root samples weighing 2.0 g were placed into a 50-ml graduated plastic test tube and homogenized in a 7-ml methanol solution (adjusted to pH = 2 with 1 mol/l HCl) using an ultrasonic homogenizer (Hielscher-UP200 Ht, Germany) at a temperature of 45 • C for a duration of 40 min.The mixture was then cooled to r oom temper atur e and diluted to a final volume of 10 ml with SDW.Following centrifugation at a speed of 10 000 r/min for 20 min, 1 ml of the resulting supernatant was filtered using a 0.22-μm pore size filter.(Mattila and Kumpulainen 2002 ).The obtained r esults wer e subsequentl y adjusted based on the weight of the dried samples.
The HPLC analysis was conducted using a SunFire TM C18 column (4.6 mm × 250 mm, 5 μm) on an Agilent 1200 instrument (Ag ilent Technolog ies , Santa Clara, C A, USA).The analysis emplo y ed an injection volume of 10 μl, a flow rate of 0.6 ml/min, a column ov en temper atur e of 30 • C, and a UV detection wavelength of 280 nm.The elution mobile phases consisted of methanol (phase A) and a 1% aqueous acetic acid solution (pH 2.5) (phase B), with retention times ranging from 0 to 30 min.The ratio of mobile phase A to B was established as 1:3.To mitigate the influence of interfering components and ensure result stability and repeatability, a 10-min delay was implemented at the conclusion of each cycle.Based on the findings of prior r esearc h (Tian et al. 2015, Bai et al. 2019 ), a subset of se v en phenolic acids wer e selected as standard samples for analysis .T hese standard samples , obtained from Sigma, consisted of coumaric acid, p -coumaric acid, caffeic acid, phydr oxybenzoic acid, v anillin, ferulic acid, and syringic acid.The quantification of phenolic acids was performed utilizing the external standard method, while the identification of samples was accomplished by assessing the retention time and peak area of the analytical standards (Bai et al. 2019 ).

Effects of phenolic acids and extracts on the m ycelium gro wth of str ain D1
The present study investigated the effects of four identified phenolic acids ( p -hydroxybenzoic acid, caffeic acid, p -coumaric acid, and ferulic acid) on the growth of F. solani D1 mycelium.To accomplish this, mycelia exhibiting equivalent growth from the periphery of the colony were extracted using a 5-mm diameter hole punch and subsequently introduced into PDA media supplemented with varying concentrations (0, 2, and 6 mg/l) of the aforementioned phenolic acids.Subsequently, the diameters of the colonies were measured after 2, 3, and 4 days of cultivation at a temper atur e of 28 • C.

Effects of LE and RE on the growth of strain D1
LE or RE wer e separ atel y added to 50 ml of PDA liquid media at four different final concentrations (0, 10, 20, and 30 g/l).The media were then inoculated with strain D1 at a final concentration of 10 6 CFU/ml.The triangular flasks containing the cultures were incubated at 28 • C and 180 r/m.Culture mixtures from each treatment were collected at intervals of 4-12 h using a pipette with 2 mm truncated tips, follo w ed b y centrifugation at 8000 r/min for 15 min.The resulting precipitates were washed with SDW and subsequently dried at 80 • C overnight until a constant weight was ac hie v ed.The dry weights of the hyphae were then measured.
The method used to investigate the effects of LE or RE on the mycelium growth of strain D1 follo w ed the same procedure as pr e viousl y described.Colon y diameter was measur ed at 84 h for each plate.Ad ditionally, spore n umbers on each plate were counted using a Sysmex-5000i automated hematology counter after spore washing with SDW.Furthermor e, spor e germination r ates wer e determined using a hemocytometer after 24 h of culture in PDB media containing filtrated LE or RE at final concentrations of 10, 20, and 30 g/l, following inoculation with the same number of spores of strain D1.

Effects of LE and RE on the activities of pa thogenesis-rela ted enzymes
To investigate the impact of LE and RE on enzymes implicated in pathogenesis, the activities of four inv asion-r elated enzymes (pectinase , cellulase , β-glucosidase , and α-amylase) were assessed and compar ed acr oss v arious tr eatments (Zhang et al. 2006, Hasan et al. 2013, Bethke et al. 2016, Huang et al. 2021 ).Utilizing the gr owth curv e determination system pr e viousl y described, a volume of 2 ml of the culture solution was tr ansferr ed via pipette into a sterile tube and subsequently centrifuged at 8000 r/min for a duration of 20 min.The resulting supernatant was then utilized as the crude enzyme for subsequent analysis.Pectinase activity was measured by quantifying the reducing sugars generated thr ough enzymatic hydr ol ysis of pectin using the DNS method (Miller 1959 ).Pectin was utilized as the substrate for determining pectinase activity.The r eaction mixtur e, consisting of an equal amount of substrate (2%) prepared in citrate buffer (0.05 mol/l, pH 4.4), and a ppr opriatel y diluted enzyme, was incubated at a temper atur e of 50 • C for a duration of 30 min in a water bath.The quantification of reducing sugars released was conducted using the DNS method, with galacturonic acid serving as the standard.The enzymatic activity of pectinase was determined as the quantity of enzyme necessary to liberate 1 μmol equivalent of galacturonic acid per minute under the specified assay conditions.The cellulase activity was conducted using a citrate buffer (0.05 mol/l, pH 4.8).The enzyme was a ppr opriatel y diluted and incubated at 50 • C for 60 min in a water bath.The activity was measured using the DNS method, with a 50-mg Whatman No. 1 filter paper strip (1.0 cm × 6.0 cm) serving as the substrate .T he cellulase enzymatic activity was defined as the quantity of enzyme necessary to reduce sugars equivalent to 1 μg of glucose per ml per minute (Ghose 1987 ).The β-glucosidase activity was carried out in an acetic acid buffer (50 mmol/l, pH 5.0).The enzyme was suitably diluted and incubated at 60 • C for 10 min in a water bath.The activity was determined by measuring the hydr ol ysis of p -nitr ophen yl βd -glucopyranoside .T he activity of β-glucosidase was quantified as the quantity of enzyme that liberates 1 μmol p -nitrophenol per min ute, re presenting 1 U of enzymatic activity (Cai et al. 1999 ).Meanwhile, the activity of α-Amylase was assessed by measuring the conversion of starch into glucose using the DNS method.One unit of α-Amylase activity was defined as the amount of reducing sugar (calculated as maltose) released from starch, equivalent to 1 mg, per ml per minute under the specified experimental conditions (Lorentz 1979 ).

Scanning electron microscopy observ a tion of the morphologies of strain D1 following different treatments
Strain D1 seed cells were evenly inoculated onto PDA plates containing v arying concentr ations of LE and RE (0 and 30 mg/l) and incubated at a temper atur e of 28 • C for a duration of 84 h.Hyphae from each plate were carefully selected and placed onto a cov erslip, whic h was subsequentl y fixed in a solution of 2.5% glutaraldehyde in 0.01 mol/l PBS for a period of 1 h at room temperature .T he co verslip was then sequentially immersed in ethanol solutions of increasing concentrations (30%, 50%, 70%, 80%, and 90%) for 10 min each, follo w ed b y dehydration with anhydrous ethanol for 10 min, repeated two to three times.Subsequently, the samples were placed onto the objective table for observation after being coated with gold, with a working distance of 6.6 mm and a voltage of 3.0 kV.

Da ta anal ysis
T he ra w data were imported into SPSS Statistics for Windows version 18.0 (WinWr a p Basic , P olar Engineering and Consulting) in order to calculate means and standard errors (SE).A phylogenetic tree was constructed using DNA sequences through the utilization of Mega X software's neighbor-joining method.Principal component analysis (PCA) was conducted in R v.3.2.1 with the gg-plot2 pac ka ge (Wic kham 2016 ), whic h r elies on the av er a ge cov ariance matrix.A tw o-w ay anal ysis of v ariance (ANOVA) pr ogr am in SPSS was utilized to examine the impact of extract types [no extr act, low extr act (LE), or high extr act (RE)] and pathogen presence (with or without) on the plant growth indices.Similarly, a tw o-w ay ANOVA w as conducted to assess the effects of extract types (LE and RE) and varying amounts (0, 10, 20, 30 g/l) on the growth and activity of pathogenesis-related enzymes of strain D1.Prior to this analysis, normality was tested using the Shapiro-Wilk normality test, and a P-value of < .05 was considered significant.Subsequentl y, Bonferr oni's post hoc tests were conducted.In each plot, different asterisk symbols ( * , * * , and * * * ) indicate significant differences between samples at significance levels of .05,.01,and .001.Significant differ ences wer e observ ed at one sampling time for certain plots, as indicated by distinct letters (Duncan's multiple range test, P ≤ .05).Permutational multivariate analysis of variance (PERMANOVA) was conducted using the command "adonis" in the "v egan" pac ka ge of R to assess the effects of the different ad diti ves on the changes in the bacterial or fungal community.The line and column charts utilized in this study were generated using Sigma Plot for Windows Version 10.0 (Systat Sofware, San J ose , C A, USA).The Shannon index was computed using Mothur1 (Schloss et al. 2011 ).

Isolation and identification of fungal pathogen causing root rot disease
Of the se v en potential pathogenic fungi associated with root rot disease, strain D1 was selected for its notable pathogenicity tow ar ds CA (Fig. 1 A).Subsequentl y, str ain D1 was v erified in accordance with Koch's postulates, and the symptom induced by D1 was found to be consistent with root rot disease (Fig. 1 B), characterized by the presence of circular or irregular light brown lesions that de v elop into dark black lesions on subterranean roots and stems, resulting in stunted growth and eventual mortality.Subsequent identification of strain D1 as a member of F. solani was ac hie v ed thr ough sequences of the thr ee r epr esentativ e genes (Fig. 1 C) and the observation of falciform spores (Fig. 5 E).

Effects of LE, RE, and D1 on seed germination
Strain D1 exhibited a significant reduction in the germination rate of seeds, with a decrease of 68.29% compared to the control group without D1 addition ( P < .001)(Fig. 2 A).Autoclave treatment of LE and RE also resulted in an inhibition of seed germination rates, with reductions of 78.05% and 68.29%, respectively.Filtr ation tr eatments of LE and RE yielded similar effects to the autoclav e tr eatments (Fig. 2 B).Both LE and RE demonstrated a synergistic effect with strain D1 on seed germination.The overall trend in the influence of LE and RE on seed germination in soil closel y mirr or ed the r esults obtained fr om the filter pa per assay (Fig. 2 C).Ne v ertheless, the adv erse effects of D1 wer e not statistically significant ( P > .05)when compared to noninoculation treatments, with the exception of the RE treatment.

Effects of LE, RE, and D1 on photosynthetic pigments in leaves of CA
On day 0 (the time period immediately following the introduction of additions within 30 min), the Chl a:b , Chl a + b , and carotenoid le v els in the D1 and LE treatments did not exhibit any significant differences when compared to the control group ( P > .05,Figs 3 A-C).Ho w e v er, the Chl a:b ratio of the LE + D1 treatment was sig-nificantly higher than that of the LE treatment ( P < .05,Fig. 3 A), which was not observed in the RE tr eatments.Additionall y, the three indices of the LE and RE treatments, with the exception of carotenoid content in the RE treatment on day 15, were found to be significantly lo w er than those of the control group on days 15 and 50 ( P < .05).The presence of strain D1 further intensified this situation.

Effects of LE, RE, and D1 on the activities of defense-related enzymes in CA
Ther e wer e no statisticall y significant differ ences observ ed among the treatments on day 0 ( P > .05,Figs 3 D-F).The activities of PAL and SOD in the control group were found to be the highest on days 15 and 50, as shown in Figs 4 (D) and (E).Both LE and RE treatments resulted in a significant decrease in the activities of these

Effects of LE, RE, and D1 on soil enzymatic activities
The additions of LE + D1, RE, and RE + D1 led to notable rises in invertase activities on day 0 ( P < .05,Fig. 4 A).T he in vertase activities of RE + D1 exhibited a statistically significant increase compared to RE on day 0 and 15 ( P < .05),but not on day 50.Furthermore, the enzymatic activity experienced a significant enhancement by LE up to day 50 ( P < .05),with a slight additional increment when combined with the mixture of LE and D1.The treatments of LE, LE + D1, and RE + D1 resulted in significant increases in urease activities compared to the control group on day 0 ( P < .05,Fig. 4 B).
By day 15, all treatments sho w ed significant increases compared to the control group ( P < .05),with D1 demonstrating the most pronounced effect.By day 50, the inhibitory effect of LE + D1 on urease activity was significant ( P < .05),whereas on days 0 and 15, it sho w ed an increase compared to the control group.A similar tr end was observ ed for alkaline phosphatase activity compar ed to invertase acti vity, exce pt that no significant differences were observed among treatments on day 50 ( P > .05,Fig. 4 C).

Effects of LE and RE on the abundances of strain D1 in soil
The LE + D1 treatment exhibited the greatest abundance of strain D1 on day 0, follo w ed b y the RE + D1 and D1 treatments (Fig. 4 D).Strain D1 was not detected in the LE and RE treatments on days 0 and 15, but a ppear ed on day 50 and r eac hed v arying abundances.The abundances of D1 in the LE + D1 tr eatments incr eased ov er time and r eac hed their peak on day 15, sur passing the abundances observed in the other treatments.

Effects of LE and RE on the growth of D1
Both LE and RE demonstrated a concentration-dependent ability to enhance the hypha weights of strain D1, with RE exhibiting a more rapid effect compared to LE (Figs 5 A and B).While LE or RE slightl y inhibited colon y diameters, the extr acts signif-icantl y pr omoted spor e numbers and the percenta ge of spor e germination ( P < .01),particularly LE at a concentration of 30 g/l (Figs 5 C and D).Through scanning electron microscopy (SEM) observation, it was evident that the extracts, particularly LE30, significantly enhanced the spore numbers and hypha diameters.Specifically, the hypha diameter experienced an ∼50% increase (Fig. 5 E).

Effects of LE and RE on activities of pa thogenesis-rela ted enzymes in D1
Both LE and RE demonstrated a concentration-dependent increase in the activities of pectinase and cellulase in D1.Notably, LE exhibited a greater promoting effect on D1 compared to RE (Figs 6 A and B).Additionally, both LE and RE were found to promote the activities of β-glucosidase and α-amylase.Ho w e v er, LE did not consistently exhibit a significantly higher effect than RE, except at the concentration of 20 g/l for β-glucosidase (Figs 6 C and D).

Contents of phenolic acids in soils and extracts
In order to ascertain the resemblance of phenolic acids to pr e vious findings, specifically in terms of heat resistance, y ear-on-y ear increment, and higher prevalence in LE compared to RE, we conducted a compar ativ e anal ysis of phenolic acid contents in soils with div erse cr opping histories of CA, as well as in LE and RE.The r esults r e v ealed an incr ease in p -hydr oxybenzoic acid and caffeic acid o ver time , while p -coumaric acid and ferulic acid exhibited greater abundance in soils subjected to continuous cropping in comparison to the control soil (Fig. 7 A).Caffeic acid, p -coumaric acid, and ferulic acid were found to be abundant in LE and absent in RE, whereas p -hydroxybenzoic acid exhibited higher levels in RE compared to LE (Fig. 7 B).The impact of these four phenolic acids on the growth of strain D1 was assessed using the index of colony diameters.Caffeic acid, p -coumaric acid, and ferulic acid wer e observ ed to stim ulate the gr owth of D1 (Figs 7 C-F).

Profiles of the microbial communities following LE and D1 additions
The control group had Mortierella and Alternaria as dominant fungi on day 0, shifting to Mortierella and Pseudeurotium on days 15 and 50 (Fig. 8 A).Filobasidium decreased in the LE treatment, while Fusarium increased in the LE + D1 treatment on days 15 and 50.The top 30 genera in the treatments are shown in Fig. S3 , with 20 belonging to the Ascomycota phylum.The Bray-Curtis dissimilarity-based PERMANOVA anal ysis r e v ealed statisticall y significant impacts on Effects of LE and RE on colonies diameters and spore numbers at 84 h (C), and spore germination rates at 24 h (D).(E) Morphologies of strain D1 following LE30 and RE30 treatments using SEM, and the pictures exhibiting the spores and mycelium of strain D1 were magnified by 2000 times and 8000 times, respectively.Different numbers of asterisk ( * , * * , and * * * ) r epr esent significant differ ences between samples at P -v alues of .05,.01,and .001. the fungal community subsequent to the addition of LE or D1 ( P < .0001,R 2 = 0.936).Ov er all, the fungal comm unities on day 0 wer e distinct from those on days 15 and 50 (Fig. 8 B).The introduction of LE resulted in a gradual return to control conditions, as evidenced by the clustering of Ctrl-15, Ctrl-50, LE15, and LE50 in the bottom right corner.The addition of D1 had a significant impact on the fungal community dynamics o ver time .Interestingly, the LE + D1 treatments on days 15 and 50 exhibited similar patterns of convergence .T he introduction of LE or D1 induced alterations in fungal diversity, with LE + D1 consistently leading to a reduction in fungal diversity (Fig. 8 C).Solely the D1 treatment exhibited a significantly lo w er fungal diversity than the other treatments on day 0. On day 15, both the D1 and LE + D1 treatments displayed significantly lo w er div ersities compar ed to the contr ol and LE tr eatment.On the 15th day, the D1 tr eatment r esulted in an increase in diversity to the level observed in the control group, while the LE + D1 treatment exhibited the lo w est diversity.
In contrast, the presence of ad diti ves and the duration of the experiment did not have any significant impact on bacterial div ersity compar ed to fungal div ersity (Fig. 8 D).Lysobacter was initially abundant in both the control and D1 treatment on day 0, but its abundance decreased over time ( Fig. S2A ).Ho w ever, this genus remained stable in the LE and LE + D1 treatments.Pseudarthrobacter , Pontibacter , Planococcus , and Paracoccus were initially abundant in the LE treatment, but their abundance significantly decreased on days 15 and 50.Both PC1 and PC2 were unable to account for the differences in treatment groups ( Fig. S2B ), indicating that ad diti v es hav e minimal influence on modifying the bacterial community composition.

Discussion
CA was extensiv el y cultiv ated in Yanc heng City, Jiangsu Pr o vince , China.Concurr entl y, the pr esence of CCO was observed in CA plantations, prompting the implementation of traditional mitigation strategies such as soil flooding, fallow periods, and crop rotation.Ho w e v er, in order to optimize these efforts, it is imperative to comprehensively understand the underlying mechanisms.Notabl y, the pr olifer ation of fungal pathogens emer ged as a significant factor contributing to CCO incidence (Chen et al. 2012 ).In r ecent decades, v arious types of fungal pathogens have been identified through the use of both culturable and unculturable strategies in the context of CCO.For instance, during the CCO of American ginseng, F. oxysporum and F. solani were observed (Liu et al. 2020 ), while Phoma eupyrena and F. culmorum were prevalent in soil subjected to continuous wheat cropping (Bateman and Kwa śna 1999 ).Additionally, Cylindrocarpon sp.emerged as a prominent fungal pathogen in the continuous cropping of fluecured tobacco (Wang et al. 2020 ).In this study, the primary fungal pathogen responsible for root rot disease in CA , specifically belonging to F. solani , was successfully isolated and confirmed.The thr ee primers wer e utilized to v erify the str ain's classification at the species le v el.While the TEF1 sequence primer did not yield as highly specific results as the ITS and RPB sequence primers, it was noted that TEF1 is commonl y r ecommended for taxonomic identification of species within the genus Fusarium (O'Donnell et al. 2018 ).Ho w e v er, it is suggested that the ITS and RPB primers may be more suitable for identifying F. solani .As a prevalent pathogenic genus found in soil, F. solani is known to cause diseases in various cr ops suc h as melon (Ku et al. 2022), pean ut (Od dino et al. 2008, Xie et al. 2017 ), soybeans (Ranzi et al. 2017 ), and so on.Our study demonstrates that the F. solani strain D1 can also act as an inv asiv e pathogen, leading to root rot disease in CA .
When examining the stress response system in CA , most of the indices exhibited significant positiv e r elationships, except for a negativ e corr elation observ ed with POD activity (Table 1 ).This finding aligns with pr e vious r esearc h on the subject (Boonsiri et al. 2007, Teixeira et al. 2017 ).Chl a : b r atio serv es as an informative pa-  rameter for assessing the functionality of pigment equipment and the adaptability of the photosynthetic apparatus to varying light conditions (Lichtenthaler and Buschmann 2001 ).Moreover, it exhibits sensitivity to w ar d envir onmental str essors suc h as dr ought (Ebrahimiyan et al. 2013 ), salt (Zhu et al. 2019 ), and high temperatur e str ess (Bahr ami et al. 2021 ).This study suggests that Chl a:b ratio can also be utilized as a reliable indicator to e v aluate the stress status in response to leaf extract (LE) or root extract (RE).
A Chl a:b ratio ranging from 0.6 to 0.8 is recommended for maintaining optimal health in plants.
Numer ous studies hav e indicated that the occurr ence of CCOs can be attributed to shifts in microbial communities (Li et al. 2020, Chen et al. 2022b ) and soil acidification (Bai et al. 2019, Li et al. 2021 ).Our r esearc h r e v ealed that the continuous cr opping of CA led to notable declines in pH over time ( Fig. S1A ), and the pH values in the LE and LE + D1 treatments exhibited significant decr eases compar ed to the contr ol ( Fig. S1B ).Ho w e v er, the ma gnitude of the decrease was < 0.24 U, and the soil pH consistently remained above 7.4 throughout the 3-year continuous cropping process.Additionall y, an y tempor ary decline in pH swiftly rebounded to le v els exceeding 7.0 ( Fig. S1B ).Ther efor e, based on the distinct primary factors contributing to CCO in various phases (Chen et al. 2020 ), it can be inferred that soil acidification may not be the primary underlying cause during this particular phase, as the observ ed c hanges in pH wer e not substantial and consistentl y r emained abov e 7.0.Furthermor e, our inv estigation did not r e v eal an y significant alter ations in bacterial div ersity and comm unity subsequent to the application of D1 and LE, whic h div er ges fr om findings reported in previous studies (Bai et al. 2019, Liu et al. 2020 ).This discrepancy could potentially be attributed to variations in cr opping dur ation and/or plant species .T he most prominent shift in the fungal community was observed in the LE + D1 tr eatment.The intr oduction of LE had a significant impact on the fungal community o ver time , particularly evident in the increased abundance of Fusarium .T his disco very suggests that LE has the potential to stimulate the growth and reproduction of strain D1, although the specific mechanism by which this occurs, whether thr ough the pr ovision of ad ditional n utrients or the promotion of colonization, r emains unclear.Furthermor e, the pr esence of LE + D1 led to a suppression of fungal diversity, indicating that strain D1 was notably encouraged by LE and potentially able to occupy more ecological niches, which could be advantageous for colonization in the rhizosphere.
This study presents a novel hypothesis, supported by field investigation and previous research, suggesting that leaf extracts ma y ha ve a significant impact on CCO.In our study, it was observed that the water extract derived from the leaf of CA exhibited greater inhibitory effects on seeds germination, seedlings growth, and soil enzymes activities in comparison to the RE.This finding contrasts with the known positive impacts of leaf litter on soil pr operties within for est ecosystems (Li et al. 2014 ) and other agricultur al envir onments (Chen et al. 2022a ).Limited r esearc h has been conducted on the identification of active compounds present in medicinal plant leaves that contribute to the de v elopment of tuber ous r oots .Our in v estigation r e v ealed a higher accum ulation of phenolic acids, specifically caffeic acid and p -coumaric acid, in the leaf tissue as opposed to the root tissue.Although the substances were found to be plentiful in both LE and RE, their degra-dation in soil occurred within a short span of a few hours (Bravetti et al. 2020 ), resulting in significantly lo w er concentrations in the soil (Fig. 7 ).Phenolic acids are widely recognized as the primary inhibitors in seed germination and plant growth processes (Lodhi 1975 ), with varying effects observed for different types of phenolic acids (Li et al. 2016b, Br av etti et al. 2020 ).While pr e vious studies hav e fr equentl y mentioned caffeic acid and p -coumaric acid, their specific interactions with the primary fungal pathogen and their impact on the microbial communities remain unclear.A recent study has identified three phenolic acids, namely chlorogenic acid, salicylic acid, and vanillic acid, as promoters of a greater relative abundance of soil-borne fungi capable of invading plant roots in a simulated rhizosphere (Clocchiatti et al. 2021 ).This finding aligns with another study that emplo y ed Illumina MiSeq sequencing technology (Li et al. 2018 ).Additionally, our research demonstrates that LE and RE can stimulate the growth of strain D1, primaril y manifested thr ough incr eased hypha weight and spor e numbers (Fig. 5 ).The presence of thicker hyphae and a higher spore count in the soil undoubtedly enhances their competitive adv anta ge within microbial communities.Although researchers have suggested that certain extracts from medicinal plants may have an antagonistic effect on Fusarium spp.(Dwivedi and Yadav 2015 ), our findings partially align with another study that demonstrated the ability of pathogenic Fusarium spp. to utilize ginsenoside, an active compound (Jiao et al. 2015 ).This discrepancy could be attributed to the use of different substances in these studies.It is worth noting that LE or RE also induced the stimulation of certain pathogenesis-related enzymes (Fig. 6 ), which might contribute to the heightened negative effects on the growth of CA seedlings when invaded by strain D1 in conjunction with LE or RE (Fig. 6 ).T his phenomenon ma y serve as a significant factor contributing to the heightened detrimental effects on the growth of CA seedlings subsequent to the invasion of strain D1 in conjunction with LE or RE.This finding aligns with prior r esearc h, as evidenced by a se parate stud y which demonstrated that the fungus pr edominantl y exhibited extr acellular biodegr adativ e enzymes (such as proteases , pectinases , and cellulases) in cultures containing litter (Schneider et al. 2010 ).Additionally, another investigation indicated that the application of agave leaf extracts could stimulate enzyme activities in fungal strains belonging to Fusarium and Lasiodiplodia (Campos-Riv er o et al. 2019 ).Given that phenolic acids in leaves have the potential to induce the growth and pr olifer ation of str ain D1, it is reasonable to comprehend the subsequent bloom of D1 in the leaf litter treatment during a later phase (Fig. 4 D).

Conclusions
In conclusion, the mechanisms underlying CCO induced by LE or RE encompass the modulation of seed germination and plant gr owth, suppr ession of the plant's immune response, augmentation of pathogens, as well as the stimulation of fungal pathogen str ain D1 gr owth and inhibition of plant pathogenesis-related enzyme activities .T hese effects ar e potentiall y attributed to the presence of caffeic acid and p -coumaric acid, particularly in LE.Consequently, it is advisable to discontinue the practice of disposing leaf litters in soil, instead opting to gather them for potential r esource utilization, suc h as weed contr ol, extr action of activ e compounds, and formulation of functional animal feeds in forthcoming endea vors .

Figure 1 .
Figure 1. Isolation and identification of a fungal pathogen causing root rot disease.(A) Disease severity index with time after inoculation with different fungal strains at the final concentration of 10 9 CFU/kg soil.(B) Symptom of root rot disease caused by strain D1 on day 11.(C) Phylogenetic tree based on the sequences of ITS region of strain D1 and its relatives by using neighbor-joining method.

Figure 2 .
Figure 2. Effects of LE and RE on seed germination using petri dishes (A, B) and pots (C) experiments.A tw o-w ay ANOVA was performed to test the effects of extract kinds (LE or RE) and D1 (with or without) on the indices, follo w ed b y Bonferroni's post hoc tests."−D1" and "+ D1" in panels (A) and (B) indicated treatments without and with the spores of strain D1 at the final concentration of 10 6 CFU/ml.Different numbers of asterisk ( * , * * , and * * * ) r epr esent significant differences between samples at P -values of .05,.01,and .001.

Figure 3 .
Figure 3. Plant stress responses to LE, RE, and/or strain D1.The changes in the contents of photosynthetic pigments of Chl a : b (A), Chl a + b (B), and carotenoid content (C) in CA plants following the addition of plant extracts and/or strain D1.The activities of defensive enzymes following the additions of plant extracts and/or strain D1(D-F).Legends shown in panel (A) apply equally to panels (B-F).Different letters at one sampling time point indicates significant differences among treatments (Duncan's multiple range test, P ≤ .05).

Figure 4 .
Figure 4. Effects of LE and RE on soil enzymes activities (A-C) and abundances of strain D1 using qPCR method (D).Legends shown in panel (A) a ppl y equally to panels (B) and (C).Different letters at one sampling time point indicates significant differences among treatments (Duncan's multiple range test, P ≤ .05).

Figure 5 .
Figure 5. Effects of LE and RE on the growth of strain D1.Hypha weights in responses to LE (A) and RE (B), and the different letters on the plots indicates significant differences among treatments at 48 h (Duncan's multiple range test, P ≤ .05).Effects of LE and RE on colonies diameters and spore numbers at 84 h (C), and spore germination rates at 24 h (D).(E) Morphologies of strain D1 following LE30 and RE30 treatments using SEM, and the pictures exhibiting the spores and mycelium of strain D1 were magnified by 2000 times and 8000 times, respectively.Different numbers of asterisk ( * , * * , and * * * ) r epr esent significant differ ences between samples at P -v alues of .05,.01,and .001.

Figure 7 .
Figure 7. Contents of the main phenolic acids in soils with different CA cropping histories (A) and plant tissues (B), and their effects on the growth of stain D1 (C-F).In panel (A), 0 a, 2 a, and 3 a indicated that soils have been planted with CA for 0 year, 2 years, and 3 years, r espectiv el y.Differ ent letters shown in panel (A) indicate significant differences in one certain phenolic acid among different cropping histories (Duncan's multiple range test, P ≤ .05).Different numbers of asterisk ( * , * * , and * * * ) shown in panel (B) r epr esent significant differences between samples at P -values of .05,.01,and .001.Different letters shown in panels (C-F) at one sampling time indicate significant differences among treatments (Duncan's multiple range test, P ≤ .05).

Figure 8 .
Figure 8. Effects of LE and/or strain D1 on the fungal community with time (A), the changes in fungal composition among different treatments during 50 days using PCA (B), and Shannon indices of fungi (C) and bacteria (D).Different letters shown in panels (C) and (D) at one sampling time indicate significant differences among treatments (Duncan's multiple range test, P ≤ .05).
and * * indicate significant correlation between the corresponding data at 0.05 and 0.01 levels, respectively. *