Beneficial rhizobacterium provides positive plant–soil feedback effects to Ageratina adenophora

SUN Yuan-yuan,ZHANG Qiu-xin,ZHAO Yun-peng,DIAO Yue-hui,GUI Fu-rong,YANG Guo-qing

1 College of Horticulture and Plant Protection,Yangzhou University,Yangzhou 225009,P.R.China

2 College of Plant Protection,Yunnan Agricultural University,Kunming 650201,P.R.China

Abstract Rhizosphere microbial communities play important roles in facilitating or inhibiting the establishment of exotic species.Since some invasive plants interact with soil microbial communities such as rhizosphere bacteria,changes triggered by rhizosphere bacteria may alter competitive interactions between exotic and native plants.This study compared the Bacillus cereus content in soils with different degrees of Ageratina adenophora invasion,and investigated the effects of A.adenophora allelochemicals on B.cereus growth and soil characteristics and the feedback effects of B.cereus on A.adenophora growth.Bacillus cereus content in the rhizosphere of A.adenophora increased with intensification of the invasion process,and newly invaded soil contained almost twice as much bacteria as noninvaded soil.When rhizosphere soil was added to the root exudates of A.adenophora,the contents of B.cereus were twice as much as the control,except on the first day.Certain soil parameters increased significantly,such as ammonium nitrogen (NH4+-N) and available phosphorus (AP),which were increased by 41 and 27%,respectively.Soil treatment with B.cereus promoted the degradation of two allelochemicals from the rhizosphere of A.adenophora,amorpha-4,7(11)-dien-8-one and 6-hydroxy-5-isopropy1-3,8-dimethyl-4a,5,6,7,8,8ahexahydraphthalen-2(1H)-one,to varying degrees;and increased the germination rate by 50%,root length by 117%,shoot length by 48% and fresh weight by 81% for A.adenophora compared to those of untreated soil.Our results confirmed that the invasion of A.adenophora will promote an increase of B.cereus,a beneficial rhizosphere bacterium,which in turn induces a positive feedback effect on A.adenophora.

Keywords:Ageratina adenophora,Bacillus cereus,feedback,allelochemicals,root exudates

1.Introduction

Exotic plant species tend to form a single dominant population in an invaded range compared to their original range (Fitter 2003).Many hypotheses about biological invasion mechanisms have been explored,including the evolution of increased competitive ability hypothesis(Callaway and Ridenou 2004),enemy release hypothesis(Keane and Crawley 2002),novel weapon hypothesis(Callawayet al.2004),and so on.Among these,allelopathy plays an important role in biological invasion (Yanget al.2008).In addition to the direct impact of allelochemicals on plant growth,the indirect effects mediated by the microbial community are persistent and important (Inderjitet al.2011).

The soil microbial community plays an important role in mediating allelopathic interactions between exotic and native plants (Inderjit 2005).Invasive plants may accumulate rhizosphere soil pathogens that are more harmful to native plants (Floryet al.2013).For example,rhizosphere soils ofChromolaena odoratacould accumulate high concentrations of generalist soil-borne fungi,thus creating negative feedback effects on some native plant species(Manglaet al.2008).Invasion of an exotic plantBromus tectorum,the host of a native seed pathogenPyrenophora semeniperda,resulted in the death of a large number of native seeds in invaded areas,as its mycorrhizal fungi destroyed the symbiotic relationships between native plants and soil microorganisms (Becksteadet al.2010).However,due to phenotypic plasticity,compensation or tolerance,pathogen accumulation may have little or no effects on invasive species (Floryet al.2013).Many invasive species possess a strong phenotypic plasticity,so a decrease in population density has little effect on either their biomass per unit area or seed yield (Vanwallendaelet al.2018).On the other hand,some beneficial rhizosphere bacteria increase under biological invasion,and positive feedback effects have been found for exotic plants (Heet al.2015).Rhizospherepromoting bacteria refers to bacteria that are beneficial to plants in the rhizosphere,including more than 20 species of nitrogen-fixing microorganisms,such asPseudomonas fluorescens,Bacillus,Rhizobium,andSerratia,among whichBacilluscan serve as biological control agents of plant diseases by producing antibiotics.The metabolically active substance produced byB.cereusCS51 andP.fluorescensCS121 has an antagonistic effect onStaphylospora curtisii(Chenet al.2003).Bacillus subtilisRR4 isolated from rice rhizosphere can induce the biosynthesis of malic acid in the rice rhizosphere,which plays an important role in the chemotaxis of beneficial bacteria to the plant rhizosphere(Rekhaet al.2017).However,factors arising along with plant invasion and involved in the changes of these functional bacteria,and their functional mechanisms need to be further investigated,such as the interactive relationships between plant secondary metabolites and bacteria in soil.

Ageratina adenophorais an invasive weed in many countries and areas,and allelopathic effects are one of the main factors facilitating its further invasion and expansion(Yanget al.2006).For example,with the addition of active carbon (AC),the negative effect ofA.adenophoraon the growth of native plants was obviously reduced (Wanet al.2010).Soil nutrient composition and the microbial community following invasion ofA.adenophorainhibited neighboring plants and partially benefited their own growth(Niuet al.2007).However,the internal relationships and mechanisms underlying these effects are still unclear.For example,could allelochemicals affect major rhizosphere microorganisms and cause positive feedbacks for their growth? Based on the analysis of major allelochemicals in the rhizosphere ofA.adenophora(Yanget al.2013),a recent study has shown that several allelochemicals in the rhizosphere have varying degrees of ‘self-toxic’ effects,and soil microorganisms can weaken their rhizospheric allelopathic effects (Liet al.2015).How does an invasive plant respond to its own allelopathy and gain a competitive advantage over native plants? Our previous field soil investigations showed that there was an obvious content ofB.cereusin sites invaded byA.adenophora.And,as a rhizotrophic bacterium,B.cereuscan inhibit the propagation of pathogenic microorganisms in soil and promote plant growth by interacting with mycorrhizal fungi (Artursson and Jansson 2003).

To address these questions,we hypothesized that the invasion ofA.adenophorawill promote the increase ofB.cereus,a beneficial rhizosphere bacterium,and the change would induce a positive feedback effect onA.adenophora.To examine this hypothesis,we comparedB.cereuscontents in rhizosphere soil ofA.adenophoraat different degrees of invasion,investigated the effects ofA.adenophoraallelochemicals onB.cereusand on soil characteristics,and determined both degradation dynamics of allelochemicals treated withB.cereus,and the partial beneficial effects ofB.cereuson the growth ofA.adenophora.

2.Materials and methods

2.1.Comparison of B.cereus in rhizosphere soil under different degrees of invasion

Four sites were selected for soil sampling in the summer of 2018 in the evergreen,broad-leaved-deciduous mixed forests in Xishan District of Yunnan Province,China,(24°58´29´´–42´´N,102°37´1´´–10´´E,with an average elevation of 2 138 m),where the rhizosphere soil was sampled in a previous study (Niuet al.2007;Yanget al.2016),and the sites were classified byA.adenophoracoverage.The four sites had similar elevation,topography,gradient,and soil types and have been supportingA.adenophoramore than 30 years.Site I (noninvaded) was covered with no plants.Site II (newly invaded) included mostly native plants and sporadicA.adenophora;the native plants includedPteridophyllum racemosum,Serissa japonica,andPieris japonica.In Site III (moderately invaded),A.adenophoracoverage was approximately 40%.Site IV (highly invaded)was dominated byA.adenophorawith a coverage of more than 60%.Each sample consisted of 16 soil cores with a diameter of 3.5 cm and a depth of 10 cm (Niuet al.2007;Yanget al.2016).Samples were randomly collected from plots of 8 m×8 m.There were three plots from each site,and the distance between plots was about 5 m.The soil was sifted through a 1-mm sieve,transferred to a plastic bag,immediately cooled,transported to the laboratory and stored at 4°C.

To quantifyB.cereusin rhizosphere soils ofA.adenophora,DNA (DNeasy®PowerSoil®Kit,Qiagen,germany) was extracted from the soil,and PCR primers (Appendix A) were designed according to the sequences ofB.cereusstrains.A Bio-Rad CFX96 touch (Bio-Rad Laboratories Incorporated,USA) was used to conduct the qRT-PCR analysis.Each 20 μL reaction contained the following:0.8 μL of each primer(10 μmol L–1),10 μL of SYBR green (TaKaRa,Japan),3.4 μL of distilled water and 5 μL of template DNA(3.27 ng μL–1).The conditions for the qRT-PCR procedure were as follows:95°C for 3 min and then 40 cycles of 95°C for 10 s and 60°C for 30 s.The PCR products were then heated to 95°C for 15 s,cooled to 60°C for 1 min and heated to 95°C for 30 s and cooled to 60°C for 15 s to measure their dissociation curves.Four independent replicates of each DNA sample were performed.

2.2.Effects of allelochemicals on B.cereus and soil characteristics

Potential allelochemicals from the root exudates ofA.adenophorawere prepared and used to test the effects onB.cereusgrowth and soil characteristics.For the root exudate preparations,a one-year-oldA.adenophoraplant with 0.6 m height was cultured by the hydroponic method.The roots were rinsed with water and wiped with alcohol to remove surface bacteria,then covered to 300 mL distilled water in a dark environment (Yanget al.2014).About 250 mL root exudates were collected after 7 days.The root exudates were filtered through four layers of filter paper,then through 0.45-μm Micro PES membranes (Tianjin Jinteng Laboratory Equipment Company,China),and stored at 4°C until use.A total of 25 g of moderately invaded soil was placed into a culture dish with a 9-cm diameter,and either 10 mL of root exudate was added,or 10 mL distilled water was added as the control.Bacillus cereuscontents in the soil were measured at 1,5,9,13,and 17 days after the treatment.There were five replications for each treatment.The qRTPCR method described above was used to determine the content ofB.cereus.

In order to investigate whether the soil properties and soil element composition were induced to change by root exudates,the content/value of ammonium nitrogen(NH4+-N),nitrate nitrogen (NO3–-N),available potassium(AK),available phosphorus (AP),pH and moisture were measured for all the treated soil samples.A Lachat Quick Chem II flow-injection analyzer was used to quantify the concentrations of NH4+-N and NO3–-N in the KCl extracts(Li and Li 2013).The Burnt-luminosity method was used to determine the AK content (Bao 2000).The colorimetric Mo-blue method was used to determine the AP content (Puet al.2013).The pH and moisture content were determined by the glass electrode method (Niuet al.2007) and drying method,respectively.

2.3.Influences of B.cereus on allelochemical degradation

Two main allelochemicals from the rhizosphere ofA.adenophora(Yanget al.2013),amorpha-4,7(11)-dien-8-one (DTD) and 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydraphthalen-2(1H)-one (HHO),were used to investigate the influences ofB.cereuson allelochemical degradation in soil.Soil from Site III was used and divided into three groups.Two of the groups were sterilized by autoclaving (121°C,0.105 MPa,1 h) three times at 24-h intervals.Bacillus cereussolution was prepared by culturing on a shaker at 180 revolutions per minute at a temperature of 30°C for 24 h,and added to one of the sterilized soil samples.Bacillus cereussolution was added at 20 µL g–1of soil (Sripontanet al.2014).The third group of soil was used as an unsterilized control.Treated soils were placed into triangle-shaped bottles (50 mL) at a rate of 30 g per bottle.A total of 100 µL of the DTD (75 mg L–1) and HHO(10 mg L–1) fromA.adenophorawas added to each bottle.Each soil treatment included four replications.Soils were incubated in the dark at ambient temperature and were sampled after 0,24,48,72,96,and 120 h for quantitative analysis of the DTD and HHO contents using HPLC.Soil samples were extracted with 30 mL ethyl acetate three times with occasional vortexing and transferred to 50 mL centrifuge tubes.The extracts were centrifuged at 1 500×g for 15 min,and the supernatant was filtered with 0.45 μm Micro PES membranes.Subsequently,all samples were dried with nitrogen prior to resuspension in 1 mL of methanol.

An L-2000 HPLC (Agilent Eclipse Plus,Palo Alto,CA)with a C18column (150 mm×4.6 mm) was used to perform HPLC at 30°C.The mobile solvent system was methanol and water (70:30,v/v) at a flow rate of 1 mL min–1,and the UV detection wavelength was 254 nm.Aliquots (10 mL)were injected into the HPLC system,and the compounds were quantified using the external standard peak area (Yanget al.2016).

2.4.The partially beneficial effects of B.cereus on the growth of A.adenophora

To determine whetherB.cereusin soil could have partially beneficial effects on the growth ofA.adenophoracompared with that of native plants,bioassay experiments were conducted withA.adenophoraand three native plants(Eupatorium lindleyanum,Trifolium repensandMedicago sativa) treated withB.cereusin soil.Soil from Site III was used and autoclaved three times for 20 min at 121.1°C on three successive days to inactivate soil microbes.All sterilized soil was then treated in one of three ways:mixed with activated carbon (AC) (25 mg) (Tianet al.2007),mixed with 200 μLB.cereus(Sripontanet al.2014),or mixed with both activated carbon andB.cereus.

Seeds ofA.adenophorawere collected in Yunnan,China,and the seeds ofE.lindleyanum,T.repensandM.sativawere purchased.All seeds were disinfected with 5% hydrogen peroxide for 15 min,washed with distilled water three times,evenly embedded in a beaker with 10 g of treated soil and watered.There were 10 seeds per replicate and three replicates per plant species.

The bioassay experiment was carried out in an incubator.The photoperiod lasted for 14 h,day and night temperatures were 28 and 20°C,respectively,with relative humidity of>85%.The number of germinated seeds was observed and recorded every 24 h for the first two weeks (with the radicle breaking through the seed coat defined as germination),and distilled water was supplemented regularly to maintain humidity.

germination percentage (%)=(Total germinated seeds/Total tested seeds)×100

germination index=∑(gt/Dt)

where Dt is germination days and gt is number of germinated seeds per day corresponding to Dt.

The root length and shoot length (mm) of seedlings were measured two weeks after treatment,and their fresh and dry weights (biomass) were also determined.

2.5.Statistical analysis

All data were analyzed by Data Processing System v7.05(DPS v7.05) Software.One-way ANOVA was used,and multiple means were compared based on Fisher’s protected LSD test (P<0.05).Two-way ANOVAs (Fisher’s protected LSD test) was used to analyze the effects of root exudates and time onB.cereusgrowth.An unpairedt-test was used to analyze soil characteristics.Prior to ANOVA analysis,data in percentages were converted into quasinormal distributions by inverse sine transformation and all data were assessed and confirmed to be in normal distributions.

3.Results

3.1.Comparison of B.cereus in rhizosphere soil under different degrees of invasion

The content ofB.cereusin rhizosphere soil ofA.adenophoraincreased with the intensification of invasion.Newly invaded soil contained almost twice as much bacteria as noninvaded soil.The content ofB.cereusin the moderately invaded soil was 69% higher than that in newly invaded soil,and the content in highly invaded soil was approximately 13% higher than that in moderately invaded soil (Fig.1).

3.2.Effects of allelochemicals on B.cereus and soil characteristics

After treatment with root exudates ofA.adenophora,the contents ofB.cereusin the soil increased significantly on the 5th day,and then there was no significant difference over time.Except for the first day,there were significant differences between treated and untreated soils,and the contents ofB.cereuswere twice as much as the control(Fig.2).

All soil characteristics except moisture content varied substantially in the soil treated with root exudates (Fig.3).NH4+-N,NO3–-N,AK and AP contents were significantly higher than those of the control group.Among them,NH4+-N and AP were 41 and 27% higher,respectively,than those of the control (Fig.3-A and D).The pH value ofA.adenophorarhizosphere soil treated with root exudates decreased significantly compared to the control (Fig.3-E).

Fig.1 The DNA copies of Bacillus cereus in the soils collected from the four sites (Site I–IV,noninvaded,newly invaded,moderately invaded,and heavily invaded,respectively) in the forest of Yunnan Province,China.Bars are mean±SE (n=3).Different letters indicate significant differences (P<0.05) among soil sources according to one-way ANOVA (Fisher’s protected LSD test).

3.3.Bacillus cereus influences allelochemical degradation

Treatments with bacteria influenced the contents of two allelochemicals (DTD and HHO) in soil to varying degrees.Bacteria-treated soil significantly promoted the degradation of DTD.The contents of DTD were 338,356 and 723%lower than those of the sterilized soil at 48,72,and 96 h after treatment,respectively (Fig.4-A).For HHO,the three treatments had little effect on its degradation.At 120 h,there was a significant difference in the content of HHO between the sterilized soil and the soil to whichB.cereuswas added (Fig.4-B).

Fig.2 The DNA copies of Bacillus cereus in the moderately invaded soil supplemented with root exudates.Bars are mean±SE (n=5).Different letters indicate significant differences(P<0.05) between the control and root exudate treatment,according to two-factor variance analysis (Fisher’s protected LSD test).

Fig.3 Physicochemical characteristics in soils supplemented with root exudates.NH4+-N,ammonium nitrogen;NO3–-N,nitrate nitrogen;AK,available potassium;AP,available phosphorus.Bars are mean±SE (n=10).Asterisks indicate the levels of significant differences between the control and root exudate treatment at P<0.05 (*),P<0.01 (**) and P<0.001(***) according to unpaired t-test.

3.4.The partial benefit of B.cereus to the growth of A.adenophora

ForA.adenophora,adding activated carbon (AC) andB.cereussignificantly increased the germination rate by 50% (Fig.5-A1),root length by 117% (Fig.5-B1),shoot length by 48% (Fig.5-C1) and fresh weight by 81%(Fig.5-D1) compared to those of untreated soil,while it had no significant effect on biomass (Fig.5-E1).In addition,E.lindleyanumshowed longer shoot length (Fig.5-C2) and heavier biomass (Fig.5-E2) in the soil with AC.The AC treatments also affected the shoot length (Fig.5-C3) and fresh weight (Fig.5-D3) ofT.repensand the root length ofM.sativa(Fig.5-B4).The results indicated thatB.cereusmay promote the growth ofA.adenophora,but had little effect on the native plants.The use of AC to eliminate the allelopathic effects was conducive to the growth of the three native plants.There was no significant difference in germination index of the four test plants under the various treatments (Appendix B).

4.Discussion

Fig.4 Degradation of DTD and HHO in soils treated with Bacillus cereus.DTD,amorpha-4,7(11)-dien-8-one;HHO,6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydraphthalen-2(1H)-one.Values are mean±SE (n=4).Different letters indicate significant differences (P<0.05) among different treatments of soil according to one-way ANOVA (Fisher’s protected LSD test).

Our results clearly indicated that with the intensification of invasion,the content of the beneficial bacteriumB.cereusin the rhizosphere ofA.adenophoraincreased.The results confirmed the findings from a previous study thatA.adenophoracould accumulate two beneficial bacteria,B.subtilisandB.megaterium,in its invaded area,while the contents of the two bacteria in the native plant area were relatively low (Niuet al.2007).Thus,the invasion ofA.adenophoracould change the composition and abundance of the soil bacterial community (Niuet al.2007),and such changes in soil microbial communities caused by invasion may lead to a positive feedback loop,promoting the success of the invasive species in the ecosystem (Hawkeset al.2005).

What explains the higher content ofB.cereusin highly invaded areas? It could be closely related to the allelochemicals secreted by the exotic plants (Inderjitet al.2011).Our laboratory experiments clearly demonstrated that treatment of the rhizosphere soil ofA.adenophorawith root exudates promoted the growth ofB.cereus.In general,different plants can form plant-specific soil microorganisms and rhizobia due to their different root exudates (Klironomos 2003).The release of plant carbohydrates increases the biomass and activity of rhizosphere soil microbes,while the plant can also regulate the structure of rhizosphere microorganisms by releasing allelochemicals (Bertin 2003).Changes in soil microbes also greatly affect the nutrients in the soil.Studies have found that with the invasion ofA.adenophora,the available soil nutrients are highly abundant (Niuet al.2007).It may be that changes in soil biota could be translated into altered soil nutritional budgets(Hawkeset al.2005).The metabolism of microorganisms and animals in the rhizosphere directly promotes or inhibits the nutrient absorption and growth of roots and can also affect the transformation of N,P,K,S and other substances in the rhizosphere soil.Our results showed that the NH4+-N,NO3–-N,AK and AP contents significantly increased in the rhizosphere soil treated with root exudates,which is consistent with previous research results (Yuet al.2005).Increased levels of these nutrients may promote the growth and reproduction ofA.adenophora.Thus,we infer that the areas dominated byA.adenophoraand their resulting soil biota changes tend to be responsible for the altered soil fertility,which may giveA.adenophoraan advantage in competition with local plants and create favorable conditions for its invasion (Yuet al.2005).

In turn,the accumulation ofB.cereusmay reduce the autotoxicity of the invasive plants.We found that with the extension of degradation time,the addition ofB.cereuscould promote the degradation of DTD and HHO to varying degrees.In addition,in the seed germination and seedling growth experiments with four recipient plants,a partial effect ofB.cereusonA.adenophorawas also observed.Therefore,A.adenophoraescapes the self-toxicity of its own DTD and HHO,gaining a competitive advantage over other native plants.It is not thatA.adenophoraadapts to the allelopathic effects of DTD and HHO,but rather thatA.adenophorahas accumulated a large population of microorganisms to degrade the allelochemicals into products that are nontoxic or only weakly toxic to themselves (Heet al.2009).Previous studies have also reported that soil biota were responsible for the reduced phytotoxicity in natural soils (Weidenhamer andCallaway 2010).The invasion of alien plants,changes in soil biota,and plant–soil feedback between invasive plants and native plants require further study and discussion (Mitchell and Power 2003).Based on these results,we propose that beneficial bacteria may mediateA.adenophorainvasion and expansion.The exotic plants changed the soil microbial community in their invaded habitats (Boudiafet al.2013) and its effects on the plant–soil interaction processes,which facilitated the growth of exotic plants while inhibiting the growth of native plants (Elgersma and Ehrenfeld 2011).Therefore,the exotic plants received belowground benefits to consolidate their own territories(Li and Li 2013).Our study reported here only focused on specific microbial groups,and the effects of soil microbial communities need to be examined in the future.Moreover,although the plant–soil biota feedback experiments used in this study are known as an ideal method for assessing plant–soil biota interactions (Bever 2002),other factors are also worth considering,including litter dynamics,soil nutrients and the other allelopathic compounds (Vivancoet al.2004),which could also affect plant–soil community interactions.

Fig.5 Seed germination and seedling growth of four test plants grown in four kinds of soil,including sterilized soil,sterilized soil with activated carbon (AC),sterilized soil with Bacillus cereus,and sterilized soil with B.cereus and AC (mean±SE,n=30).Different letters indicate significant differences (P<0.05) according to one-way ANOVA (Fisher’s protected LSD test).

5.Conclusion

In this study,we investigated the effects ofA.adenophoraon the growth ofB.cereusand the positive feedback effects ofB.cereusonA.adenophora.The results confirmed our hypothesis thatthe accumulation of beneficial bacteria in the rhizosphere helpsA.adenophorato disperse further,which offers scientific bases for improving the soil microecological environment and replacingA.adenophorawith native plants.

Acknowledgements

This research was funded by the National Natural Science Foundations of China (31772229 and 31660546).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm