Wen-qing YU，F(xiàn)ang-hao WAN，Xin-hua HE，Wen-zhi LIU，Wan-xue LIU*，Li-li ZHANG

1State Key Laboratory for Biology of Plant Diseases and Insect Pests，Institute of Plant Protection，Chinese Academy of Agricultural Sciences，Beijing 100193，China;2Heilongjiang Academy of Land Reclamation Sciences，Jiamusi，Heilongjiang 154007，China;3Xinjiang Production and Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin，College of Life Sciences，Tarim University，Alar，Xinjiang 843300，China;4State Centre of Excellence for Ecohydrology，University of Western Australia，WA 6009 and Edith Cowan University，WA 6207，Australia

INTRODUCTION

Some special soil microbes alter the interaction between exotic and native plants (Batten et al.，2008;Callaway et al.，2004a，2008)，whilst some exotic plants also alter soil microbial communities and their functioning (Kao-Kniffin ＆ Balser，2008;Kourtev et al.，2002;Stinson et al.，2006;Walling ＆ Zabinski，2004).The soil microbe-plant interaction affects the co-existence of exotic and native plants (Klironomos，2002)，and the invasion-induced soil microbial community changes can enhance the spread of exotic plants (Carey et al.，2004;Reinhart ＆ Callaway，2006;Stinson et al.，2006;Wardle et al.，2004).On the other hand，arbuscular mycorrhizal fungi (AMF)，an important component of soil microbes，affects plant succession and ecological domination by altering plantplant interactions and nutrient availability (Karasawa＆ Takebe，2001;Marler et al.，1999;van der Heijden＆ Horton，2009).AMF also enhances plant tolerance to unfavorable conditions (Beltrano ＆ Ronco，2008;Cartmill et al.，2008)，maintains plant diversity and plant resource distribution (Dhillion ＆ Gardsjord，2004)，which promotes ecosystem stability and resilience (van der Heijden et al.，1998).Approximately～80% of higher plants including almost all invasive species are able to form AMF，resulting in the transfer of nutrients and water from the fungi to the host plant(Brundrett，2009;Wang ＆ Qui，2006).Therefore，AMF may regulate resource competition between native and exotic plants and mediate the successful invasion of exotic plants.For instance，AMF from soils dominated by exotic plants can counterbalance the negative influence of the exotic species (Kisa et al.，2007)or facilitate the competition of invasive plants in invaded areas (Callaway et al.，2004b;Fumanal et al.，2006).Several mechanisms of AMF affecting plant invasion have been suggested:(1)Resistance Hypothesis;(2)Enhanced Mutualisms Hypothesis;(3)Mutualisms Hypothesis;and (4)Degraded Mutualisms Hypothesis (Shah et al.，2009).However，limited literature exists on how the growth of native and exotic plant species could be influenced by soils from invaded or non-invaded areas which hold different AMF communities.

Ageratina adenophora Sprengel (or Eupatorium adenophorum Sprengel)，an exotic plant native to Mexico，has rapidly spread，since 1930's，to southeastern Asia，eastern Australia，New Zealand，and southwestern Africa (Cronk ＆ Fuller，1995;Wang，2005).A.adenophora was firstly found in China in 1935 in Yunnan Province，from where it has rapidly spread to southwestern China.Presently，it has invaded 80% of Yunnan，and has become one of the worst invasive exotic species in Guizhou，Sichuan，Guangxi，Xizang，Chongqing and Hubei Provinces (Lu ＆Ma，2006;Xie et al.，2001).A.adenophora is expanding northwards at an annual rate of 20 km in those regions，leading to serious loss of native plants (Wang＆ Wang，2006).A.adenophora invasion affects both soil bacterial (Niu et al.，2007;Yu et al.，2005)and AMF communities (Yu et al.，2012).However，information on how a changed microbial community could affect the competition between A.adenophora and native plants is limited.The present research，therefore，aimed to study in greenhouse pot experiments the effect of distinct microbial communities，from freshlyinvaded and not invaded forest soils，on A.adenophora and native plants，as well as their interactions in greenhouse pot experiments.

MATERIALS AND METHODS

Soil collection

Collections of rhizosphere soils and roots were made according to Sigüenza et al.(2006).In the middle of July 2009，soils at 0 ～20 cm depth were collected under two vegetation types (500 ～2000 m away from each other)in a coniferous and broad-leaved mixed forest，located in Kunming，Yunnan，China(25°03'N and 102°52'E，～1980 m above sea level).The area has been severely invaded by A.adenophora for at least 15 years.Kunming has a sub-tropical monsoon climate，with an annual mean temperature of 15 ℃and precipitation of 1100 mm.The vegetation type Ⅰwas dominated by native plants (Setaria viridis，Medicago falcata，Stellaria chinensis)without the invasion of A.adenophora，so that the collected soil was referred to as non-invaded soil (NS).The vegetation typeⅡwas dominated by A.adenophora with a ten-year invasive history so that the collected soil was referred to as invaded soil (IS).The soil type was typical red soil in southern China (Hapludult，US system).The soil at 0 ～20 cm has pH 7.7，39.3 g·kg-1DW organic carbon，1.9 g·kg-1N，25.0 mg·kg-1available P and 132.8 mg·kg-1available K.

The soil samples were randomly collected under each vegetation type within an area of 100 m× 300 m from 10 plots.Each plot had a size of 9 ～25 m2and were 15 ～20 m away from each other.Soil samples about 3 kg·plot-1from each vegetation type was collected and stored in plastic bags with dry ice plates and immediately transported to the glasshouse for the pot experiments.

Plant species and experimental design

To test the effects of soil inocula on the growth of native and exotic plants，three species were selected:the invasive A.adenophora，and the native plants S.viridis and M.falcata.Seeds of the three species were collected from the different vegetation types and planted as monocultures:A.adenophora (A)，S.viridis(S)or M.falcata (M)as well as in combination:A.adenophora with S.viridis (A +S)or with M.falcata(A+M).

Each of these seed combinations were planted into four different soils:NS and IS，with or without autoclave sterilization (105 ℃，1.5 h)，giving a total，of 20 treatments.Each treatment was replicated 12 times.We used 2 L tubby pots(6 cm bottom diameter，9 cm top diameter，11 cm high)，containing 1.8 kg growth substrate (1:4 perlite/sterile sand mix，v/v)，and 10 g sterilized or non-sterilized fresh soil that was buried 0.5 cm beneath the substrate surface (Klironomos，2002).Seeds were sown in the soil inoculation.Plants were grown between 21 September 2009 and 20 November 2009 under natural light conditions and controlled temperature of 32/18 ℃ (day/night)，which was similar to the summer weather in the soil collecting area，in a glasshouse on the campus of Chinese Academy of Agricultural Sciences，Beijing，China.The pots were randomly arranged and their position changed weekly or biweekly，watered every other day with tap water，and fertilized biweekly with 100 mL Hoagland's solution.Four (two for each species in the two combinations)seedlings in each pot were maintained after two weeks of sowing.

The plants were harvested after 2 months (including shoots and roots)and were oven-dried at 70 ℃for 60 h.To measure AMF root colonization，fresh fibrous 1 cm root segments of A.adenophora were cleared with 10% KOH for 25 min，rinsed with 2% hydrochloric acid for 5 min，and stained with 0.01% acid fuchsin-lactic acid-glycerin dye at 90 ℃for 25 min (acid fuchsin 0.1 g，lactic acid 875 mL，glycerin 63 mL，distilled water 300 mL)，followed by immersion in 10～20 mL 99% lactic acid until AMF root colonization had been scored.AMF root colonization was scored by light microscope (http:∥invam.caf.wvu.edu/;Biermann ＆ Linderman，1981).Microbial responsiveness，the relative response of plant growth to soil microbes was calculated with the following formula:RD=(BB0)/B×100%，in which B and B0were dry biomass of plant with un-sterilized and sterilized soil inocula，respectively (Harner et al.，2010).

Data analysis

Effects of soil inoculum source on root AM colonization，plant biomass production and microbial responsiveness were subjected to ANOVA and significant differences of data (means ±SE)between treatments were compared with LSD under P ＜0.05 with General Linear Model.Data were analyzed using the software SPSS version 13.0 (SPSS Inc.，Chicago，USA).

RESULTS

Effects of soil inocula on the root colonization rates of A.adenophora

At harvest，no obvious AMF structures were observed in any plant roots grown in sterilized soil inoculum，whereas all plants grown in the non-sterilized inoculum were colonized by AMF.The rates of AMF root colonization of A.adenophora ranged from 81.7% to 97.7%.The mycorrhizal colonization rate of A.adenophora was not affected by inoculum sources (P=0.163).However，A.adenophora in A+S (A/A +S)and A.adenophora in A+M (A/A+M)inoculated with soil inculum from IS had a higher AMF colonization rate than that from NS (P1=0.020，P2＜0.0001)(fig.1)，indicating that AMF in invaded soil preferred to colonize A.adenophora while AMF in native soil did not.

Fig.1 AMF root colonization rate (means±SE)of A.adenophora

Effect of microbial inoculum source，sterilization and neighboring plant on plant growth

Soil inocula from different sources had variable effects on the growth of A.adenophora and native plants.The total biomass of A.adenophora sown with non-sterilized inoculum from IS was 17.4% higher than that inoculated with NS，but inoculum source had no effect on growth of the two native plant species.Sterilization retarded the growth of both A.adenophora and native plants.The neighboring plants accelerated the growth of A.adenophora，but it retarded the growth of the two native species.The interaction of inoculum source and sterilization on the growth of all plants was non-significant.The interaction of inoculum source and neighbor plant was significant for the native plants，but it was non significant for A.adenophora.The interaction of sterilization and neighbor plants was significant for A.adenophora，but non significant for native plants.The interaction of soil source，sterilization and neighbor plants was significant for native plants，but non significant for A.adenophora (table 1 ～2).

Microbial inoculum source

Sterilization had no effect on plant biomass when inoculated NS and IS in all planting combinations except for S.viridis planted alone.But in the non-sterilized treatment，the plant biomass was higher for IS than NS except M.falcata planted with A.adenophora(table 1).

Effect of sterilization

In NS，sterilization accelerated the growth of all plants except A.adenophora planted with S.viridis;While sterilized IS retarded the growth of A.adenophora when planted with S.viridis，but facilitated the growth of the native plants (table 1).

Table 1 Dry biomass (g)per plant with different inoculum sources and planting combination

Table 2 Summary of ANOVA of the effects on plant biomass of soil source (invaded，non-invaded)，sterilization (sterilized or nonsterilized)，and their interactions

Effect of neighbouring plants

S.viridis facilitated the growth of A.adenophora especially with non-sterilized IS inoculum，but M.falcata did not have such an effect.A.adenophora retarded the growth of the two native plants，especially in the IS (table 1).

Soil inocula from A.adenophora invaded area(IS)facilitated the growth of A.adenophora when planted with the two native plant species as neighbors.The growth of the native plants was retarded by the neighbor A.adenophora when soil inoculum from IS was used.The results were opposite when NS soil inoculum was used.

Effect of inoculum source and sterilization on plant biomass

Plant biomass per pot of the five planting combinations was A ＞A +S (P ＜0.0001)＞A + M(P =0.028)＞M(P ＜0.0001)＞S (P =0.004).The inoculum source and sterilization had no effect on plant biomass in A and A+M.The inoculum from IS resulted in higher biomass than NS in the A+S treatment，but sterilization decreased the biomass.The inoculum from IS yielded lower biomass than NS for S，and sterilization decreased this biomass.The inoculum of IS resulted in higher biomass of M.falcata than of NS，it was not affected by sterilization，but significant interaction was found between them (fig.2).

Response of A.adenophora and native plant species to microbial inoculum

A.adenophora exhibited varied response to soil inocula from different sources.When sown with inoculum from NS，A.adenophora showed a negative response in monoculture and when grown with M.falcata，but a positive response with S.viridis.A.adenophora showed strong positive response for IS (in monoculture:P ＜0.0001;in mixture of A +S:P =0.05;in mixture of A+M:P ＜0.0001).The response of S.viridis (in monoculture:P ＜0.0001;in mixture of A+S:P ＜0.0001)and M.falcata (in monoculture:P ＜0.0001;in mixture of A+M:P ＜0.0001)to microbes was also different.The native S.viridis yielded a stronger negative response to the inoculum from NS than that from IS，and M.falcata showed negative response to the inoculum from NS but positive response to the inoculum from IS in monoculture，while it yielded a strong negative response when planted with A.adenophora (fig.3).

Fig.3 Microbial responsiveness (means±SE)of plant growth

DISCUSSION

Invaded soil microbial community changes the competitive interactions between exotic and native plants

During invasion，A.adenophora releases allelochemicals that strongly affect the composition of the soil microbial community (Niu et al.，2007;Yang et al.，2006，2008)，and altering the AMF community is an important invasive mechanism for A.adenophora(Yu et al.，2012).In the present study，A.adenophora responded positively to the changed soil microbial community that became more favorable to A.adenophora than to the native species inoculum from native soil had no effect on the root colonization rates of A.adenophora in different planting combinations but adding invaded soil resulted in higher AMF colonization rate of A.adenophora in polyculture with S.viridis or M.falcata，even when compared to monoculture plantings.And inocula from native and invaded soils had different effects on the growth of A.adenophora and the native plants suggesting that microbes in invaded soil including AMF，changes the competitive interactions between these plants (Dhillion ＆ Gardsjord，2004).

M.falcata showed stronger resistance to A.adenophora invasion.Its growth was not retarded by A.adenophora when inoculum from native soil was used.S.viridis showed no ability of resisting the invasion，but it grew better with inoculum from invaded soil than native plant dominated soil，so this native plant can be considered as the substitute plant in the restoration of A.adenophora invaded ecosystem.

Exotic plants exploit the resources of native plants via AMF

The growth of A.adenophora was also accelerated by inoculum from invaded soil when grown in polyculture with S.viridis，which resulted in a positive feedback loop and further spreading of A.adenophora.However，this positive feedback loop only happens when exotic plants are grown with neighboring native plants，a situation where the exotic plants exploit the resources of native plants (Callaway et al.，2004a;Marler et al.，1999;Zabinski et al.，2002).After the AMF community had been altered，the AMF colonization rate increased in A.adenophora grown in polyculture with S.viridis or M.falcata.

Non-sterilized soil inocula enhanced AMF colonization rates and growth of A.adenophora.The effects of AMF on plant growth are stronger if nutrition is not enough (Harner et al.，2010).In our experiment we had to fertilise the plants.Each plant species in different planting combinations had the same biomass when they were inoculated with sterilized inoculum (both NS and IS)or inoculated with non-sterilized inoculum from soil dominated by native plants，which served as a negative control，showing that regular soil fungi do not change the invasive weed's growth.The non-sterilized inoculum from A.adenophora invaded soil，however，significantly contributed to the vigorous growth of A.adenophora and its further spread.The mutualistic enhancement of invasive plants by AMF most likely causes changes to the outcome of competitive interactions between exotic and native species.These changes may come from the impacts of AMF on the uptake and exchange of nutrients (Shah et al.，2009).Future studies could focus on the effect of environmental factors on the interactions among exotic plants，soil microbes and native plants，to better understand the interactions between non-native vegetation and soil microbial communities，along with their feedbacks to the native plants in highly invaded areas.The synergistic effects of the plant growth promoting rhizobacteria(PGPR)and the AMF on exotic plants and native plants should also be investigated (Khan ＆ Zaidi，2007).

ACKNOWLEDGEMENTS:We thank Professors Gabor L.L?vei (Aarhus University，Denmark)，Dan L.Johnson (University of Lethbridge，Canada)，and Imtiaz Ali Khan (The University of Agriculture，Peshawar，Pakistan)for their valuable comments and linguistic corrections of the manuscript.

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