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Applied Soil Ecology 61 (2012) 85– 91

Contents lists available at SciVerse ScienceDirect

Applied Soil Ecology

journa l h o me page: www.elsev ier .com/ locate /apsoi l

ystemic modification of cotton root exudates induced by arbuscular mycorrhizalungi and Bacillus vallismortis HJ-5 and their effects on Verticillium wilt disease

uoyi Zhang, Wasim Raza, Xiaohui Wang, Wei Ran ∗, Qirong Sheniangsu Key Lab for Organic Solid Waste Utilization, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China

r t i c l e i n f o

rticle history:eceived 10 November 2011eceived in revised form 2 February 2012ccepted 2 February 2012

eywords:acillus vallismortis HJ-5xudatelomus versiforme

a b s t r a c t

Verticillium wilt caused by Verticillium dahliae Klab. limits the production of cotton worldwide. An effortwas made to identify the local and systemic plant phenolic acids produced in the presence or absenceof an antagonistic strain of Bacillus vallismortis HJ-5 (HJ-5) and an arbuscular mycorrhiza fungus Glomusversiforme (AM) during the incidence of the wilt to explore better ways to control the disease. When V.dahliae and HJ-5 were applied to AM-inoculated soil, the cotton plant disease was decreased especiallywith the co-inoculation of HJ-5 and AM and the disease index decreased up to 63.3% compared with thecontrol. Four phenolic acids were found in the cotton root exudates, and all the phenolic acids at lowconcentrations stimulated germination of V. dahliae spores, while higher concentrations were inhibitory.

ycorrhizahenolic aciderticillium dahliae

The phenolic acid concentrations in the root exudates decreased significantly with the application ofHJ-5 and AM. A split-root system verified that the alterations of the exudation pattern in HJ-5, AM andV. dahliae-inoculated cotton roots were not only local but also systemic. The change in the levels ofcotton root exudate phenolic acids partially revealed the mechanism for pathogenesis in Verticilliumwilt disease. Our results would be useful in the development of methods to better control cotton wilt

disease.

. Introduction

Verticillium wilt, a destructive vascular disease of cotton causedy the fungal phytopathogen Verticillium dahliae Klab. (Vd), occursampantly worldwide and leads to serious economic losses. Thencidence of Verticillium wilt of cotton is closely related to the

icro-ecological environment of the soil and is primarily influ-nced by the root exudates in addition to the physical and chemicalroperties of the soil (Luo et al., 2010). A small change in rootxudates will lead to large changes in the microbial populationsf the soil. Root exudates initiate and manipulate the biologicalnd physical interactions between the micro-ecological environ-ent of the soil and the roots; therefore, they play an important

ole in root-microbe communication (Broeckling et al., 2008; Lingt al., 2010). De-la-Pena et al. (2008) have shown that the pro-ein composition of root exudates changes in the presence of aiven microbial neighborhood. Root exudates of non-mycorrhizalucumber plants and that of from mycorrhizal cucumber plantsehaved very differently on the arbuscular mycorrhizal (AM) fungi

o colonization of roots (Vierheilig et al., 2003). Many pheno-ic compounds from root exudates of crops were considered toave positive allelopathic effects on many crop pathogens (Ling

∗ Corresponding author. Tel.: +86 2584396824; fax: +86 2584396824.E-mail address: ranwei@njau.edu.cn (W. Ran).

929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.apsoil.2012.02.003

© 2012 Elsevier B.V. All rights reserved.

et al., 2010). Wu et al. (2008a) found that root exudate phenolicacids such as p-hydroxybenzoic, phthalic, gallic, coumaric, cin-namic, ferulic, salicylic, and sinamic acids were antifungal andcould effectively destroy plant pathogens. They also found thatlow concentrations (<50 mg L−1) of the root exudates stimulatedthe conidial germination of pathogenic fungi, and when the con-centration reached 200 mg L−1, an inhibitory effect was observed.However, there is little information on the influence of phenolicroot exudates on the population of V. dahliae in the rhizosphere ofcotton.

Bacillus vallismortis, whose identification was reported in 1995,promoted the growth of plants and was effective in controllingthe Verticillium wilt disease of cotton (Roberts et al., 1996; Zhanget al., 2008). The bacterium induced plant systemic resistance topathogens and excreted antibiotics that suppressed the pathogens(Zhao et al., 2010). AM fungi are regarded as natural biocontrolagents. They are symbiotic soil fungi that colonize the roots ofapproximately 80% of vascular plants. They can compensate for rootdamage, compete for colonization sites, change the rhizospheremicrobial populations, and activate plant defense mechanisms(Azcón-Aguilar and Barea, 1996). Among all the tested AM species,Glomus versiforme was the most effective in controlling the cot-

ton wilt disease caused by V. dahliae (Liu, 1995). In addition, thepre-inoculation with AM fungi increased the early nutrient con-centration and growth of field-grown leeks under high productivityconditions (Idoia et al., 2004).

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The composition of phenolic chemicals in the root exudatesf a crop can be systematically changed by the response of thelant to either the application of biocontrol agents or the infec-ion by phytopathogens (Ling et al., 2010), or it can be directly

odified by the availability of resources for which microorgan-sms compete (Badri and Vivanco, 2009). Vierheilig (2004) reportedhat the changed exudation pattern of mycorrhizal plants is par-ially involved in the changed susceptibility of mycorrhizal plantsoward soil-borne microorganisms. Clarifying the regulation of rootxudates by soil microflora, including plant pathogens, may helps understand the mechanisms of pathogenesis and preventionf Verticillium wilt and further propound practices for the con-rol of the disease. The positive allelopathic effects of phenolic rootxudates on plant pathogens may be biologically regulated by theommonly used arbuscular mycorrhizal fungi or other biologicalontrol agents. Therefore, it was reasonable to speculate that thelant root secretions may be changed in response to a given neigh-oring microbe for a particular purpose (Badri and Vivanco, 2009;anoue et al., 2010).

We hypothesized that the root exudates might be altered inesponse to the inoculation with an AM fungus G. versiforme BGCD01C (AM), an antagonistic strain of B. vallismortis HJ-5 (HJ-5) and

Verticillium wilt pathogen strain V. dahliae CGMCC no. 33757 (V).he aim of this study was to examine the effects of cotton rootxudates on the growth and spore germination of V. dahliae, toetermine the changes of phenolic acids present in cotton root exu-ates under different treatment conditions and to examine whetherhe root exudate secretion is systemic or local. New information onhe regulation of root exudates by soil microflora, including plantathogens, will help to clarify the mechanisms of pathogenesis andrevention of Verticillium wilt and will guide us toward better waysf controlling wilt diseases.

. Materials and methods

.1. Microbial strains

.1.1. Bacterial strain and the preparation of its cultureB. vallismortis HJ-5 was previously isolated and identified

y our laboratory. The strain HJ-5 was antagonist toward thehytopathogen V. dahliae with an 87.4% inhibition rate, and a prepa-ation of solid fermented organic fertilizers containing the strainJ-5 was effective in controlling cotton Verticillium wilt (Zhangt al., 2008). An overnight culture of HJ-5 was inoculated in 200 mlf beef extract peptone liquid culture (5 g beef extract, 10 g peptone,

g NaCl in 1000 ml of sterilized water, pH 7.0–7.2) and incubatedt 170 rpm at 30 ◦C. After two days, the culture was centrifuged at000 × g for 15 min, and the cell pellets were collected and resus-ended in sterile distilled water. An aliquot (0.1 ml) of the HJ-5uspension was plated on beef extract peptone agar plates usinghe dilution method (10−5, 10−6, 10−7). The germinated coloniesere counted, and the spore concentration of HJ-5 in the suspen-

ion was adjusted to 109 spores per ml by diluting it with sterileater.

.1.2. Arbuscular mycorrhizal fungi culture preparationThe G. versiforme (Karsten) Berch inocula (BGC GD01C), a mix-

ure of soil, spores, hyphae and root material, was purchased fromhe Bank of Glomales (BGC) in China, Institute of Plant Nutrition and

esources, Academy of Agricultural and Forestry Sciences, Beijing,hina. Pots of the AMF propagula were produced by the inoculationf maize seedlings with the GD01C mixture in autoclaved soil-sandedium after three months of growth.

cology 61 (2012) 85– 91

2.1.3. V. dahliae culture preparationThe cotton Verticillium wilt pathogen V. dahliae Kleb was pro-

vided by the China General Microbiological Culture CollectionCenter (CGMCC no. 33757). The pathogen strain was grown on PDAmedium and incubated in the dark at 25 ◦C for 14 days. The steriledistilled water was spread on the Petri plate, and the spores werescraped from the culture surface using a fine brush. The suspensionwas then filtered through three layers of sterile cheesecloth, and theconidia concentration was determined using a hemocytometer.

2.2. Greenhouse pot experiment

The seeds of a cotton cultivar (Gossypium hirsutum Linn. Xin-luzao 8), provided by Shihezi University, Shihezi, Xinjiang, China,were surface-sterilized in 10% H2O2 for 30 min, rinsed three timesin sterilized distilled water, and then germinated at 30 ◦C in 9-cm plates covered with sterile wet filter paper. Each seedling wasgrown in pots (450 ml in volume) containing autoclaved vermi-culite. Later, 30 g of AM propagula was added as the AM treatment,and autoclaved AM propagula were used as controls. After growthfor 30 days, 1 × 108 CFU/ml of HJ-5 and 1 × 105 CFU/ml of V. dahliaespores were applied to the corresponding pots. The pot experi-ment had five treatments groups: (1) CK (control); (2) V (plantsinoculated with V. dahliae); (3) V + AM (plants inoculated with V.dahliae and arbuscular mycorrhizal fungi); (4) V + HJ-5 (plants inoc-ulated with V. dahliae and B. vallismortis HJ-5); and (5) V + AM + HJ-5(plants inoculated with V. dahliae, arbuscular mycorrhizal fungi andB. vallismortis HJ-5). All the pots were arranged randomly, and eachtreatment was replicated thirty times. Plant and soil samples werecollected after 60 days of growth in pots to collect root exudates, tomeasure the HJ-5 population in the soil and roots and to measurethe AM colonization rate.

2.3. Split-root system experiment

The split-root system consisted of two compartments, whichwere glued together and separated by a plastic plate. This plasticplate was impermeable to roots and microbes. The selected cottonseeds were grown as described above. Three days after seeding,the main roots of the plantlets were cut. The remaining cottonplant root systems were each divided into two equal parts andplanted in two split-root compartments containing autoclaved ver-miculite treated with AM as described above. After growth for 30days, 109 CFU/ml of HJ-5 and 106 V. dahliae spores were applied toeach side. The split-root system experiment had four treatments, inwhich one compartment of each split-root system was inoculatedwith V. dahliae, while the other compartments were inoculatedwith the following treatments: (1) un-inoculated (CK/V); (2) AM(AM/V); (3) HJ-5 (HJ-5/V); and (4) HJ-5 and AM (AM + HJ-5/V). Allthe pots were arranged randomly, and each treatment was repli-cated thirty times. Sixty days after inoculation, the cotton plantswere harvested, and the root exudates from each side were col-lected separately as described below.

2.4. Biological analysis

2.4.1. Shoot and root dry weightThe roots were separated from the soil, washed with sterile

water and dried in the oven at 70 ◦C. After three days, the dryweights were calculated.

2.4.2. Arbuscular mycorrhizal colonizationAfter careful rinsing with tap water, the roots were cleared in a

10% KOH solution for 40–60 min at 90 ◦C in a water bath. The rootswere washed and bleached in alkaline H2O2, placed in a 4% HCl

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olution for 3 min, and stained with a glycerol-trypan blue solu-ion (Kormanik et al., 1980). Using 50 root segments that were cutnto 1 cm long pieces, the levels of AM colonization were quantifiedccording to the method of Giovannetti and Mosse (1980).

.4.3. HJ-5 CountA semi-selective medium was used for counting the Bacillus

ubtilis-like group HJ-5 (Turner and Backman, 1991; Kinsella et al.,009). Ten grams of fresh rhizosphere soil was suspended in 90 mlf sterile water and shaken at 170 rpm for 30 min. An aliquot0.1 ml) from each dilution series (10−5, 10−6, 10−7) was introducednto Petri dishes and incubated in the dark at 30 ◦C. After five days,he colonies were counted (Ausher et al., 1975).

.4.4. Disease indexThe severity of Verticillium wilt in cotton plants was assessed

sing a leaf wilt index (LWI) at harvest stage. The degree of leafilt development in each plant was rated on a scale of 0–4, where

indicates that the whole plant was healthy; 1 indicates slightarginal chlorosis; 2 indicates moderate marginal chlorosis; 3 indi-

ates moderate wilt and visible necrosis; and 4 indicates severeilt and defoliation (Huang et al., 2006). The disease index was

alculated by the following formula:

Disease index

=∑

the number of plants in this range × the disease rangetotal plants investigated × the highest range

× 100.

.4.5. V. dahliae countThe V. dahliae numbers were estimated using an improved selec-

ive medium (1 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4, 0.01 g sodiumthylenediaminetetraacetate, 0.05 g pentachloronitrobenzene, 2 g-asparagine, 0.5 g ox bile salt, 2 g l-sorbose, 1 g sodium tetrabo-ate, and 1 L deionized H2O at pH 5.4; 0.3 ml of 1% streptomycintock solution was added after medium sterilization) for V. dahliaeAusher et al., 1975). Ten grams of fresh rhizosphere soil was sus-ended in 90 ml of sterile water and shaken at 170 rpm for 30 min.n aliquot (0.1 ml) from each dilution series (10−2, 10−3, 10−4) waspread on Petri dishes and incubated in the dark at 25 ◦C. After fiveays, the colonies were counted.

.5. Collection of root exudates

For the collection of root exudates, the plants were removedy gently washing the vermiculite off the roots with tap water.he roots of the plants from five pots were submerged in a beakerontaining 200 ml of sterile double-distilled water three times. Allhe plants were placed in a plant growth chamber for 24 h (16 hight/8 h dark) at 28–32 ◦C. The root exudates were subsequentlyreeze-dried, dissolved in 5 ml spectral-grade methanol and storedt −70 ◦C until they were used in the bioassay and HPLC analysis.

.6. Effect of root exudates from the normal and split-rootystems on conidial germination

To determine the effect of the root exudates on conidial germi-ation, 1 ml of the concentrated root exudates of each treatmentas mixed with 2% water agar and poured into Petri plates. The

onidial suspension of V. dahliae was diluted with sterile distilled

ater to no more than 1000 conidia per milliliter, and the diluted

uspension (0.1 ml) was spread onto Petri plates and incubated at8 ◦C. After five days, the number of colonies was counted (Wu et al.,008b).

cology 61 (2012) 85– 91 87

2.7. Identification and quantification of phenolic compoundsfrom the normal and split-root systems in the root exudates

The standard phenolic compounds gallic acid and 4-hydroxybenzoic acid were supplied by Alfa Aesar (Ward Hill,MA, USA), and vanillic acid, p-coumaric acid, syringic acid and caf-feic acid were purchased from Sigma–Aldrich (Steinem, Germany).The stock solutions were prepared in methanol.

The standard phenolic compounds were analyzed using anHPLC system (Agilent 1200, USA) with an XDB-C18 column(4.6 mm × 250 mm, Agilent, USA). The mobile phase consisted of2% acetic acid (A) and acetonitrile (B) with a gradient elution. TheUV detector wavelength was set at 275 nm. The standard phenoliccompounds from each fraction were identified by their retentiontimes (Banwart et al., 1985).

2.8. Effect of exogenous phenolic acids on conidial germinationand on V. dahliae colony growth

The four standard phenolic acids, gallic acid, �-hydroxybenzoicacid, vanillic acid and syringic acid, were diluted to 0, 6.25, 12.5,25.0, 50.0 and 100.0 �g/ml. The conidial germination test was con-ducted as described above. Each treatment was replicated threetimes. For the evaluation of V. dahliae colony growth, four stan-dard phenolic acids (gallic acid, �-hydroxybenzoic acid, vanillicacid and syringic acid) were diluted to 0, 12.5, 25.0, 50.0, 100.0and 200 �g/ml. The phenolic acids were filter-sterilized (0.22 �mpore size) and then added into the 2% water agar media on Petriplates. The V. dahliae colonies were counted after five days at 28 ◦C.

2.9. Statistical analysis

The data were analyzed using the statistical program SPSS forWindows, version 16 (SPSS, Inc., Chicago, IL). The data were ana-lyzed using the analysis of variance (ANOVA) method, and themeans were separated by Tukey’s HSD tests at P ≤ 0.05.

3. Results

3.1. AM colonization and shoot and root dry weights

The data showed that V. dahliae significantly reduced the plantshoot dry weights; however, the reduction of the root dry weightsby V. dahliae was not significant compared with the control. Theapplication of the AM or HJ-5 caused similar shoot and root dryweights; however, those were significantly higher than the weightsin the control and V. dahliae treatment groups. The treatment withV + AM + HJ-5 showed the highest shoot and root dry weights whencompared with other treatments. These data confirmed that theaddition of the biocontrol strains could promote plant growth,especially with the co-inoculation of AM and HJ-5. The coloniza-tion of AM was slightly higher in the V + AM treatment than in theV + AM + HJ-5 treatment, but the differences were not significant(Table 1).

3.2. The numbers of HJ-5 and V. dahliae in the rhizosphere soiland the disease index

The number of HJ-5 in the rhizosphere revealed that the pres-ence of AM significantly increased the number of HJ-5 in therhizosphere (Table 2). The number of V. dahliae in the rhizosphere

decreased with the inoculation with biocontrol agents. However,the effect of AM was more significant compared with HJ-5. Themost significant decrease in the number of V. dahliae pathogens wasobtained with the co-inoculation of AM and HJ-5 when compared

88 G. Zhang et al. / Applied Soil Ecology 61 (2012) 85– 91

Table 1AM colonization and the dry weights of the shoots and roots of cotton plants after 60 days of growth in pots under control conditions in a green house.

Treatments Shoot dry weight (g) Root dry weight (g) AM colonization (%)

CK 1.17 ± 0.12b 0.40 ± 0.02b –V 0.85 ± 0.13c 0.33 ± 0.01b –V + AM 1.22 ± 0.11b 0.54 ± 0.06a 76.08 ± 3.15aV + HJ-5 1.21 ± 0.10b 0.39 ± 0.02b –V + AM + HJ-5 1.57 ± 0.27a 0.61 ± 0.05a 68.96 ± 5.78a

The treatment groups included control (CK), and plants inoculated with V. dahliae (V), V. dahliae and arbuscular mycorrhizal fungi (V + AM), V. dahliae and B. vallismortis HJ-5(V + HJ-5), and V. dahliae, arbuscular mycorrhizal fungi and B. vallismortis HJ-5 (V + AM + HJ-5). Data are expressed as mean ± standard error. Data in a column with a differentletter differed significantly at P = 0.05 based on Tukey’s HSD test.

Table 2Disease index of tested cotton plants and the abundance of V. dahliae and HJ-5 in the rhizosphere soil after growth of cotton plants for 60 days.

Treatments Disease index (%) V. dahliae in rhizosphere soil (×103 CFU/g) HJ-5 in rhizosphere soil (×106 CFU/g)

CK – – –V 70.00 ± 5.00a 28.00 ± 4.36a –V + AM 43.6 ± 5.72b 15.67 ± 2.08b –V + HJ-5 45.00 ± 5.00b 20.00 ± 5.00ab 11.00 ± 1.50bV + AM + HJ-5 25.67 ± 4.04c 11.33 ± 1.53b 16.00 ± 1.40a

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he treatment groups included control (CK), and plants inoculated with V. dahliae (VV + HJ-5), and V. dahliae, arbuscular mycorrhizal fungi and B. vallismortis HJ-5 (V + Aetter differed significantly at P = 0.05 based on Tukey’s HSD test.

ith other treatments. The disease index showed that all the bio-ontrol treatments decreased the disease index significantly whenompared with the treatment with the pathogen alone; however,M and HJ-5 each had similar anti-pathogen effects. The minimumisease index was obtained with the co-inoculation of AM and HJ-5.

.3. Effect of cotton root exudates on the conidial germination of

. dahliae

The root exudates collected from plants inoculated with only. dahliae showed the highest germination number of V. dahliaehen compared with the four other treatment groups (Fig. 1). The

ddition of root exudates from the biocontrol agent-inoculatedreatments could suppress the germination of V. dahliae spores,nd the maximum suppression of V. dahliae spore germination was

btained with the co-inoculation of AM and HJ-5. The root exudatesrom the V + HJ-5 treatment group decreased spore germination by1.9%, while the root exudates from the V + AM + HJ-5 treatment

ig. 1. Effects of root exudates from different treatments on the conidial germi-ation of V. dahliae. Root exudates were obtained from different treatments at theime of harvesting cotton plants grown in the normal system (“normal” needs to beefined). The treatment groups included CK (control), V (plants inoculated with V.ahliae), V + AM (plants inoculated with V. dahliae and arbuscular mycorrhizal fungi),

+ HJ-5 (plants inoculated with V. dahliae and B. vallismortis HJ-5), and V + AM + HJ-5plants inoculated with V. dahliae, arbuscular mycorrhizal fungi and B. vallismortisJ-5). The data were subjected to Tukey’s HSD ANOVA test. The bars represent show

he standard error of the mean. The histogram columns with different letters areignificantly different at P = 0.05 according to Tukey’s HSD test.

ahliae and arbuscular mycorrhizal fungi (V + AM), V. dahliae and B. vallismortis HJ-5J-5). Data are expressed as mean ± standard error. Data in a column with a different

group decreased spore germination by 31.85% when comparedwith root exudates that had been treated solely with V. dahliae.

3.4. Concentrations of phenolic compounds in cotton rootexudates

Gallic acid, �-hydroxybenzoic acid, vanillic acid and syringicacid were found in cotton root exudates (Fig. 2). The maximalconcentration of phenolic acids was found in the plant root exu-dates inoculated only with V. dahliae. Gallic acid was found in alarge proportion of cotton root exudates in each treatment, and itwas most concentrated (50 �g/ml) in the exudates that had beentreated with only V. dahliae. Syringic acid was only found in twotreatment groups, V and V + AM, and its concentration was lowerthan the other three phenolic acids. The application of HJ-5 to theV. dahliae-treated exudates significantly decreased the concentra-tions of the phenolic acids when compared with the plants that

had been inoculated with only V. dahliae. The V + AM + HJ-5 treat-ment group showed the lowest level of phenolic acid levels whencompared with the other four treatment groups.

Fig. 2. The concentrations of different phenolic acids in the root exudates, detectedby HPLC, from different treatment groups at the time of harvest. The treatmentgroups included CK (control), V (plants inoculated with V. dahliae), V + AM (plantsinoculated with V. dahliae and arbuscular mycorrhizal fungi), V + HJ-5 (plants inoc-ulated with V. dahliae and B. vallismortis HJ-5), and V + AM + HJ-5 (plants inoculatedwith V. dahliae, arbuscular mycorrhizal fungi and B. vallismortis HJ-5). The data weresubjected to Tukey’s HSD ANOVA test. The bars show the standard errors of themean. The histogram columns with different letters are significantly different atP = 0.05 according to Tukey’s HSD test.

G. Zhang et al. / Applied Soil Ecology 61 (2012) 85– 91 89

Fig. 3. Effects of exogenous phenolic acids (�g/ml) on the spore germination num-ber of V. dahliae (after seven days; unit: CFU). Four kinds of exogenous phenolic acidswith six concentrations (from 0 �g/ml to 100 �g/ml) affected the spore germinationnsa

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Fig. 5. Effect of root exudates from different treatments on the conidial germinationof V. dahliae in the split-root system. CK/V represents one side inoculated with V.dahliae and the other side inoculated with nothing. AM/V represents one side inoc-ulated with V. dahliae and the other side inoculated with arbuscular mycorrhizalfungi. HJ-5/V represents one side inoculated with V. dahliae and the other side inoc-ulated with B. vallismortis HJ-5. AM + HJ-5/V represents one side inoculated with V.dahliae and the other side inoculated with arbuscular mycorrhizal fungi and B. val-

umber of V. dahliae. The data were subjected to Tukey’s HSD ANOVA test. The barshow the standard errors of the mean. The histogram columns with different lettersre significantly different at P = 0.05 according to Tukey’s HSD test.

.5. Effects of exogenous phenolic acids on V. dahliae conidiaermination

Low concentrations of phenolic acids (0–25 �g/ml) enhancedpore germination of V. dahliae, while higher concentrations of phe-olic acids (25–100 �g/ml) decreased spore germination (Fig. 3).allic acid, �-hydroxybenzoic acid, vanillic acid and syringic acidt a concentration of 25 �g/ml all generated the highest V. dahliaepore germination. However, 50 �g/ml concentrations of galliccid, �-hydroxybenzoic acid, and vanillic acid did not significantlyecrease spore germination when compared with treatments with5 �g/ml phenolic acid.

.6. Effects of exogenous phenolic acids on V. dahliae colonyrowth

The colony diameter of V. dahliae increased with 0–50 �g/mloncentrations of �-hydroxybenzoic acid, vanillic acid and syringiccid, and 25 �g/ml gallic acid generated the maximum colonyiameter. Higher concentrations of phenolic acids (50–200 �g/ml)ecreased the colony diameter (Fig. 4). Syringic acid did not have aignificant effect on the colony diameter of V. dahliae at concentra-ions of 12.5–100 �g/ml. High concentrations of �-hydroxybenzoiccid and vanillic acid caused a decrease in V. dahliae colony diam-ter when compared with syringic acid and gallic acid.

.7. Effect of root exudates from the split-root system on conidialermination of V. dahliae

Like the normal system, the root exudates that were collected

rom the biocontrol agent-inoculated side of the split-root systemeduced spore germination more than the pathogen-inoculatedide; however, the differences were only significant betweenhe side inoculated with different biocontrol agents, while the

ig. 4. Effects of exogenous phenolic acids (�g/ml) on the colony diameter of. dahliae. Four kinds of exogenous phenolic acids with six concentrations (from

�g/ml to 100 �g/ml) affected the colony diameter of V. dahliae. The data wereubjected to Tukey’s HSD ANOVA test. The bars show the standard errors of theean. The histogram columns with different letters are significantly different at

= 0.05 according to Tukey’s HSD test.

lismortis HJ-5. The data were subjected to Tukey’s HSD ANOVA test. The bars showthe standard errors of the mean. The histogram columns with different letters aresignificantly different at P = 0.05 according to Tukey’s HSD test.

differences between the two sides of a system were not significantexcept in the AM + HJ-5/V treatment group. In the split-rootsystem, when the biocontrol agents were applied to one side,the exudates collected from the other side caused a reduction inV. dahliae spore germination when compared with the exudatesfrom the control side or the side that was only inoculated with V.dahliae (CK/V). The maximum decrease in conidia germination wasobserved in the treatment where one side was inoculated withAM + HJ-5 and the other side was inoculated with V. dahliae (Fig. 5).

3.8. Concentration of phenolic compounds in split-root exudates

In the split-root incubation system (Table 3), only gallic acid,�-hydroxybenzoic acid and vanillic acid were found. In each treat-ment group, the concentrations of the phenolic acids excreted fromthe pathogen-inoculated sides were significantly higher than thebiocontrol strain-inoculated sides, except for gallic acid, where thedifferences were not significant. However, the concentrations of thephenolic acids were significantly lower in both sides of all treat-ment groups when compared with the control side and the sidethat was inoculated with only V. dahliae (CK/V). The inoculation ofAM, HJ-5 or AM + HJ-5 on one side systemically decreased the con-centrations of the phenolic acids in the cotton root exudates of theother sides, which were inoculated with V. dahliae.

4. Discussion

The results showed that the pre-inoculation with AM and thepost-inoculation with HJ-5 of pathogen-infested soil significantlypromoted plant growth and effectively suppressed cotton Verti-cillium wilt disease by decreasing the number of V. dahliae in therhizosphere. The co-inoculation with AM and HJ-5 further effec-tively suppressed Verticillium wilt disease and improved cottongrowth. The AM compensates for root damage, competes for colo-nization sites, and activates the plant defense mechanisms, whilethe Bacillus strains have proven to be effective biocontrol agents bysolubilizing soil phosphorous and producing hydrolytic enzymes,antibiotics, and plant growth hormones (Abdel-Fattah et al., 2011;Castellanos-Morales et al., 2010; Garmendia et al., 2005; Huanget al., 2003). Both strains were more effective when co-inoculated,which is consistent with previously reported results (Felici et al.,

2008).

In the trial, the root exudates of cotton were changed by theinoculation with biocontrol strains and V. dahliae. The conidial ger-mination of V. dahliae was decreased by incubation with the root

90 G. Zhang et al. / Applied Soil Ecology 61 (2012) 85– 91

Table 3Concentrations of phenolic compounds in the root exudates of cotton plants grown in split-root systems.

Treatments Gallic acid (�g/ml) �-Hydroxybenzoic acid (�g/ml) Vanillic acid (�g/ml)

Left Right Left Right Left Right

CK/V 2.21 ± 0.25a 2.72 ± 0.10a 0.41 ± 0.04a 0.54 ± 0.02a 0.46 ± 0.01a 0.69 ± 0.02aAM/V 1.83 ± 0.11ab 1.96 ± 0.08b 0.33 ± 0.02b 0.38 ± 0.02b 0.15 ± 0.01c 0.28 ± 0.02bHJ-5/V 1.49 ± 0.03bc 1.51 ± 0.10c 0.33 ± 0.01b 0.36 ± 0.01b 0.21 ± 0.02b 0.26 ± 0.04bAM + HJ-5/V 1.20 ± 0.10c 1.51 ± 0.15c 0.14 ± 0.02c 0.24 ± 0.01c 0.11 ± 0.00d 0.26 ± 0.03b

CK/V represents the split-root system where the left side was not inoculated and the right side was inoculated with V. dahliae. Similarly, AM/V represents the system where thel oculata rhizale ding t

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eft side was inoculated with arbuscular mycorrhizal fungi and the right side was innd V. dahliae. AM + HJ-5/V represents the system inoculated with arbuscular mycorrror. Data in a column with a different letter differed significantly at P = 0.05 accor

xudates from AM- and HJ-5-inoculated plants and increased byncubation with the root exudates from plants inoculated solely

ith V. dahliae. These results were consistent with the reportshat the root exudates from pathogen-challenged tomato andatermelon plants caused a stimulation of microconidial germi-ation, while exudates inoculated with antagonistic strains causedhe suppression of conidial germination of the plant pathogenSteinkellner et al., 2008). The effect of root colonization by AMn the degree of root colonization by pathogenic and symbioticungi has been reported in several studies (Caron et al., 1986).he root exudates of non-mycorrhizal cucumber plants stimulatedathogen root colonization, whereas root exudates of mycor-hizal cucumber plants suppressed root colonization by pathogensVierheilig et al., 2003). The growth and invasion of V. dahliae arenfluenced by the micro-ecological environment of the soil. Otherhan the environmental conditions of soil, the micro-ecologicalnvironment is influenced primarily by the root exudates (De-a-Pena et al., 2008; Ling et al., 2010). Scheffknecht et al. (2006)eported results that contrasted with ours. They showed that thehanges in the root exudates of tomato plants upon inoculation byycorrhiza were favorable to Fusarium oxysporum f. sp. Lycopersici

onidia germination.Four types of phenolic acids were detected in the cotton root

xudates in our experiment. Gallic acid was dominant in the cottonoot exudates, and its levels were significantly different in all thereatment groups. Intriguingly, syringic acid was only detected inhe V. dahliae treatment group. In the rhizosphere, the colonizationf antagonistic strains lowered the colonization rate of V. dahliaend decreased the secretion of phenolic acids. The highest concen-ration of phenolic acid was 50 �g/ml, which was obtained in thereatment with only V. dahliae. In normal field conditions, the con-entrations of phenolic acids in the rhizosphere are barely higherhan 30 �g/ml. The rhizosphere is very complex, and the phenoliccids can be adsorbed or fixed by the soil, diffused in a soil solution,nd deposited by other microorganisms (Bais et al., 2006; Bertint al., 2003). Therefore, at the rhizosphere level, relatively high phe-olic acid concentrations stimulated the conidial germination of V.ahliae, and low phenolic acids concentrations were not as effectiven conidial germination.

Wu et al. (2008a) and Hao et al. (2010) reported theiological effects of phenolic acid on the pathogen. Phthalic, p-ydroxybenzoic, gallic, coumaric, cinnamic, ferulic, salicylic, andinamic acids were antifungal phenolic acids. At relatively high con-entrations, they inhibited the conidial germination of pathogens.ur results were similar and showed that four kinds of exogenoushenolic acids stimulated V. dahliae conidia germination at loweroncentrations and started to suppress V. dahliae conidia germina-ion at concentrations higher than 50 �g/ml.

Our results showed that the secretion of phenolic acids might be

nduced by V. dahliae which was an important mechanism for thelant to control Verticillium wilt. When the plants were exposed to. dahliae, they excreted more exudates with more phenolic acidshat promoted the growth of the pathogens; the concentration of

ed with V. dahliae. HJ-5/V represents the system inoculated with B. vallismortis HJ-5 fungi plus B. vallismortis HJ-5 and V. dahliae. Data are presented as mean ± standardo Tukey’s HSD test.

phenolic acids was not high enough to inhibit V. dahliae growth.In the presence of antagonistic strains, the number of pathogencolonies did not increase to a level that can cause wilt, possiblycausing the root exudates to have lower levels of phenolic acids.The pathogen that causes Verticillium wilt could damage plantcell membrane and enhance the leakage of compounds (Gapilloutet al., 1995; Tai and Xu, 2006), which could be the reason thatthe concentration of phenolic acids in the root exudates collectedfrom the V. dahliae-inoculated plants was significantly higher thanthe controls in this study. Therefore, severe pathogenic invasionmight lead to high phenolic acid concentrations in the plant rhi-zosphere, and a higher phenolic acid concentration, especially forgallic acid, might also result in a severe pathogenic invasion. Gal-lic acid could also indirectly exert detrimental effects by triggeringoxidative stress, thereby predisposing cotton plants to infection bypathogens (Ye et al., 2006). This would cause a harmful cycle, whichwould eventually lead to symptoms of Verticillium wilt or evendeath. However, the destructive cycle might not take place in therhizosphere of V. dahliae-challenged cotton plants that are inoc-ulated with AM or HJ-5 because AM and HJ-5 could systemicallydecrease the phenolic acid secretion of the roots, as we showed inthis study. Additionally, Bacillus strains form biofilms in the rhizo-sphere and produce antibiotics and hydrolytic enzymes that protectthe roots from pathogenic invasion (Raza et al., 2009). This couldreduce the autotoxicity of cotton plants and thus promote plantgrowth and suppress the incidence of Verticillium wilt in cotton(Ling et al., 2010). In addition to the production of antibiotic andhydrolytic enzymes (Raza et al., 2009), the regulation of phenolicacid levels might also be an important mechanism for the controlthe Verticillium wilt by AM and HJ-5.

The results from the split-root system experiment showedthat the phenolic acid concentrations and conidial germinationdecreased in the root exudates collected from the V. dahliae-inoculated side when the other side was inoculated with AM and/orHJ-5. The pathogen changed the root exudates for its own benefit,whereas its challengers, HJ-5 and AM, prevented this advantage andgenerated poor conditions for conidial germination of the pathogenV. dahliae. We suggest that inoculation with HJ-5 and AM with orwithout the pathogen V. dahliae not only locally but also systemi-cally affected the root exudates, which subsequently impacted theconidial germination of V. dahliae in cotton. The induction of plantactions by microbes has been reported in several studies (Duttaet al., 2008; Scheffknecht et al., 2006; Vierheilig et al., 2003). Fur-thermore, the alterations of the exudation pattern were not limitedto AM-, HJ-5- and/or V. dahliae-colonized roots, but also occurredin the non-colonized roots of a colonized root system through aplant-mediated mechanism.

This study clearly showed that the altered exudation pat-tern of cotton plants by the inoculation with AM, HJ-5 and/or V.

dahliae could contribute to different bioactive effects on V. dahliaeconidial germination, and this might be an important biologicalphenomenon. Moreover, the alterations of the root exudates fromcotton plants inoculated with AM, HJ-5 and/or V. dahliae were not

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nly local but also systemic. Gallic acid might play a specific role in. dahliae conidial germination. Therefore, the evaluation of cottonoot exudates inoculated with or without the pathogen V. dahliaer antagonistic microorganisms such as AM and HJ-5 will reveal theicro-ecological environmental conditions that affect and control

athogenesis.

cknowledgments

This work was supported by the National Basic Researchrogram of China (Grant no. 2011CB100503) and the Nationalepartment Public Benefit Research Foundation of the Ministry ofgriculture of China (Grant no. 201103004).

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