R E S E A R C H L E T T E R

Isolationand characterizationof endophytic and rhizospherebacterial antagonists of soft rot pathogen fromPinellia ternataXiufang Hu, Qionglou Fang, Shixiao Li, Jinguang Wu & Jishuang Chen

Institute of Bioengineering, Zhejiang Sci-Tech University, Hangzhou, China

Correspondence: Jishuang Chen, Institute

of Bioengineering, Zhejiang Sci-Tech

University, Road 2, Xiasha, Hangzhou

310018, China. Tel.: 186 571 8684 3200;

fax: 186 571 8684 3196; e-mail:

chenjs@zstu.edu.cn

Received 29 August 2008; accepted 16

February 2009.

First published online 9 April 2009.

DOI:10.1111/j.1574-6968.2009.01558.x

Editor: Bernard Paul

Keywords

biological control; soft rot disease;

Pectobacterium carotovorum; Pinellia ternata;

endophytic and rhizosphere antagonists.

Abstract

Pinellia ternata, a traditional Chinese herb that has been used in China for over

1000 years, is susceptible to a soft rot disease, which may cause major loss of yield.

The use of bacteria as potential antagonists against Pectobacterium carotovorum

SXR1, the causal agent of the disease on P. ternata, was evaluated. Altogether, 1107

candidate bacteria were isolated from the rhizosphere and surface-sterilized plants

of P. ternata. In Petri dish tests, 55 isolates inhibited the growth of strain SXR1, and

21 of these reduced the disease development on P. ternata slices by over 50%.

Four selected antagonists significantly reduced the disease incidence on tissue

culture seedlings, and also prevented the disease on the transplants. Agonist P-Y2-2

yielded a good prevention level of 81.9%. The four antagonists rapidly colonized

the tissue culture seedlings and transplants, whereas greater populations of the

antagonists (107–109 CFU g�1 fresh tissues) were observed in the seedlings and in

the preinoculated transplants than in those inoculated during transplanting. The

use of pathogen-free tissue culture seedlings pre-inoculated with antagonist may

provide a strategy for production of P. ternata plantlets resistant to soft rot disease.

This is the first report on the efficacy of biocontrol agents against pathogens on

P. ternata.

Introduction

Pinellia ternata (Thumb.) Breit. (Araceae) is a perennial

medicinal herb that has been used in Chinese medicine for

over 1000 years (Hu & Tao, 2005). To meet increasing

demand and to protect this natural resource, P. ternata has

been cultivated on a large scale since the 1970s (Mao & Peng,

2002). However, a soft rot disease caused by Pectobacterium

carotovorum has recently been reported on cultivated

P. ternata (Hu et al., 2008). This pathogen induces water-

soaked lesions, soft rot and finally, collapse of the whole

plant. The disease generally occurs in summer and spreads

widely, causing significant loss of yield. Traditional control

measures include crop rotation, use of certified tuber seed

and healthy, noncontaminated transplants, proper disposal

of infected plant debris and application of copper or other

bactericides (Fu & Wen, 2006). However, no strategy is

currently available to completely protect this plant from

damage. Therefore, an alternative approach is needed to

protect P. ternata from soft rot disease.

Biological control, especially the use of bacteria, has been

a focus of recent research (Weller, 1988). Plant growth-

promoting rhizobacteria (Kloepper, 1983), fluorescent Pseu-

domonas strains (Altin & Bora, 2001) and antagonistic

bacteria isolated from the rhizosphere (Chard et al., 1991)

are common biological control options for controlling soft

rot pathogens. Recently, the use of endophytic bacteria as

biological control agents for soil-borne root diseases has

been of very high interest due to their ability to colonize

healthy plant tissue and produce antibiotics in situ (Hallman

et al., 1997). Several bacterial endophytes have been re-

ported to support growth and improve the health of plants,

and therefore may be important sources of biocontrol agents

(Smith et al., 2003; Kavino et al., 2007). Pectobacterium

cartovorum is inhibited by numerous endophytic bacteria,

including strains of Pseudomonas sp., Curtobacterium

luteum and Pantoea agglomerans (Sturz et al., 1999).

Although the results of these previous studies are promising,

few applicable biocontrol agents that suppress soft rot have

been identified to date. Moreover, no bacterial antagonists

FEMS Microbiol Lett 295 (2009) 10–16c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

have been reported to suppress the soft rot pathogen on

P. ternata.

Biocontrol is an emerging trend aimed at reducing

chemical input in plant production, while increasing plant

fitness, productivity and resistance to diseases in the context

of sustainable horticulture. Biocontrol measures using an-

tagonists provide the capability of preplanting protection,

which is crucial for the production of the traditional herb

P. ternata. The objective of this study was to select endophy-

tic and rhizosphere bacterial antagonists and to evaluate

their efficacy to enhance resistance against soft rot pathogen

on P. ternata.

Materials and methods

Collection of candidate antagonists

Plants and rhizosphere soil samples were collected from

healthy P. ternata and P. ternata infected by soft rot patho-

gens in Zhejiang and Shandong provinces in China. Soil

samples (5 g) were shaken in 150 mL sterile distilled water

for 1 h at 200 r.p.m. Suspensions were diluted four to six

times at a 1/10 ratio and plated on nutrient agar (NA)

medium for bacterium development. The entire plants were

washed in tap water, surface-sterilized with 70% ethanol for

1 min and with 1% sodium hypochlorite for 3 min, and

finally washed three times in sterile-distilled water. The

external plants were placed on NA plates to validate the

efficacy of the sterilization procedure. Roots, tubers and

stems (including leaves) of the treated plants were separately

ground with a mortar and pestle and diluted with phosphate

saline buffer. A series of dilutions were spread onto NA

plates. After incubation at 28 1C for 48 h, morphologically

different colonies were selected as candidate antagonists.

In vitro antagonistic activity

On Petri dishes

Pectobacterium carotovorum SXR1, isolated and identified

in our previous experiments (Hu et al., 2008), was used

as the soft rot pathogen. A suspension of strain SXR1

(105 CFU mL�1) was spread on NA medium, and each of

the candidate bacteria (107 CFU mL�1) was then spot inocu-

lated as three replicates. After an incubation period of 48 h at

28 1C, antagonistic activities were evaluated by measuring

the widths of the clear zones surrounding the spot cultures.

The percentage of the efficiency was calculated using the

Abbott formula (Abbott, 1925).

On P. ternata slices

The potential antagonistic bacteria selected from Petri

dishes were tested for their ability to suppress soft rot caused

by strain SXR1 on P. ternata slices. Five pieces of P. ternata

tuber slices 15–20 mm in diameter (sterilized as above) were

placed on a moist sterile filter paper in Petri dishes, and were

inoculated with 20mL of candidate antagonist solution

(105 CFU mL�1) and 20mL of the pathogen (103 CFU mL�1).

The inoculated slices were then incubated at 28 1C. Slices

inoculated with strain SXR1 or the candidate antagonist

were used as controls. The development of the resulting

disease symptoms was evaluated 1 week after inoculation.

In vivo antagonistic activity

On tissue culture seedlings

Antagonists with over 60.0% protection of P. ternata slices

from soft rot were further tested on the 21-day-old tissue

culture seedlings. Inoculation of the antagonists was per-

formed with droplets (20 mL) of bacterial suspension

(107 CFU mL�1) deposited on each stem base of the seed-

lings in five tissue-cultured bottles, and after 24 h, strain

SXR1 (105 CFU mL�1) was drip-inoculated. Negative and

positive controls were inoculated only with sterile water and

strain SXR1, respectively. The inoculation was performed

under aseptic conditions. The seedlings were grown in a

growth chamber conditioned at 25 1C with 12 h of 3000 lux

illumination per day. The development of the disease

symptoms was evaluated at 5-day intervals.

On transplants

Antagonists with over 60.0% suppression of soft rot on

seedlings were tested on 4-week-old tissue culture plantlets

of P. ternata. The plantlets were drip-inoculated with the

antagonist, either 7 days before, or during, transplantation.

The roots of the plantlets were then dipped into the

pathogen suspension for 15 min. Sterile water and strain

SXR1 were used as negative and positive controls, respec-

tively. Five replicates of the treated plantlets were planted in

pots containing field soils; each replicate contained two

plantlets. The replicates were maintained in a climate-

controlled room at 25 1C with a humidity of 80–90%. The

development of the disease symptoms was evaluated 2 weeks

later.

Colonization of the antagonists

The colonization of antagonists was investigated on tissue

culture seedlings and the resulting transplants. The inocula-

tion procedure and growth method were similar to those of

the in vivo assay described above. The inoculation on

transplants was performed 7 days before or during trans-

planting, as five replicates. Seedlings were sampled for

population estimation at days ranging from 0.5 to 30 days

after inoculation. The roots, stems and leaves of each

FEMS Microbiol Lett 295 (2009) 10–16 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

11Biocontrol of soft rot on Pinellia ternata

treatment were collected and separately ground with a

mortar and pestle, and the resulting homogenate was serially

diluted and placed on NA plates to estimate the population

of the antagonist. Population counts were also performed

using the uninoculated controls.

The transplants were sampled 4 weeks after transplanting.

The leaves were washed in tap water, surface sterilized with

70% ethanol for 1 min and with 1% sodium hypochlorite

for 3 min and finally washed three times in sterile-distilled

water. Sterilization was validated by placing the leaves on NA

plates. The sterilized leaves were used as above to estimate

the population of the antagonist.

Evaluation and statistical analysis

The evaluation of antagonistic activity was performed using

a 0–3 scale. The scale for tuber slices was: 0 = no symptom,

1 = 0–2/5 soft rot of slice, 2 = 2/5–4/5 soft rot of slice and

3 = complete soft rot of slice. The scale for seedlings and

plants was: 0 = no symptom, 1 = water-soaked lesion on the

base of stem, 2 = water-soaked lesion on stem and leaf and

3 = collapse of plant. The percentage of the efficiency was

calculated using the Abbott formula (Abbott, 1925). The

experimental data were statistically analyzed by a t-test using

the program DPS 7.05 (Tang & Feng, 2007).

Results

Collection of candidate antagonists

Seven hundred and ninety-one bacterial isolates out of 4859

colonies were obtained from the stems, tubers and roots of

P. ternata plants, and 318 isolates out of 1805 colonies from

the rhizosphere soils. Two of the isolates proved to be

pathogenic to the tubers of P. ternata. Thus, the rest of the

total 1107 isolates (212 from stems, 382 from tubers, 196

from roots and 317 from rhizosphere) were used as candi-

date antagonists in the subsequent studies (Table 1).

In vitro antagonistic activities

On Petri dishes

The antagonistic effects of the candidates on the pathogen

SXR1 are shown in Table 1. Fifty-five out of the 1107 isolates

revealed antagonistic activity with the antibiosis 6–28 mm in

diameter, whereas the others showed little or no activity. Of

the 55 potential antagonists, 18, 14, 13 and 10 isolates were

derived from the stems, tubers, roots and rhizosphere,

respectively. The proportion of antagonists from a specific

location on the plants ranged from 3.2% to 8.5%, indicating

that the antagonists were distributed throughout the plant.

On P. ternata slices

The efficacy of several antagonists on P. ternata slices is

given in Table 1. Seven out of the 55 potential antagonists

(Y1-9, W6S1, GJ1-11, L1R2-2, P-WXS2-3, H2T3, L1-5 and

L3R2-1) strongly inhibited (100%) the disease incidence

on P. ternata slices (Fig. 1a). Antagonists H2T1, GJI-11

and Y2S5-2 resulted in a reduction of the disease ranging

from 71.4% to 82.1%. Eleven antagonists (L1S2-2, L3R3-1,

P-Y2-2, P-H2T1-2, P-Y11T3-2, Y2S3-2, GJI-8, WXS2-1,

P-H2T1-1, H2T1-2 and H2T1-3) prevented 50.0–64.3% of

slices from soft rot whereas 15 antagonists prevented disease

development at a ratio o 50.0%. The other antagonists

showed no suppression of the disease. The 21 antagonists

with 50.0% or greater disease prevention were used in the

following studies.

In vivo antagonistic activities

On tissue culture seedlings

All the tested antagonists, with the exception of H2T1-3 and

L1-5, delayed and reduced the incidence of soft rot on the

P. ternata seedlings (Table 2). Only one of the antagonists

(P-Y2-2) completely inhibited the disease. GJ1-8 and

P-Y11T3-1, the second best antagonists in this study,

reduced the disease development at ratios of 93.3% and

80.0%, respectively. Symptom development on the seedlings

was inhibited at a ratio in the range of 67.0–53.3% by

antagonists L3R3-1, P-WXS2-3 and P-H2T1-2. Seven an-

tagonists (Y2S3-2, WXS2-1, L1R2-2, P-H2T1-1, H2T1-2,

Y2S3-2 and H2T1) prevented the development of the disease

with a ratio of 46.6% or lower. No efficacy was observed for

the other antagonists 15 days after the inoculation. No

symptoms were observed on the negative control seedlings,

whereas 100.0% of the seedlings collapsed 3–4 days after

inoculation in the positive controls (Fig. 1b). The antago-

nists with 60.0% or greater antagonistic activity on the

seedlings were further tested on the transplants.

Table 1. Numbers of bacterial isolates and antagonists selected using

in vitro tests

Location

Candidate

isolates

Selected

antagonists

(ratio%) on

Petri dishes

Selected antagonists with

different scale of disease

severity on tuber slices

0 1 2 3

Endophyte

Stem 212 18 (8.5) 3 4 5 6

Tuber 382 14 (3.7) 1 6 2 5

Root 196 13 (6.6) 1 1 6 5

Rhizosphere 317 10 (3.2) 2 3 3 3

Total 1107 55 (5.0) 7 14 16 16

0 = no symptom, 1 = 0–2/5 soft rot of slice, 2 = 2/5–4/5 soft rot of slice,

3 = complete soft rot of slice.

FEMS Microbiol Lett 295 (2009) 10–16c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

12 X. Hu et al.

On transplants

The effects of the four antagonists P-Y2-2, GJ1-8, L3R3-1

and P-Y11T3-1 on the transplants can be seen in Table 3 and

Fig. 1c. It is evident that the four antagonists reduced the

occurrence level of the disease on the transplants. GJ1-8,

L3R3-1 and P-Y11T3-1 provided 63.5–81.9% disease reduc-

tion on the preinoculated transplants, and 48.0–68.6% on

the transplants inoculated during transplanting. Good pro-

tection on the preinoculated transplants and on those

inoculated during transplanting (81.9% and 68.6%, respec-

tively) was achieved by P-Y2-2 in this study. No symptoms

were present on negative control plants. Higher reductive

levels of the soft rot disease were obtained on the preinocu-

lated transplants, indicating that preinoculation with the

antagonists on the transplants improved their antagonism.

Colonization of the antagonists

On tissue culture seedlings

As shown in Table 2, all the tested antagonists could readily

colonize the seedlings of P. ternata, and their populations

were maintained in the range of 106–108 CFU g�1 fresh

leaves. The population dynamics of the four antagonists

with higher efficacy (GJ1-8, P-Y11T3-1, P-Y2-2 and L3R3-1)

in the seedlings were further investigated. As shown in

Fig. 2a, colonizations were observed on the leaves of the

seedlings just 12 h postinoculation. The populations of the

antagonists gradually increased after inoculation, and the

highest populations of 108–109 CFU g�1 fresh leaves were

observed about 3 days after inoculation. The populations

then remained in the range of 107–108 CFU g�1 fresh leaves

NC(a)

(b)

(c)

T PCANC

PCT

NC A T PC

Fig. 1. Photographs showing in vitro and in vivo protection of the antagonists against the soft rot of Pinellia ternata. (a) Protection of the P. ternata

slices by the antagonists. (b) Protection of the seedlings by the antagonists. (c) Protection of the transplants by the antagonists. NC, treatment with

sterile-distilled water; T, treatment with antagonists and strain SXR1; A, treatment with antagonists only; PC, treatment with strain SXR1.

FEMS Microbiol Lett 295 (2009) 10–16 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

13Biocontrol of soft rot on Pinellia ternata

over the subsequent 27 days. Comparably, the total antago-

nists recovered in the roots and tubers were significantly

higher than those in the stems and leaves 7 days after

inoculation (Fig. 2b). In fact, antagonist populations in

leaves and stems remained lower than those in tubers and

roots throughout the experiment (data not shown). No

difference in the magnitude was observed between the

populations of the rhizosphere and endophyte antagonists.

No bacteria were isolated from the control seedlings.

On transplants

Bacteria were also isolated from surface-sterilized leaves of

the transplants (Fig. 2c). The only predominant morpho-

type of the inoculated antagonists was obtained in the

preinoculated transplants with a frequency of 106–107 CFUg�1

fresh leaves. However, more than one type of predominant

bacteria, as well as lower populations (102–103 CFUg�1 fresh

leaves) of the antagonists, were observed in those transplants

inoculated during transplanting. It can be seen that antago-

nists colonized more readily under the tissue culture condi-

tions, which is likely due to the reduced possibility of

microbial competitors in the tissue culture seedlings com-

pared with the transplants inoculated during transplanting.

Discussion

The soft rot P. carotovorum is a pathogen of many plant

species, affecting crops in subtropical and temperate areas

worldwide (Toth et al., 2003). Recently, P. ternata cultivation

has been seriously affected by soft rot induced by

P. carotovorum, and the prevalence of the disease has steadily

increased year by year. About 30–90% of surveyed cultivated

Table 2. Effect of the antagonists against strain SXR1 and their colonizations on the seedlings of Pinellia ternata

Antagonists

Disease reduction (%) postinoculation (days) Populations postinoculation (107 cell g�1 fresh leaves) (days)

5 15 5 15

Rhizosphere

P-Y2-2 100.0� 0.0 100.0� 0.0 21.2�1.2 27.8�1.5

GJ1-8 93.3� 5.2 93.3� 5.2 23.6�2.2 6.4�0.2

Y1-9 63.3� 4.5 0.0� 0.0 6.7�0.5 2.1�0.1

GJ1-11 55.0� 3.1 0.0� 0.0 19.2�1.3 40.8�2.3

L1-5 0.0� 0.0 0.0� 0.0 6.2�0.3 0.6�0.0

Endophyte

Root

L3R3-1 86.7� 6.4 66.7� 5.5 30.4�1.6 14.4�1.1

L1R2-2 80.0� 4.8 26.6� 3.3 0.2�0.0 60.0�4.1

L3R2-1 18.3� 2.5 0.0� 0.0 36.5�1.7 146.0�5.3

Tuber

P-Y11T3-1 95.0� 7.5 80.0� 5.6 17.1�0.8 79.6�4.6

P-H2T1-2 86.6� 4.7 53.3� 6.2 19.0�1.0 85.1�6.2

P-H2T1-1 41.7� 3.2 20.0� 2.4 21.9�1.2 6.9�0.5

H2T1-2 23.3� 5.4 13.3� 1.5 88.0�4.5 28.5�3.7

H2T1 43.3� 3.3 6.6� 1.2 0.0�0.0 0.9�0.0

H2T3 48.3� 5.1 0.0� 0.0 0.0�0.0 0.1�0.0

H2T1-3 1.6� 1.2 0.0� 0.0 166.0�6.9 2.5�0.2

Stem

P-WXS2-3 63.3� 5.0 55.0� 4.5 0.2�0.0 1.2�0.1

Y2S3-2 73.3� 5.3 46.6� 4.7 114.0�6.9 0.8�0.0

WXS2-1 70.0� 8.1 33.3� 2.2 70.5�4.5 1.2�0.1

Y2S5-2 63.3� 3.4 0.0� 0.0 0.0�0.0 33.8�0.0

L1S2-2 60.0� 3.2 0.0� 0.0 32.0�2.2 21.1�1.6

W6S1 23.3� 1.7 0.0� 0.0 8.4�0.5 11.9�0.3

Positive control 0.0� 0.0 0.0� 0.0 0.0�0.0 0.0�0.0

Negative control 100.0� 0.0 100.0� 0.0 100.0�0.0 100.0�0.0

Table 3. Effect of the antagonists against strain SXR1 on the transplants

of Pinellia ternata

Antagonist

Disease reduction (%) on transplants

Inoculated during

transplanting Preinoculated

P-Y2-2 68.6� 3.8 81.9�5.6

L3R3-1 56.5� 2.2 69.1�3.3

P-Y11T3-1 51.7� 2.6 63.5�4.3

GJ1-8 48.0� 1.8 66.4�2.7

Positive control 0.0� 0.0

Negative control 100.0�0.0

FEMS Microbiol Lett 295 (2009) 10–16c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

14 X. Hu et al.

tubers were found to carry the pathogen, which can rapidly

cause disease symptoms when conditions are favorable (Hu

et al., 2008). The pathogenicity of P. carotovora is regulated

by quorum sensing, which is a population density-depen-

dent modulation of the bacterial phenotype (Swift et al.,

1996). Because the pathogen invades the inner part of the

plants, the conventional chemical bactericides such as

copper may not provide adequate control of the disease.

Therefore, the disease may be efficaciously inhibited by an

endophyte maintaining the pathogen population below the

level that is required for pathogenicity. In the present study,

good prevention of the disease was indeed obtained using

endophytic and rhizosphere antagonists P-Y2-2, GJ1-8,

L3R3-1 and P-Y11T3-1, which were phenotypically and

phylogenetically identified as Pantoea ananatis, Mynoides

odoratimimus, Enterobacter asburiae and Pseudomonas aeru-

ginosa, respectively (data not shown). Petri dish antagonism

does not necessarily correlate with efficacy on a plant, but

the antagonism on P. ternata tuber slices and seedlings

correlated well with the observed efficacy on this plant.

Endophytes represent a promising source of biocontrol

strains, and their use may be more successful than the use of

rhizosphere bacteria due to less competition with other

bacteria in the apoplast. Our data presented here showed

that both endophytes and rhizosphere antagonists could

become endophytically established in roots, stems and

leaves, confirming that strains could be both rhizosphere

inhabitants and tissue colonizers of hosts (Tjamos et al.,

2004). Established, thriving and stable microbial endoplant

communities may induce disease resistance through synth-

esis of structural compounds, the inhibition of bacterial

penetration, the induction and expression of general mole-

cular-based plant immunity and a simple exclusion of other

organisms by niche competition (Sturz et al., 2000). Our

results suggest that P-Y2-2, the rhizosphere antagonist,

prevented 81.9% of the P. ternata transplants from soft rot

caused by pathogen SXR1 in climate-controlled experi-

ments, indicating that it may be useful for disease manage-

ment in China. The contribution of the antagonists requires

further research using field tests.

The present study demonstrates that preinoculation of

antagonists results in a greater population in P. ternata

plantlets and a higher efficacy in preventing soft rot disease.

Nowak & Pruski (2002) found that bacterized potato plant-

lets were greener, had elevated levels of cytokinins, phenyla-

lanine ammonia-lyase, free phenolics and contained more

lignin. Upon exposure to stress, prebacterized plantlets

adapt better and faster than controls (Conrath et al., 2002).

Microbial inoculation of tissue culture plantlets may lead to

developmental and physiological changes enhancing resis-

tance to bacterial infections, which could be the reason why

enhanced antagonism was achieved in preinoculated trans-

plants compared with transplants inoculated during trans-

planting. Tubers are commonly used as seeds in the planting

of P. ternata, but they are usually infected at various levels by

soft rot pathogen (Hu et al., 2008). Because tuber seeds

containing soft rot pathogen are hazardous in P. ternata

planting, preinoculation of an antagonist in pathogen-free

seedlings via apical meristem cultures may provide a con-

venient strategy for the production of P. ternata plantlets or

tuber seeds resistant to soft rot on a large scale.

In conclusion, four endophytic and rhizosphere antago-

nists were obtained, which provided good prevention of soft

rot disease on tissue culture seedlings and subsequent trans-

plants of P. ternata. Pathogen-free seedlings preinoculated

with antagonists exhibited effective antagonism towards soft

rot. These results may provide a more environmentally sound

and economically feasible alternative to the control of soft rot

disease on P. ternata by bactericidal compounds, whose

efficacy has proven to be unsatisfactory.

Acknowledgement

This research was financially supported by the National

High Technology Research and Development Program of

China (Project No. 2006AA10Z428).

6

8

10

0.5 1 3 5 10 15 20 25 30

Days after inoculation

Log

pop

ulat

ion

of a

ntag

onis

t(c

ells

g–1

leav

es)

(a)

**** ****

** * *

5

7

9

11

P-Y11T3-1 L3R3-1 GJ1-8 P-Y2-2

Antagonist

Log

popu

latio

n of

ant

agon

ist

(cel

ls g

–1)

(b)

**

**

**

**

0

2

4

6

8

P-Y11T3-1 L3R3-1 P-Y2-2 GJ1-8

Antagonist

Log

pop

ulat

ion

of a

ntag

onis

t(c

ells

g–1

leav

es)

(c)

**

Fig. 2. Colonization of endophytic and rhizosphere antagonists on the seedlings and the resulting transplants of Pinellia ternata. (a) Populations of the

four antagonists on the seedlings of P. ternata after inoculation. (b) Populations of the four antagonists in different tissues of the seedlings 7 days after

inoculation. (c) Populations of the four antagonists in two types of inoculated plants 4 weeks post-transplanting. �P4 0.05; ��Po 0.01.

FEMS Microbiol Lett 295 (2009) 10–16 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

15Biocontrol of soft rot on Pinellia ternata

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