Applied Soil Ecology 129 (2018) 136–144

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Biological control tobacco bacterial wilt and black shank and root T
colonization by bio-organic fertilizer containing bacterium Pseudomonas
aeruginosa NXHG29
⁎
Li Maa,c, ,1, Hao-Yu Zhangb,1, Xing-Kui Zhoua,d, Cheng-Gang Yanga,c, Shuai-Chao Zhenga,c,
⁎
Jin-Ling Duoa,c, Ming-He Moa,e,
a
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming 650091, PR China
b
College of Agriculture, South China Agricultural University, Guangzhou 510642, PR China
c
Key Laboratory of Microbial Diversity in Southwest China, Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, PR China
d
Biocontrol Engineering Research Center of Plant Disease & Pest, Yunnan University, Kunming 650091, PR China
e
Biocontrol Engineering Research Center of Crop Disease & Pest, Yunnan Province, Kunming 650091, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Tobacco bacterial wilt (TBW) and tobacco black shank (TBS) are two of the most devastating tobacco soil-borne
Biological control diseases worldwide. In this study, Pseudomonas aeruginosa NXHG29 exhibited dually antagonistic activities
Bio-organic fertilizer against TBW and TBS in vitro assays. Pot experiments were performed to evaluate the capability of a novel bio-
Colonization organic fertilizer (BOF) consisting of organic fertilizer with NXHG29 to control TBW and TBS. The results
Tobacco bacterial wilt
showed that application of BOF could more effectively decrease the disease incidence of TBW and TBS than the
Tobacco black shank
Pseudomonas aeruginosa
direct application of NXHG29. Higher amounts of BOF application (0.5% and 1% amendment) resulted in the
more suppressive effects on tested pathogens when compared with a low amount of BOF application (0.1%
amendment). To determine the antagonistic mechanism of NXHG29, we investigated the colonization pattern of
NXHG29 on tobacco roots in a sand system and a natural soil system by tagging NXHG29 with a GFP-marked
plasmid. Similar observations were obtained in the two systems. The results indicated that GFP-tagged NXHG29
colonized first the differentiation zones followed by the elongation and maturation zones of the primary roots
and subsequently around the junctions of primary and lateral roots. The population dynamics of GFP-tagged
NXHG29 on tobacco roots and in the rhizosphere were also monitored. The development of the BOF using dually
antagonistic bacteria might provide new options for control strategies, especially with respect to managing both
diseases simultaneously in the host plant, which should be more effective in the long term.

1. Introduction
Tobacco (Nicotiana tabacum L), is an economically important crop
worldwide. Tobacco bacterial wilt (TBW) and tobacco black shank
(TBS), respectively caused by Ralstonia solanacearum (Yabuuchi et al.,
1995), and Phytophthora nicotianae (Lucas, 1975), are two of the most
devastating soil-borne diseases resulting in severe losses in yield and
quality of tobacco. However, traditional control methods such as agri-
cultural and chemical control do not always show positive effects.
Therefore, more recently, the use of microbial antagonists has been
considered a promising strategy for the management of these soilborne
diseases (Ling et al., 2010; Wei et al., 2011; Yang et al., 2011; Zhang
et al., 2011; Zhao et al., 2011). In many tobacco-producing regions of
China, bacterial wilt often co-occurred with black shank in the same
plant and results in a disease complex that causes greater economic
losses and renders the disease control more difficult than either pa-
thogen acting alone (Liu et al., 2007; Wang et al., 2010). In general, a
single biocontrol agent is usually used for biocontrol of plant disease
against a single pathogen (Wilson and Backman, 1999). However,
controlling just one of the pathogens might not fully solve the problem
since different pathogens often co-occur on the same plant and result in
a disease complex causing synergistic yield losses. An environmentally
friendly alternative could be the use of an individual strain of micro-
organism with a broad spectrum of antagonism against multiple-pa-
thogens, which might provide new options for control strategies for
soilborne diseases. Consequently, recent research has focused on the

⁎
Corresponding authors at: The No.2 Cuihubei road, Kunming 650091, PR China.
E-mail addresses: westgirlml@163.com (L. Ma), minghemo@163.com (M.-H. Mo).
1
Authors Li Ma and Hao Yu Zhang contributed equally to this work.

https://doi.org/10.1016/j.apsoil.2018.05.011
Received 19 June 2017; Received in revised form 30 January 2018; Accepted 14 May 2018
Available online 21 May 2018
0929-1393/ © 2018 Elsevier B.V. All rights reserved.
L. Ma et al.
discovery of biocontrol agents (BCA) with broad-spectrum activity to
effectively control multiple plant pathogens in the same host plant.
Dually antagonistic bacteria (DAB) refers to those bacteria in which one
strain (or one species) of bacterium acts antagonistically against two
diverse, co-occurring pathogens (Huang et al., 2015). The potential of
DAB agents against disease complexes has been well demonstrated
(Adam et al., 2014; Siddiqui and Ehteshamul-Haque, 2000; Son et al.,
2009). However, no information is available concerning DAB agents
against R. solanacearum and P. nicotianae in tobacco.
Directly deploying BCAs in the soil is the usual mode of application,
but this mode may lead to poor activity of the functional agents. Many
recent reports have shown that a combination of BCAs and organic
amendments (e.g., compost, manure, plant solid waste) to create bio-
organic fertilizers (BOFs) is a novel alternative method for biocontrol of
soil-borne diseases (Luo et al., 2010; Saravanan et al., 2003; Yang et al.,
2011) and is more efficient in inhibiting disease than using than using
single BCAs (Huang et al., 2011; Liu et al., 2013). It is a promising
strategy to control soilborne diseases which may be applied im-
mediately for certain known functional microbial strains and organic
amendments and may be further developed in the future. BOFs are not
only a source of organic matter in soil which promote plant growth by
improving soil structure, fertility, quality and nutrient use efficiency
(Rivera-Cruz et al., 2008; Shen et al., 2013) but also supply nutrients for
antagonists, allowing effective colonization and thus improving the
suppressive capacity towards pathogens (Ling et al., 2010; Zhang et al.,
2011). Studies have documented that the application of BOFs improved
crop yields and successfully decreased bacterial disease incidences in
many crops such as banana (Zhang et al., 2011), cucumber (Huang
et al., 2011; Zhang et al., 2008), watermelon (Ling et al., 2010; Wu
et al., 2009), tomato (Wei et al., 2011), tobacco (Ren et al., 2012),
pepper (Wu et al., 2015), sweet melon (Zhao et al., 2011), and cotton
(Lang et al., 2012). Nonetheless, reports on the effect of BOFs con-
taining a combination of DAB strains with organic amendments as
biocontrols for TBW and TBS are rare.
Significant progress in the understanding of the mechanisms for
biocontrol of soil-borne pathogens by BCAs has been made in the past
two decades (Compant et al., 2005). Root colonization by BCAs is
considered a prerequisite for successful biological control and is di-
rectly related to their efficacy in controlling soil-borne diseases (Maurer
et al., 2013). Therefore, research on the colonization pattern of an-
tagonistic agents and their survival in the rhizosphere when introduced
into soil is needed to get positive results in the biological control of soil-
borne pathogens. Currently, biocontrol of soil-borne diseases using
plant-growth-promoting rhizobacteria (PGPR) is a promising research
area (Bhattacharyya and Jha, 2012). PGPR strains of Pseudomonas
aeruginosa have been widely used in the control of soil-borne diseases
because of their excellent root colonizing ability, catabolic versatility,
and ability to produce a wide range of metabolites that favor the plant
to stand under varied biotic and abiotic stresses (Kumar et al., 2009;
Illakkiam et al., 2013; Sathyapriya et al., 2012). These abilities make
strains of P. aeruginosa excellent candidates for sustainable biocontrol.
An antagonistic strain, P. aeruginosa NXHG29, was isolated from the
rhizosphere of cucumber by State Key Laboratory for Conservation and
Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming,
China and showed strong antifungal activity against banana wilt dis-
ease caused by Fusarium oxysporum f.sp. cubense (Li et al., 2012). The
present study was, therefore, carried out to evaluate the antagonistic
activity of P. aeruginosa NXHG29 towards R. solanacearum causing TBW
and P. nicotianae causing TBS in vitro. In addition, we developed a novel
bioorganic fertilizer (BOF) by fermenting mature compost with P. aer-
uginosa NXHG29, and the capability of the BOF to control TBW and TBS
of tobacco was evaluated in greenhouse experiments. Finally, the P.
aeruginosa NXHG29 strain was tagged with GFP, and the colonization
pattern of this bacterium on tobacco roots in a sand system and a
natural soil system was studied by using confocal laser scanning mi-
croscopy (CLSM). This is the first report on the investigation of the
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Applied Soil Ecology 129 (2018) 136–144
colonization by GFP-tagged P. aeruginosa of tobacco roots.
2. Materials and methods
2.1. Strains, plasmids and culture conditions
P. aeruginosa NXHG29 (GenBank accession number HQ844486) was
previously isolated and identified as well as stored in our laboratory (Li
et al., 2012). This bacterium was grown in NB medium (L−1: beef ex-
tract 3 g, peptone 10 g, NaCl 5 g, distilled water 1000 mL, pH 7.0–7.2)
on a shaker at 180 rpm and 37 °C for 48 h. The cultures of this bac-
terium were maintained on nutrient agar (NA) plates and stored at
−80 °C in nutrient agar broth (NB) +25% glycerol. Pathogens P. ni-
cotianae HD1 and R. solanacearum K1 were kindly provided by Yunnan
Tobacco Agriculture Science Research Institute of China, and cultured
routinely using potato dextrose agar medium (PDA) and Kelman's tet-
razolium chloride (TTC) agar medium (Kelman, 1954), respectively. A
green fluorescent protein (GFP)-expressing plasmid, pSMC2, which
constitutively expressed a bright mutant of GFP and stably maintained
in Pseudomonas strains, was kindly provided by Dr. Guido V. Bloemberg
(Department of Microbiology and Molecular Genetics, Harvard Medical
School, Boston, Massachusetts) (Bloemberg et al., 1997).
2.2. Bio-organic fertilizer (BOF) preparation
The preparation of the organic fertilizer (OF) used for the BOF
preparation was carried out following the procedure described pre-
viously (Ling et al., 2010; Wu et al., 2009; Zhang et al., 2008), with
minor modification. The OF used for the preparation of BOF consisted
of a mixture of amino acid fertilizer and cattle manure compost (1:1, w/
w). The amino acid fertilizer was made from Sacha Inchi (Plukenetia
volubilis L) seed oil residues that were microbe-enzymatically hydro-
lyzed for 8 days to obtain a mixed amino acid fertilizer. This amino acid
fertilizer contained 43.50% organic matter, 10.14% total amino acids,
small molecular peptides and oligopeptides, 4.87% nitrogen (N), 3.02%
P2O5 and 0.94% K2O. P. aeruginosa NXHG29 was served as a biocontrol
strain and its cell concentration of the suspension was
1.0 × 109 CFU mL−1 in the BOF preparation. The cattle manure com-
post was made by composting cattle manure at a temperature range of
30–66 °C for 25 days. This compost contained 45.5% organic matter,
3.35% N, 2.44% P2O5 and 1.2% K2O. The BOF product used in this
experiment was prepared according to Ren et al. (2012) and Zhang
et al. (2011). The preparation was as follows: 1000 mL suspension of
1 × 109 colony-forming units (CFU) of P. aeruginosa NXHG29 per mil-
liliter and 5 kg OF were thoroughly mixed in a 600 × 400 × 200-mm
plastic container for secondary fermentation. The mixture was main-
tained at 40–45% moisture by adding sterile water at room temperature
(25–32 °C) for 6 days and manually turned every day. On the 7th day,
the mixture was air-ventilated in an aerated room at 19–24 °C for 2 days
to decrease the moisture to less than 30%. During the secondary fer-
mentation stage, the temperature and bacterial density of the substrate
were monitored every day. The density of P. aeruginosa NXHG29 in the
finished BOF product was more than 1 × 109 CFU g−1 dry weight (DW)
of BOF. The BOF product was stored at room temperature prior to use in
pot experiments.
2.3. Plant material and growth conditions
Tobacco variety Hongda was used in the experiments, which is
susceptible to bacterial wilt and black shank disease. Tobacco seeds
were provided by Yunnan Tobacco Agriculture Science Research
Institute, Yunnan Province, China and were surface-sterilized with 2%
household sodium hypochlorite for 3 min, rinsed four times using
sterile distilled water, and then germinated on a 90-mm sterile plate
covered with sterile moistened filter papers at 25 °C for 48 h. The ger-
minated seeds were sown in floating polystyrene tray. The trays were
L. Ma et al.
floated in a plastic trunk containing Hoagland’s nutrient culture
(Hoffland et al., 1989) and kept at 25–28 °C and 60% relative humidity
for about 40 days. Then, tobacco seedlings were directly transplanted to
pots (diameter 30 cm, height 25 cm) containing 3 kg soil for the pot
experiments, and one seedling was maintained in each pot.
2.4. Design of pot experiments
The soils used for the pot experiments were collected from Yuxi,
Yunnan province, China, which had the following properties: pH 6.33,
30.12 g kg−1 of total C, 1.86 g kg−1 of total N, 1.17 g kg−1 of total P,
102.01 mgkg−1 of available N, 23.05 mgkg−1 of available P, and
79.44 mgkg−1 available K, 45.37% of sand, 43.41% of silt, and 11.22%
of clay. The suspension of P. aeruginosa NXHG29 was prepared as
above.
In pot experiment 1 (Exp. 1), R. solanacearum was cultured using
TTC liquid medium on a rotary shaker for 48 h at 37 °C and 180 rpm.
Cultures were harvested by centrifugation at 12,000 rpm for 10 min and
the pellets were resuspended in distilled water, and then were mixed
thoroughly with soils. The concentration of R. solanacearum was ad-
justed to 108 CFU g−1 soil. In pot experiment 2 (Exp. 2), Zoospores of P.
nicotianae with a concentration of about 10, 000 zoospores mL−1 were
prepared following the description of Huang et al. (2015). The soil was
inoculated with the P. nicotianae zoospores suspension to obtain a
concentration of 106g−1 soil. Both experiments (Exp. 1 and 2) were
designed as follows: (1) CK1 (health control), soil without inoculation
with pathogen; (2) CK2 (diseased control), soil inoculated with pa-
thogen; (3) OF1, pathogen-inoculated soil was mixed with 0.1% (w/w)
organic fertilizer (a mixture of amino acid fertilizer and cattle manure
compost (1:1, w/w)); (4) OF2, pathogen inoculated soil was mixed with
0.5% (w/w) organic fertilizer (a mixture of amino acid fertilizer and
cattle manure compost (1:1, w/w)); (5) OF3, pathogen-inoculated soil
was mixed with 1.0% (w/w) organic fertilizer (a mixture of amino acid
fertilizer and cattle manure compost (1:1, w/w)); (6) ANT1, pathogen-
inoculated soil was mixed with the suspension of P. aeruginosa NXHG29
to obtained a final concentration of 108 CFU g−1 soil; (7) ANT2, pa-
thogen-inoculated soil was mixed with the suspension of P. aeruginosa
NXHG29 to obtained a final concentration of 109 CFU g−1 soil; (8)
BOF1, pathogen-inoculated soil was mixed with 0.1% (w/w) BOF made
with P. aeruginosa NXHG29 at the concentration of 108 CFU g−1 soil; (9)
BOF2, pathogen-inoculated soil was mixed with 0.5% (w/w) BOF made
with P. aeruginosa NXHG29 at the concentration of 108 CFU g−1 soil;
(10) BOF3, pathogen-inoculated soil was mixed with 1.0% (w/w) BOF
made with P. aeruginosa NXHG29 at the concentration of 108 CFU g−1
soil; (11) BOF4, pathogen-inoculated soil was mixed with 0.1% (w/w)
BOF made with P. aeruginosa NXHG29 at the concentration of
109 CFU g−1 soil; (12) BOF5, pathogen-inoculated soil was mixed with
0.5% (w/w) BOF made with P. aeruginosa NXHG29 at the concentration
of 109 CFU g−1 soil; (13) BOF6, pathogen-inoculated soil was mixed
with 1.0% (w/w) BOF made with P. aeruginosa NXHG29 at the con-
centration of 109 CFU g−1 soil. Each treatment was replicated three
times, and each block contained ten pots. One tobacco seedling was
transplanted into each pot (diameter 30 cm, height 25 cm) which was
filled with 2 kg of mixed soil. The seedlings were grown in a greenhouse
(25 ± 3 °C, 60% relative humidity, 10–12 h daylight). The same nu-
trient levels in all treatments were adjusted by the application of che-
mical fertilizer.
2.5. Disease incidence
Observation for disease incidence and severity were conducted from
the day of transplantation to the 45th day. The disease incidence was
recorded at the end of the experiment. The disease severity for TBW was
scored based on a scale of 0–4 (Liu et al., 2013) in each treatment:
where: 0 = no visible disease symptoms, 1 = a few flecks or small le-
sions on the stem and a little wilting, 2 = moderate black lesions
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Applied Soil Ecology 129 (2018) 136–144
showed up on the stem but not on the top and more than 50% of leaves
wilting on the lesion side, 3 = large lesions extending up to the top of
stem and more than two thirds of leaves wilting on the lesion side, and,
and 4 = death of plants. While, the disease severity for TBS was rated
on a 0–5 scale (Huang et al., 2015): 0 = no visible disease symptoms,
1 = leaves slightly wilted, with brownish lesions beginning to appear
on stems, 2 = 30–50% of plant foliage wilted, 3 = 50–70% of plant
foliage wilted, 4 = 70–90% of plant foliage wilted, 5 = plant dead. The
disease incidence was calculated as previously described by Xue et al.
(2009). Disease incidence (DI) = [Σ (Disease index × The number of
diseased plants in this index)/(Highest disease index × total number of
plants investigated) ] × 100%.
2.6. Construction of GFP-tagged P. aeruginosa NXHG29
Following the protocols of Choi et al. (2006), competent cells of P.
aeruginosa NXHG29 were prepared and electroporated using the
plasmid pSMC2. The transformed cells were immediately transferred
into 1 mL of LB medium. After incubation at 180 rpm for 1 h at 30 °C,
the transformed cells were plated onto selective medium (LB agar
medium supplemented with 400 μg mL−1 carbenicillin and
200 μg mL−1 ampicillin). Bacterial colonies harboring pSMC2 were
identified under fluorescence microscopy (Olympus DP80) with an
excitation wavelength of 488 nm.
2.7. Plasmid stability
The assay for plasmid stability was performed as described by
Compant et al. (2005). Furthermore, the colony and cell morphologies
and growth patterns as well as antagonism of the gfp derivatives were
compared to those of the wild-type strain in NA and NB liquid medium.
2.8. In vitro antagonism assay
An antibacterial bioassay was performed by the agar-diffusion
method (Lemessa and Zeller, 2007). Briefly, 200 μL fresh culture of R.
solanacearum with a concentration of 108 CFU mL−1 was mixed with
250 mL of molten NA (temperature not higher than 50 °C) and poured
into plates. On each plate, six wells with diameters of 5 mm were made
using a puncher. 200 μL fresh culture of wild type or GFP-tagged P.
aeruginosa NXHG29 with a given cell concentration (107, 108, or
109 CFU mL−1) was added to each well. An equivalent volume of NB
medium in place of the bacterial culture was used as a control. Ten petri
dishes were prepared for each treatment and all treatments were per-
formed in triplicate. After 48 h at 37 °C, diameters of inhibition zone
(IZ) were measured. The antibacterial activity of antagonistic strain can
be classified into three levels (Rota et al., 2008): weak activity
(IZ ≤ 12 mm), moderate activity (12 mm < IZ < 20 mm) and strong
activity (IZ ≥ 20 mm). An antifungal activity was monitored following
the Oxford cup method (Wang et al., 2009). The pathogenic P. nico-
tianae was grown on PDA plates at 28 °C for 4–5 days. An aliquot of
200 μL culture of wild type or GFP-tagged P. aeruginosa NXHG29 with a
given cell concentration (107, 108, or 109 CFU mL−1) was added into an
Oxford cup 5 mm in diameter which was previously placed in the center
of a PDA plate. Two 5 mm-diameter mycelial plugs of P. nicotianae from
a colony actively growing on PDA were placed beside the cup at a
distance of 2 cm. An equivalent volume of NB medium without the cell
suspension was used as a control. Ten petri dishes were prepared for
each treatment and all treatments were performed in triplicate. The
plates were incubated at 28 °C and inhibition of fungal growth was
monitored for 4–7 days. The antifungal activities were expressed as the
inhibition rate using the formula inhibition rate = (DC-DT)/
DC × 100%, where DC and DT respectively represent the colony dia-
meters of the pathogen for the control and the treatments.
L. Ma et al. Applied Soil Ecology 129 (2018) 136–144

2.9. Colonization of tobacco roots by P. aeruginosa NXHG29

The GFP-tagged P. aeruginosa NXHG29 was grown overnight in

250 mL of NB medium containing 400 μg mL−1 carbenicillin and
200 μg mL−1 ampicillin at 37 °C under vigorous shaking. Then the
bacterial cells were harvested by centrifugation at 12,000 rpm for
10 min. The harvested cells were washed twice in 10 mM phosphate-
buffered saline buffer (PBS buffer, pH 7.2) and resuspended in PBS
buffer to a final concentration of 6.75 × 108 CFU mL−1. Tobacco seeds
were surface-sterilized and germinated as described above. Well-ger-
minated tobacco seedlings were soaked in the cell suspension of the
GFP-tagged NXHG29 for 2 h at 30 °C. The control treatment was to soak
seedlings in sterilized PBS buffer. Subsequently, seedlings that had been
treated with GFP-tagged NXHG29 were transferred to container with
quartz sand, or natural soil. The plants of both the sand and the soil
systems were maintained in a climate-controlled growth chamber at
25 ± 3 °C, with 70% relative humidity and day/night cycle: 16/8h. In
both of the two systems, the tobacco seedlings were irrigated with one
half Hoagland medium. Un-inoculated seedlings were also prepared as
controls.
To visualize the spatio-temporal course of colonization of the GFP-
tagged P. aeruginosa NXHG29 on tobacco roots, samples of fresh roots
were collected at days 1, 3, 6, 9, and 12 after bacterial inoculation.
Roots were rinsed clean with sterile water and mounted on a glass slide
with PBS under a coverslip. Bacterial distribution along the roots was
observed using Olympus FV1000 confocal laser-scanning microscopy
(CLSM, Olympus, Tokyo, Japan) with an excitation wavelength of
488 nm to induce green fluorescence. Images were captured and pro-
cessed using FV10-ASW Version 2.1. Final pictures were processed with
Photoshop 4.0 software (Adobe Systems Inc., San Jose, CA).

2.10. Population dynamics of GFP-tagged P. aeruginosa NXHG29 on the

tobacco roots and in the rhizosphere soil

In the soil system, the population of GFP-tagged P. aeruginosa

NXHG29 on tobacco roots and in the rhizosphere soil was determined at
days 0, 3, 6, 9, 12, and 20 after inoculation. Five plant samples were
analyzed per sampling time. The tobacco seedlings were carefully taken
out of the soil and gently shaken to remove all loosely adhering bulk
soil. Then, the roots were soaked in distilled water and shaken to obtain
rhizosphere soil samples. The soil suspension was serially diluted and
plated onto NA medium containing 100 μg mL−1 cycloheximide,
400 μg mL−1 carbenicillin and 200 μg mL−1 ampicillin. For the collec-
tion of root samples, 1 g roots were homogenized in 9 mL PBS buffer
using an autoclaved mortar and pestle. The homogenates were 10-fold
serially diluted and plated onto NA medium containing 100 μg mL−1
cycloheximide, 400 μg mL−1 carbenicillin and 200 μg mL−1 ampicillin.
After incubation for 2–3 days at 37 °C, bacterial colonies were detected
by fluorescence microscopy (Olympus DP80), and the number of co-
lonies emitting green florescence was counted.
2.11. Statistical analysis
Population density data estimated by using CFU were subjected to
logarithmic transformation before statistical analysis with one-way
analysis of variance. Differences among treatments were calculated and
statistically assessed by one-way ANOVA and Duncan’s multiple range
tests (P < .05). All statistical analyses were performed with SPSS
ver.13.0 statistical software (SPSS, Inc., Chicago, USA).
3. Results
3.1. In vitro antagonistic activity of P. aeruginosa NXHG29
The antibacterial ability of P. aeruginosa NXHG29 towards R. sola-
nacearum was assayed in vitro by the agar diffusion method. Results
Fig. 1. Inhibitory effect of wild-type and GFP-tagged Pseudomonas aeruginosa
NXHG29 against Ralstonia solanacearum (A, B, and C) and Phytophthora nico-
tianae (D, E and F) in an in vitro assay. A and D, equivalent volume of NB
medium was used as control. B and C, antibacterial activity against R. solana-
cearum by wild-type and GFP-tagged P. aeruginosa NXHG29, respectively. E and
F, antifungal activity against P. nicotianae by wild-type and GFP-tagged P.
aeruginosa NXHG29, respectively.
indicated strain NXHG29 inhibited the growth of the pathogen with an
obvious inhibition zone as shown in Fig. 1B. Further, the strain
NXHG29 displayed a variable degree of antibacterial activity with the
inhibition zone diameters ranging from 17.31 to 20.46 mm, depending
on the concentration (107 to 109 CFU mL−1) of strain NXHG29
(Table 1). The strain NXHG29 at the concentration of 109 CFU mL−1
showed the highest activity with zones of inhibition larger than 20 mm,
which was statistically (P < .05) significantly higher than the con-
centration of 107 CFU mL−1 (17.31 mm). Meanwhile, the antifungal
activity of P. aeruginosa NXHG29 against P. nicotianae was tested with
the Oxford cup assay system. It is remarkable that strain NXHG29 was
able to inhibit the growth of pathogenic fungi tested in vitro as evi-
denced by the presence of inhibition zones (Fig. 1E). As shown in
Table 1, the NXHG29 strain has variable levels of antifungal activity
against tested fungi with inhibition rates ranging from 65.18% to
71.21%, depending upon the different concentrations of the treatment
(107 to 109 CFU mL−1). The highest inhibition rate observed (71.21%)
was recorded at a concentration of 109 CFU mL−1 of NXHG29 followed
by 70.45% at 108 CFU mL−1. Interestingly, an application of NXHG29
at the concentrations of 108 and 109 CFU mL−1 caused significant
(P < .05) differences in antimicrobial activity when compared to the
107 CFU mL−1 concentration tested, but, no significant difference in
antimicrobial activity was observed between the concentrations of 108

139
L. Ma et al. Applied Soil Ecology 129 (2018) 136–144

Table 1
In vitro antagonistic activity of wild-type and GFP-tagged P. aeruginosa NXHG29 against R. solanacearum and P. nicotianae.
P. aeruginosa NXHG29 Diameter of inhibition zone (mm) Inhibition rate (%)

−1* −1* −1*

7
10 CFUmL 8
10 CFUmL 9
10 CFUmL 107 CFUmL−1* 108 CFUmL−1* 109 CFUmL−1*

Wild strain 17.31 ± 1.21 a A 20.35 ± 2.37 b B 20.46 ± 1.18 b B 65.18 ± 1.24 c C 70.45 ± 2.51 d D 71.21 ± 2.05 d D
GFP-tagged strain 17.28 ± 1.43 a A 20.44 ± 1.42 b B 20.62 ± 2.15 b B 64.04 ± 1.14 c C 70.57 ± 2.37 d D 70.34 ± 1.94 d D

Note: * represents the cell concentration of P. aeruginosa NXHG29 inoculation. Values are the means ± standard deviations based on triplicate measurements. The
different capital letters indicate the significant differences between the wild type and GFP-tagged NXHG29 strain in the same concentration treatment according to
Duncan’s test (P < .05). The different lowercase letters indicate the significant differences between different concentrations treatments in the wild type or GFP-
tagged NXHG29 strain according to Duncan’s test (P < .05).

Fig. 3. Effect of different treatments on the disease incidence of tobacco black

Fig. 2. Effect of different treatments on the disease incidence of tobacco bac-
terial wilt. Notes: CK1 (healthy control), soil without inoculation with pa-
thogen; CK2 (diseased control), soil inoculated with pathogen; OF1-3, pa-
thogen-inoculated soil was mixed with cattle manure compost (1:1, w/w) and
0.1%, 0.5%, and 1.0% (w/w) organic fertilizer (a mixture of amino acid ferti-
lizer), respectively; ANT1 and ANT2, pathogen-inoculated soil was mixed with
the suspension of P. aeruginosa NXHG29 to obtain a final concentration of 108
and 109 CFU g−1 soil, respectively; BOF1-3, pathogen-inoculated soil was
mixed with 0.1%, 0.5%, and 1.0% (w/w) BOF made with P. aeruginosa NXHG29
at the concentration of 108 CFU g−1 soil, respectively; BOF4-6, pathogen-in-
oculated soil was mixed with 0.1%, 0.5%, and 1.0% (w/w) BOF made with P.
aeruginosa NXHG29 at the concentration of 109 CFU g−1 soil, respectively. All
values are the means of three blocks. Each block contained ten pots and one
tobacco seedling was transplanted into each pot. Bars with different letters
indicated statistical differences among treatments according to Duncan’s test
(P < .05);
and 109 CFU mL−1 (Table 1).
3.2. Disease incidence of tobacco bacterial wilt and tobacco black shank
The data showed that the application of ANTs (ANT1 and ANT2)
and BOFs (BOF1, BOF2, BOF3, BOF4, BOF5, BOF6) all significantly
(P < .05) reduced disease incidence of TBW and TBS on tobacco in the
pot experiments (Exp. 1 and 2) when compared to CK2 and OF treat-
ments (OF1, 2, 3, Figs. 2 and 3). Application of OF was also shown to
decrease the disease incidence, but there were no significant differences
between the CK2 and OF treatments in the pot experiment Exp. 1 and 2,
respectively. The disease incidence was 46.62% and 43.05% as well as
45.14% and 40.64% for the ANT (ANT1 and ANT2) treatments in pot
experiments Exp. 1 and 2, respectively. While the disease incidence in
tobacco plants treated with BOFs (BOF1-6) ranged from 16.68% to
34.37% in the Exp. 1 and from 14.74% to 31.45% in the Exp. 2, sig-
nificantly lower than the disease incidence of the ANT treatment in
both experiments Exp. 1 and 2. These data showed that the application
of the organic fertilizer combined with the antagonist had a better
disease suppression effect than the application of the antagonist alone.
shank. Notes: CK1 (healthy control), soil without inoculation with pathogen;
CK2 (diseased control), soil inoculated with pathogen; OF1-3, pathogen-in-
oculated soil was mixed with cattle manure compost (1:1, w/w) and 0.1%,
0.5%, and 1.0% (w/w) organic fertilizer (a mixture of amino acid fertilizer),
respectively; ANT1 and ANT2, pathogen-inoculated soil was mixed with the
suspension of P. aeruginosa NXHG29 to obtain a final concentration of 108 and
109 CFU g−1 soil, respectively; BOF1-3, pathogen-inoculated soil was mixed
with 0.1%, 0.5%, and 1.0% (w/w) BOF made with P. aeruginosa NXHG29 at the
concentration of 108 CFU g−1 soil, respectively; BOF4-6, pathogen-inoculated
soil was mixed with 0.1%, 0.5%, and 1.0% (w/w) BOF made with P. aeruginosa
NXHG29 at the concentration of 109 CFU g−1 soil, respectively. All values are
the means of three blocks. Each block contained ten pots and one tobacco
seedling was transplanted into each pot. Bars with different letters indicated
statistical differences among treatments according to Duncan’s test (P < .05).
Even better suppression of the disease was achieved with the applica-
tion of more highly concentrated BOF (0.5% and 1%, w/w), with the
disease incidence being 20.29% and 17.28% in the Exp. 1 as well as
18.76% and 15.58% in the Exp. 2, compared to lower concentrations of
BOFs (0.1%, w/w). However, there was no significant difference in
disease incidence between the concentrations of 108 and 109 CFU mL−1
of strain NXHG29 in BOF treatments (BOF1-6, Figs. 2 and 3).
3.3. GFP-tagging of P. aeruginosa NXHG29 and plasmid stability
The GFP-tagged strain of P. aeruginosa NXHG29 was constructed by
transferring pSMC2, a plasmid containing the gfp gene, into this bac-
terium. GFP-tagged cells were readily visualized by their GFP fluores-
cence under fluorescence microscopy (Fig. 4A), and fluorescence re-
mained stable for several days. The plasmid pSMC2 was stably
maintained in NXHG29 for approximately 6 weeks by plating aliquots
of the bacterial culture on NA without selective antibiotics. The mor-
phology and growth pattern of the GFP-tagged P. aeruginosa NXHG29
strain on NB medium were similar to those of the wild type (data not
shown). Furthermore, the GFP-tagged P. aeruginosa NXHG29 strain
showed antagonistic activity (Fig. 1C and F; Table 1) similar to that of
the wild-type strain against TBW and TBS, indicating that the presence

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L. Ma et al. Applied Soil Ecology 129 (2018) 136–144

Fig. 4. CLSM images of GFP-tagged P. aeruginosa NXHG29 colonizing tobacco roots in soil system. GFP-tagged P. aeruginosa NXHG29 cells visualized by fluorescence
microscopy (A). The control tobacco root sections (B, D, F, L, Q, and S). The bacterial cells colonized the differentiation zone and root tip of the primary roots 1 day
after inoculation (C), and the zones of elongation (E) and maturation (G) of primary root as well as proliferated on the differentiation zone (H and I) by day 3 after
inoculation. The bacterial cells congregated in the elongation zone and progressed towards the junctions of primary roots (J and K) and lateral roots (M) as well as
aggregated on the tissues of the lateral roots (N, O and P) by days 6 and 9 after inoculation. The bacteria were found at the junctions between the roots and the stems
(R) and progressed from the roots towards the stems. The bacteria had colonized the stems (T) by day 12 after inoculation. Scale bar represents 50 µm in all panels
except in A where it represents 100 µm.

of pSMC2 did not interfere with the normal metabolism of P. aeruginosa
NXHG29.
3.4. Colonization of tobacco roots by GFP-tagged P. aeruginosa NXHG29
the root hairs. In addition, the CLSM images showed that spatial-tem-
poral pattern of colonization of GFP-tagged P. aeruginosa NXHG29 on
tobacco roots grown in the quartz sand system was consistent with the
results in the soil system.

The colonization pattern of this bacterium on tobacco roots was
visualized by observing the fluorescent cells of the GFP-tagged strain
under CLSM at different times after inoculation with GFP-tagged P.
aeruginosa NXHG29 in the natural soil system. The images revealed that
bacteria were predominantly located on the tobacco root differentiation
zone and root cap of the primary roots one day after inoculation
(Fig. 4C). Subsequently, the bacteria progressed towards the zones of
elongation and maturation (Fig. 4E and G), and proliferation of bac-
terial cells in the differentiation zone was obvious (Fig. 4H and I),
whereas only a few cells had accumulated in the zones of elongation
and maturation by the third day. During days 6 and 9 after inoculation,
the bacteria were congregated in the elongation zone (Fig. 4 J and K)
and was observed at the junctions of the primary and lateral roots
(Fig. 4M) as well as in cell aggregates in the tissues of the lateral roots
(Fig. 4N, O, and P). Eventually, by day 12, the bacteria were found to
have formed dispersed microcolonies at the junctions between the roots
and the stems (Fig. 4R) and along the surface of the stems (Fig. 4T),
indicating that the bacteria had finished migrating from the roots to the
stems within 12 days. Notably, the bacteria were not found to colonize
3.5. Population dynamics of GFP-tagged P. aeruginosa NXHG29 on tobacco
roots and in the rhizosphere soil
The population dynamics of GFP-tagged P. aeruginosa NXHG29 on
tobacco roots and in the rhizosphere soil were monitored at 0, 3, 6, 9,
12, and 20 days after inoculation (DAI) and the data were listed in
Table 2. Immediately after inoculation by soaking, the bacterial popu-
lation was 2.05 × 108 CFU g−1 of fresh root on the roots. At 3 DAI, the
GFP-tagged NXHG29 population decreased to 7.7 × 106 CFU g−1 of
fresh root, and the rhizosphere soil contained 5.25 × 105 CFU g−1 of
soil. The bacterial populations recorded at 6 DAI showed an increase
from 7.7 × 106 to 5.37 × 107 CFU g−1 of fresh root, meanwhile, the
cell counts in the rhizosphere soil achieved a peak of
4.38 × 107 CFU g−1 of soil. Subsequently, the abundance began to
decrease on the roots and in the rhizosphere soil on 9 DAI. After that,
between 12 and 20 DAI, the bacterial population of GFP-tagged
NXHG29 ultimately stabilized at 6.2 × 105 −4.5 × 105 CFU g−1 of
fresh root on the roots, and 3.42 × 106 − 1.18 × 106 CFU g−1 of soil in
the rhizosphere soil.

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L. Ma et al.
Table 2
The population of GFP-tagged P. aeruginosa NXHG29 on tobacco root surface and in the rhizosphere soil.
Treatments Tissue observed Population of GFP-tagged P. aeruginosa NXHG29 (log10 CFU g−1)
0 day 3 days
Un-inoculated Root surface 0 0
Rhizosphere soil 0 0
GFP-tagged NXHG29 inoculated Root surface 8.31 ± 0.78 6.89 ± 0.85
Rhizosphere soil 0 5.40 ± 1.13
Notes: Each treatment had five replicates of one plant. Values in the column were expressed as the mean ± standard error.
4. Discussion
The constantly increasing threats caused by tobacco bacterial wilt
(R. solanacearum), and tobacco black shank (P. nicotianae) have become
a matter of great concern in tobacco production areas throughout the
world. The two diseases usually co-occur on the same plant host and
result in a disease complex causing synergistic yield losses (Liu et al.,
2007; Wang et al., 2010). Traditionally, a single biocontrol agent is
used for biocontrol of plant disease against a single pathogen (Wilson
and Backman, 1999). However, controlling just one of the pathogens
might not fully solve the problem caused by a disease complex of two
different co-occurring diseases on the same plant. Dually antagonistic
bacteria (DAB) might provide new options for control strategies for soil-
borne diseases, especially with respect to co-occurring disease com-
plexes (Adam et al., 2014; Huang et al., 2015). Therefore, based upon
this strategy, we focused our attention on DAB strains for their potential
as BCAs against soil-borne disease complexes in tobacco, and dis-
covered that a strain, P. aeruginosa NXHG29, displayed dually antag-
onistic activities towards TBW and TBS in in vitro assays (Fig. 1B and E;
Table 1). In additionally, the P. aeruginosa NXHG29 strain has also
shown antifungal activity against wilt disease caused by Fusarium
oxysporum f.sp. cubense on banana (Li et al., 2012), indicating that P.
aeruginosa NXHG29 strain has a broad spectrum in the control of plant
soil-borne diseases. Therefore, P. aeruginosa NXHG29 strain is selected
as the promising candidate as a BCA against soil-borne diseases. P.
aeruginosa has been considered as a promising biocontrol agent to a
range of soil-borne pathogens (Babu and Paramageetham, 2013;
Illakkiam et al., 2013; Singh et al., 2010), but, to our knowledge, this is
the first report on P. aeruginosa screened as a DAB strain against TBW
and TBS. Here we have extended the antagonistic activity of P. aeru-
ginosa toward two new pathosystems on tobacco: P. nicotianae and R.
solanacearum.
In the present study, we tested the effect of novel bio-organic fer-
tilizer (BOF), which combined an amino acid fertilizer and mature
cattle manure compost with the DAB strain P. aeruginosa NXHG29, on
the suppression of TBS and TBW of tobacco in pot experiments. The
incidence of TBS and TBW diseases was significantly suppressed by the
application of BOF and the antagonist alone, compared to the control
(Figs. 2 and 3). The more effective control of TBS and TBW was
achieved with BOF application compared with the application of the
antagonist NXHG29 alone, which was consistent with previous studies
which reported that the combination of antagonist with organic ferti-
lizer (OF) better suppressed the pathogens than the antagonist appli-
cation alone (Huang et al., 2011; Liu et al., 2013). These findings
suggest that combination of a DAB antagonist with a suitable organic
fertilizer (OF) is a good alternative for controlling TBS and TBW in
tobacco. As far as we know, this is the first report on the biocontrol
effects of TBS and TBW by a novel BOF, which combined the DAB strain
P. aeruginosa NXHG29 with an amino acid fertilizer as well as mature
cattle manure compost. The combination of an antagonist with a sui-
table OF may provide a unique advantage due to the fact that the OF
can provide sufficient nutrients for the antagonist to ensure high levels
of inoculum in the treated soil and on the plant root surfaces, promoting
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Applied Soil Ecology 129 (2018) 136–144
6 days 9 days 12 days 20 days
0 0 0 0
0 0 0 0
7.73 ± 0.94 6.74 ± 1.04 5.79 ± 0.88 5.65 ± 1.31
7.64 ± 0.67 7.25 ± 0.71 6.53 ± 1.24 6.07 ± 0.92
the root colonization ability of the antagonist, and thus preventing the
invasion of the pathogens (Huang et al., 2011; Lang et al., 2012; Wei
et al., 2011; Wu et al., 2015). Thus, the development of the BOF using
DAB might provide new options for control strategies, especially with
respect to managing both diseases at the same time in the host plant,
which should be more effective in the long term. Additionally, different
amounts of BOF application led to different suppressive effects on TBS
and TBW in tobacco. Higher amounts of BOF application resulted in
more suppressive effects on tested pathogens (Figs. 2 and 3). Therefore
in fields with a history of tobacco crops heavily diseased with TBS and
TBW, the higher rate of BOF application (1% BOF amendment) is re-
commended, whereas the 0.1% BOF amendment is recommended for
fields with a history of slightly diseased plants, with the view of de-
creasing the cost of BOF field applications.
Root colonization is considered a prerequisite for successful biolo-
gical control and plays an important role in the suppression of soil-
borne pathogens (Bloemberg et al., 2000; Compant et al., 2005; Maurer
et al., 2013). To further study the biocontrol mechanism of TBS and
TBW of tobacco plants by DAB strain P. aeruginosa NXHG29, we tagged
the strain with a GFP-marked plasmid pSMC2 and investigated its co-
lonization pattern on the tobacco roots. We generated GFP-tagged P.
aeruginosa NXHG29 and confirmed that the GFP was stable and con-
stitutively expressed with a plasmid stability assay. Our GFP-tagged P.
aeruginosa strain NXHG29 showed antagonistic activity against the
pathogens tested similar to that of the wild type strain as shown by the
inhibition zone in the plate assay (Fig. 1C and F; Table 1). These results
suggested that GFP-tagged P. aeruginosa strain NXHG29 was suitable for
use in the observation of its colonization on tobacco roots. Here we
showed the patterns of root colonization by GFP-tagged P. aeruginosa
strain NXHG29. It could successfully colonize the surfaces of tobacco
roots. The colonization occurred first in defined regions of the differ-
entiation zones and root tips of the primary roots, then progressed to-
wards the elongation and maturation zones as well as around the
junctions of primary and lateral roots, and finally occurred along the
stems by 20 days after inoculation (Fig. 4). The colonization pattern
was similar to that observed for other P. aeruginosa strain (Sathyapriya
et al., 2012). To our knowledge, this is the first detailed report on the
colonization pattern on tobacco roots by P. aeruginosa. Previous studies
have shown that different BCAs differ in the way they colonize plant
surfaces (Buddrus-Schiemann et al., 2010; Neveu et al., 2007). The
importance of root hairs in the colonization of olive roots by beneficial
Pseudomonas spp. has been clearly demonstrated (Prieto et al., 2011).
However, root hair colonization was not observed by GFP-tagged P.
aeruginosa NXHG29 strain in this study. Root colonization is a com-
plex phenomenon under the influence of many parameters, of which
root exudates is the most important factor (Badri and Vivanco, 2009,
Compant et al., 2010). The different colonization patterns may be the
consequence of the different chemotaxis of different bacterial strains
toward different root exudates (Bais et al., 2006; Zhang et al., 2014).
Higher populations of the BCAs are usually required for biological
control so as to have a better competition for nutrients and niches. To
investigate further the distribution of GFP-tagged P. aeruginosa
NXHG29 in the soil, the bacterial populations on the root surfaces and
L. Ma et al.
in the rhizosphere were monitored. The density of GFP-tagged P. aer-
uginosa NXHG29 ranged from 105 to 108 CFU g−1 of fresh root and 105
to 107 CFU g−1 of soil during the whole sampling period (Table 2).
These values were consistent with and reached the previously described
threshold required for antagonism to take place (Timmusk et al., 2005;
Zhang et al., 2011). Additionally, the population dynamic of coloniza-
tion of GFP-tagged P. aeruginosa NXHG29 on tobacco roots and in the
rhizosphere soils (Table 2) was also similar to that previously described
for other plant root systems with Pseudomonas spp strains (Pliego et al.,
2008; Prieto et al., 2011; Zhang et al., 2011).
To summarize, this study was the first report on using the DAB
strain P. aeruginosa NXHG29 as a potential BCA to control TBW and
TBS, and also provided the first detailed description of the spatial-
temporal pattern of colonization of GFP-tagged P. aeruginosa on tobacco
roots, and demonstrated that GFP-tagged P. aeruginosa NXHG29 can
effectively colonize and survive on tobacco roots and in the rhizosphere
soil under a natural soil system. Our results demonstrated that the BOF,
combination of organic fertilizer with the DAB strain P. aeruginosa
NXHG29, effectively suppressed TBW and TBS. Further field experi-
ments are needed, and more research is needed to better understand the
underlying mechanism.
Conflict of interest
No conflict of interest declared.
Acknowledgements
This research was financially supported by the NSFC (31200086,
31660023, 31460028, 31570108), Department of Science and
Technology of Yunnan Province – China (2017FB020, 2017FA016) and
Yunnan of CNTC-YN2018. We are very grateful to Dr. Jianping Xu for
his editorial assistance and advice.
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