Accepted Manuscript

Monitoring the efficiency of Streptomyces galilaeus strain KPS-C004 against

root knot disease and the promotion of plant growth in the plant-parasitic nem-
atode infested soils

Pongrawee Nimnoi, Neelawan Pongsilp, Pornthip Ruanpanun

PII: S1049-9644(17)30183-4
DOI: http://dx.doi.org/10.1016/j.biocontrol.2017.08.016
Reference: YBCON 3640

To appear in: Biological Control

Received Date: 24 May 2017

Revised Date: 18 July 2017
Accepted Date: 27 August 2017

Please cite this article as: Nimnoi, P., Pongsilp, N., Ruanpanun, P., Monitoring the efficiency of Streptomyces
galilaeus strain KPS-C004 against root knot disease and the promotion of plant growth in the plant-parasitic
nematode infested soils, Biological Control (2017), doi: http://dx.doi.org/10.1016/j.biocontrol.2017.08.016

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 Monitoring the efficiency of Streptomyces galilaeus strain KPS-C004

against root knot disease and the promotion of plant growth in the plant-

parasitic nematode infested soils

Pongrawee Nimnoi1, Neelawan Pongsilp2 and Pornthip Ruanpanun3*

1
Department of Microbiology, Faculty of Liberal Arts and Sciences, Kasetsart University,

Kamphaeng Saen Campus, Nakhon Pathom 73140, THAILAND

2
Department of Microbiology, Faculty of Science, Silpakorn University-Sanam Chandra

Palace Campus, Nakhon Pathom 73000, THAILAND

3
Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart

University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, THAILAND

1
and 3contributed equally to this work

E-mail: umco_perra@hotmail.com1, pongsilp_n@su.ac.th2, fagrptr@ku.ac.th3

*Corresponding author: Pornthip Ruanpanun

Tel: +66 34 351890; Fax: +66 34 351890

1
Abstract
Streptomyces galilaeus strain KPS-C004 obtained from the plant-parasitic nematode

infested soil suppressed up to 58% of root knot disease of chili caused by Meloidogyne

incognita in a greenhouse experiment. The strain promoted plant growth by increasing

biomass, shoot and root length by 81%, 46% and 100%, respectively. The same trend was

observed with plant nutrients, increases of nitrogen, phosphorus, potassium, calcium,

magnesium and iron in chili in the treatments inoculated with spores of the strain KPS-C004

were up to 27%, 27%, 40%, 18%, 37% and 137%, respectively. The highest control

efficiency of the strain was recorded when inoculated its spores in the vicinity of chili roots

before nematode invasion. In addition, S. galilaeus strain KPS-C004 was capable of

surviving and proliferating in the nematode infested soils throughout the entire 45 days of a

cultivation period. However, reinoculation at every 15 days is recommended in order to

achieve better control potential of the strain. Besides its biological control potential and plant

growth-promoting attributes, the strain did not affect soil bacterial community. Altogether, its

beneficial characteristics suggested that S. galilaeus strain KPS-C004 could be a potential

biocontrol agent for being integrated in the root knot disease management program.

Keywords:
Streptomyces galilaeus, Meloidogyne incognita, biocontrol, plant growth promoter, chili

1. Introduction
Root-knot nematodes (RKNs, Meloidogyne spp.) are sedentary endoparasitic

nematodes with a broad host range across a number of important tropical and temperate crop

species. In general, yield losses of crops caused by RKNs had been estimated to be about

11%, and the losses in economically important crops (vegetables, fruits and non-edible field

crops) were about 14%, for a total of over 80-157 billion US dollars worldwide annually (Sun
2
et al., 2006; Caillaud et al., 2008; Singh et al., 2015). Among RKN species, Meloidogyne

incognita is found mostly and has spread throughout agricultural areas in north, northeastern,

central and south regions of Thailand (Cliff and Hirschmann, 1984; Handooet al., 2005;

Ruanpanun et al., 2010; Ruanpanun and Khun-In, 2015).

In the course of our searching program for nematicidal microorganisms, we isolated

an actinomycete isolate KPS-C004, later identified as Streptomyces galilaeus, which showed

high ability to decrease egg hatch rate by 80% and increase juvenile mortality rate of M.

incognita by 79% (P. Ruanpanun, unpublished data). Of additional interest to its

antinematode, this strain had ability to promote plant growth by producing indole-3-acetic

acid (IAA) and ammonium at concentrations of 20.46 µg/ml and 50.83 µg/ml, respectively

(P. Nimnoi, unpublished data).

S. galilaeus had been previously reported as a favorable host for the development of

hybrid anthracyclines that used to treat acute leukemias and non-Hodgkin’s lymphomas in

Japan and France (Kunnari et al., 1997; Rätyet al., 2002 ). Castillo et al. (2016) presented that

S. galilaeus, a tropical strain CFFSUR-B12, was active against an ascomycete,

Mycosphaerella fijiensis Morelet, which is a causal agent of black sigatoka disease in banana

by producing antifungal extracellular chitinases. However, there appears to be no record in

the literature about antinematode and plant growth-promoting ability of S. galilaeus. In this

study we described the potential of S. galilaeus, a tropical strain KPS-C004, for controlling

root knot disease of chili caused by M. incognita and promoting plant growth in the RKN

infested soils. It is known that the microbial community is also critical for the health of land

plants and the processing of soil organic matter (Breidenbach et al., 2016) and therefore the

ultimate benefit of the use of biocontrol agents is not only based on their biocontrol ability

and plant growth-promoting attributes, but also their microbial community friendliness. Thus,

we determined the effects of this strain on soil microbial community as well as monitored its
3
activity and survival in the RKN infested soils. The gained information is important for the

consideration for the success of any inoculation procedure.

2. Materials and methods

2.1 Preparation of inoculum of S.galilaeus strain KPS-C004

S. galilaeus strain KPS-C004 (GenBank accession No. LC202902) was provided from

Plant Nematology Laboratory, Department of Plant Pathology, Faculty of Agriculture at

Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom,

Thailand. The strain was isolated from the chili field infested with a root-knot nematode in

Muang District, Nakhon Pathom Province, Thailand, using the method described by

Ruanpanun et al. (2010). Stock cultures of the strain were maintained on Hickey-Tresner

(HT) slant agar and kept in 20% (v/v) glycerol suspensions at -20°C.

Spores of S. galilaeus strain KPS-C004 were propagated on rice seeds that had been

cooked in an electric rice cooker (2:1 rice seeds to water) following the method described by

Ruanpanun and Chamswarng (2016). The 10 5 spores of the strain suspended in 5 ml of sterile

water were inoculated into a sterile plastic bag (size 15 × 23 cm2) containing 200g of cooked

rice. Each bag was closed with a rubber band, then punctured 30 air flow holes all around the

bag. The bags were incubated at room temperature (25 ± 3⁰C) for 7–14 days. After that, the

spores were removed from rice seeds by washing with distilled water and filtering through a

sieve. The spore suspension was used as inoculum in a greenhouse experiment.

2.2 Propagation of M. incognita (RKN)

Hatching second-stage juveniles (J2) of RKN, which was identified as M. incognita,

were propagated by inoculation in the vicinity of roots of chili that had been planted in a

greenhouse for 40-45 days (Hunt and Handoo, 2009). The egg masses (contained 300-500

eggs) on root surfaces were detached and surface-disinfected with 1% (v/v) NaOCl, followed
4
by washing three times with sterile distilled water to remove residual NaOCl. The egg masses

were put in water for 48 h or more until the eggs were hatched. Hatching M. incognita

juveniles were carefully removed and used for a greenhouse experiment.

2.3 Planting of chili in the RKN infested soils under a greenhouse experiment

The antinematodal potential of S. galilaeus strain KPS-C004 on chili infected with

RKN was evaluated twice in March (dry season) and July (wet season) 2016 in a greenhouse.

Four-week-old chili seedlings were planted in pots (15 cm wide, 10 cm long and 15 cm high)

containing the RKN infested soil. The RKN infested soil was collected from the chili field in

Muang District, Nakhon Pathom Province, Thailand (lat. 15°51’N and long. 99°51’E) and

used for a greenhouse experiment at Kasetsart University, Kamphaeng Saen Campus,

Nakhon Pathom, Thailand within 2 days after collection. Soil physical and chemical

characteristics were studied initially according to the methods described below in the section

2.5.2. The experiment was performed under a greenhouse condition similar to that of the field

environment. Watering was performed around 1.5 mm/day and temperature range was around

27.1-38°C.

Fifteen repetitions of each treatment (75 pots) were undertaken. The treatments were

as follows. (1) A1: 106 spores of S. galilaeus strain KPS-C004 were inoculated in close

vicinity to chili roots 48 h before inoculation with 1,000 J2 of RKN. (2) A2: 1,000 J2 were

inoculated in close vicinity to chili roots 48 h before strain inoculation. (3) A3: 106 spores of

the strain and 1,000 J2 were simultaneously inoculated in close vicinity to chili roots. (4) A4:

chili seedlings were cultivated in the RKN infested soil containing 1,000 J2 only (without

strain inoculation). (5) A5: chili seedlings were cultivated in the RKN infested soil (without

both strain and RKN inoculations). A total of 75 pots were arranged in a randomized block

design, and plants were grown in a greenhouse for 45 days. After cultivation, plants of 15

5
repetitions of each treatment were detached carefully from the soils and their roots were

washed under running tap water.

2.4 Evaluation of control efficiency

The numbers of egg mass on chili roots and J2 present in the soils were estimated.

Percentage of root system with galls was estimated to determine a galling index as described

by Ruanpanun and Chamswang (2016). The control efficiency was analyzed following the

formula described by Barker (1985).

2.5 Plant growth-promoting activity of S. galilaeus strain KPS-C004 assay

After determination on gall formation, 45-day-old roots and shoots of chili were

washed in deionized water. Root and shoot length were measured. Plant dry weight was

determined after drying in an oven for approximately 72 h at 65⁰C until a constant weight

was obtained (Nimnoi et al., 2014).

2.5.1 Plant nutrient analysis

Dried plants were ground in a mortar. The ground plant materials were subjected to

digestion with a mixture of H2SO4, Se and Na2SO4 modified from the Bergersen (1980)

method. The Kjeldahl (wet oxidation) method was used for N analysis. Quantity of P was

determined spectrophotometrically using the vanadate-molydate-yellow procedure. Amounts

of K, Ca, Mg and Fe were measured by an atomic absorption spectrometer (AAS; Perkin-

Elmer 3100) using the AOAC method (Walinga et al., 1989; Helmer and Sparkers, 1996).

2.5.2 Soil physical and chemical property analysis

After 45 days of planting, soil samples were air-dried, sieved (2-mm/10-mesh) and

subjected to soil physical-chemical characteristic study. The samples were analyzed for

particle-size distribution following the method described by Bouyoucos (1962). Organic

matter content was analyzed as described by Walkley and Black (1934). Amounts of P and

Ca were analyzed by the Bray II method (Bray and Kurtz, 1945).Amounts of K and Mg were
6
determined by the flame photometric method (Jackson, 1958). Quantity of available N was

also estimated as described by Mctaggart and Smith (1993). pH and electrical conductivity

(ECe) values were also measured (Jackson, 1967).

2.6 Monitoring of S. galilaeus strain KPS-C004 in the RKN infested soils

2.6.1 Denaturing gradient gel electrophoresis (DGGE) and Reverse

Transcription (RT) PCR-DGGE analyses

The effects of S. galilaeus strain KPS-C004 on soil bacterial community, the

persistence and traceability as well as the activity of the strain during 45 days after being

introduced into the RKN infested soils were determined by using DGGE and RT PCR-DGGE

techniques. Soils from each treatment (3 replicates/each of 5 collection times) were collected

after being planted for 5, 10, 15, 35 and 45 days and subjected to total DNA and RNA

extractions. The total DNA and RNA were extracted by using PowerSoilTM DNA Isolation

Kit (MoBio, CA) and RNA PowerSoilTM Total RNA Isolation Kit (MoBio, CA),

respectively, according to the manufacturer’s instructions. After extraction, purity and

quantity of DNA and RNA were determined by using DS-11FX+ Spectro/Fluorometer

(DeNovix, DE) with dsDNA and RNA calculative applications, respectively. To amplify 16S

rDNA, the forward primer F341 together with GC-clamp and the reverse primer R534 were

used (Muyzer et al., 1993). PCR reactions and conditions were performed as described in

Nimnoi et al. (2011).

For complementary DNA (cDNA) synthesis, total RNA extracted from each sample

was used as a template. One-step RT-PCR was performed using the SuperScript® III On-Step

RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen, CA) together with the

forward primer F341 and the reverse primer R534. PCR reactions and conditions were

performed as described in Nimnoi et al. (2017). The PCR fragments were separated by using
7
DGGE performed with the BioRad DCodeTM Universal Mutation Detection System (Bio-

Rad, CA). For DNA PCR-DGGE, 25 µl of PCR products were applied to 8% polyacrylamide

gel with a liner gradient of 20-50% denaturant (100% denaturant corresponds to 40%

[vol/vol] of formamide plus 7 M urea). Electrophoresis was performed at 130 V for 7 h at a

constant temperature of 60 oC. For cDNA PCR-DGGE, 25 µl of PCR products were applied to

8% polyacrylamide gel with a liner gradient of 15-45% denaturant. Electrophoresis was

performed at 130 V for 5 h at a constant temperature of 60oC. Gels were then stained with

GelStar® Nucleic Acid Gel Stain (Cambrex Bio Science, ME) for 30 min and visualized.

2.6.2 Cloning and sequencing of DGGE bands

Dominant bands were excised from the gel, eluted in 30 µl of Nuclease free water

(Promega, WI) and stood overnight at 4 oC. Eluted DGGE bands were used as templates for

reamplification with primers F341 (non GC-clamp) and R543. Two µl of the products were

cloned using the pCR®8/GW/TOPO® TA Cloning kit with One Shot® Mach™1-T1R

Chemically Competent Cells (Invitrogen, CA) and positive clones were screened by colony-

PCR with vector-specific primers according to the manufacturer’s instruction. The 16S rDNA

fragments amplified from clones were loaded on the same DGGE gel in order to examine

whether the cloned 16S rDNA fragments migrated at the same positions with the bands of

interest of the corresponding community patterns. Plasmids containing the cloned fragments

which possessed the electrophoretic mobility identical to the original bands were extracted

using NucleoSpin® Plasmid QuickPure (Macherey-Nagel, Germany) and sequenced by 1 st

Base Inc., (Selangor Darul Ehsan, Malaysia). These sequences were also tested for PCR

generated chimeras using automated tests for recombination of RDP-II. The 16S rRNA gene

sequences were aligned with known nucleotide sequences in the database of the National

Center for Biotechnology Information (NCBI) using the BLAST search option.

8
2.7 Statistical analysis

The experimental data (regarding control potential, plant growth promotion and soil

elements) were subjected to an analysis of variance (ANOVA) using Tukey’s test (P<0.05)

of the program SPSS version 17.0 (SPSS Inc., Chicago, IL).The cluster analysis and the

dendrogram generation were carried out by the TL101 1D analysis Software (Totallab,

Germany). The cluster analysis was performed according to the presence and absence of

bands that occurred in DNA-based DGGE gels. The presence or absence of a nucleic acid

band with equal migration in each lane was marked with a 1 or 0, respectively. The

similarities between the DGGE patterns were analyzed by using the Pearson correlation

coefficient and displayed graphically as a dendrogram based on UPGMA algorithms.

Multiple dimensions of microbial community structure were performed by Principle

coordinate analysis (PCA).

3. Results

3.1 Potential of S. galilaeus strain KPS-C004 to control root knot disease in chili under a

greenhouse experiment

Results obtained from antinematodal microorganisms screening program indicated

that S. galilaeus strain KPS-C004 had ability to significantly decrease egg hatch and increase

juvenile mortality rate of M. incognita in vitro. Biocontrol efficiency of the strain against root

knot disease of chili was evaluated twice in March (dry season) and July (wet season) 2016 in

a greenhouse. The results from both plantings were not significantly different from each

other. The representative data from the one planted in March 2016 presented that S. galilaeus

strain KPS-C004 significantly reduced numbers of egg masses by 27.70%-54.04% when

compared to the control without strain inoculation (the treatment A4) (Table 1, Column 2).

Meanwhile, the strain decreased numbers of J2 in the RKN infested soils by 59.99%-66.05%
9
(Table 1, Column 3). Nematode control efficiency of the strain was between 41.70% and

58.33% (Table 1, Column 5). The most effective treatment in decreasing root galls of chili

was the treatment A1 (with prior inoculation of the strain) (Table 1, Column 4). Fig. 1

presents a comparative decrease of gall formation on chili roots between the treatment A1

and the control without strain inoculation (the treatment A4).

3.2 Effects of S. galilaeus strain KPS-C004 on plant growth

To determine effects of S. galilaeus strain KPS-C004 on the growth of chili, the data

were recorded in 3 categories including growth yield, soil physical and chemical properties

and quantities of elements in plants. The results showed that the strain KPS-C004 promoted

plant growth by increasing dry weight, shoot and root length of chili when applied to chili

seedlings 48 h before RKN inoculation (the treatment A1). In contrast, the strain had no

stimulatory effect on the growth of chili when applied to chili seedlings, either

simultaneously or sequentially, with RKN inoculation (Table 1, Column 6-8). As interpreted

from the results in Table 2, the significant increases of all elements, except total Mg, were

observed in the soils from the treatment A1 (with prior inoculation of the strain) and the

treatment A3 (with simultaneous inoculations of the strain and RKN), as compared to the

control without strain inoculation (the treatment A4). Organic matter content and quantities

of all elements, except total P, were significantly increased in soil from the treatment A2

(with prior inoculation of RKN). Overall, the treatments with strain inoculation (the

treatments A1-A3) conferred the increases of organic matter and all elements in the RKN

infested soils. The highest percentages of the increases in organic matter, total N, P, K, Ca

and Mg were 31.79%, 202.56%, 41.94%, 57.46%, 20.70% and 12.28%, respectively, as

compared to the control without strain inoculation (the treatment A4). In addition, soil

physical property in terms of percentages of sand, slit and clay were similar to those of the

initial soil. Some changes were observed with electrical conductivity value that remarkably
10
decreased in all treatments and pH value that slightly increased in all treatments, regardless of

both strain and RKN inoculations. The results from analysis of quantities of elements in chili

are presented in Table 3. Total N was the only one element that increased significantly in all

treatments with strain inoculation, regardless of the inoculation order, as compared to both

controls (the treatments A4 and A5). The treatment A1 (with prior inoculation of the strain)

was the only one treatment that conferred the significant increases of all elements including

total N, P, K, Ca, Mg and Fe. The highest percentages of the increases in total N, P, K, Ca,

Mg and Fe were 27.48%, 27.39%, 40.25%, 18.70%, 37.20% and 137.03%, respectively, as

compared to the control without strain inoculation (the treatment A4).

3.3 Establishment of S. galilaeus strain KPS-C004 after being introduced into the RKN

infested soils

Total soil DNA and RNA of each treatment mentioned in materials and methods were

extracted. The establishment of S. galilaeus strain KPS-C004 after being introduced into the

RKN infested soils for 5, 10, 15, 35 and 45 days was analyzed by using DGGE technique. In

this study, we first compared the DGGE patterns among 3 replicates of each treatment. As the

DGGE patterns of replicates were identical, only one DGGE pattern for each treatment was

represented without replication. The ideal of DGGE is employed for the separation of the

fragments that are identical in length, but different in sequence. Fragments that are identical

in both length and sequence migrate the same distance on DGGE gel. As shown in Fig.2, we

used 16S rDNA of pure culture of S. galilaeus strain KPS-C004as the reference strain for

verification of the species-specific migration position on DGGE gel. DGGE profiles of each

treatment were first identified by comparing their relative migration positions on the gel with

the reference strain. PCR-DGGE analysis of all treatments during 45 days of cultivation

showed the presence of 16S rDNA bands that migrated at the same position of the reference

band. These results indicated that S. galilaeus strain KPS-C004 inhabited the RKN infested
11
soils throughout a cultivation period. To confirm this finding, we excised the 16S rDNA

bands (B5, B12, B15, B17, B21, B24, B27, B31 and B33) which had the same motility on

DGGE gel as the reference band and submitted to cloning and sequencing. The sequencing

results showed that all presumptive bands represented S. galilaeus strain KPS-C004 (Table

4). The results also confirmed that, after being introduced into the RKN infested soils, S.

galilaeus strain KPS-C004 was capable of surviving and proliferating in the soils throughout

the entire 45 days of a cultivation period.

Moreover, the activity of S. galilaeus strain KPS-C004 during 45 days after

inoculation was examined by using RT PCR-DGGE of 16S rRNA gene. RNA-based DGGE

profiles (Fig. 3) revealed the difference in metabolically active bacteria in the RKN infested

soils. DGGE fingerprints of RNA-based analyses showed a large number of fainter bands

representing less metabolic activity. However, RNA-based DGGE profiles of each treatment

showed that 16S cDNA bands migrated at the same position with the reference band. These

results indicated that S. galilaeus strain KPS-C004 was metabolically active in the RKN

infested soils during 45 days of cultivation. To confirm this finding, 16S cDNA bands (C3,

C7, C9, C12, C14, C15, C17, C19, C22 and C23) that migrated the same distance on DGGE

gel as the reference band were excised from gel and submitted to cloning and sequencing.

Analysis of 16S cDNA sequences showed that all presumptive bands represented S. galilaeus

strain KPS-C004 (Table 4). These results also indicated that S. galilaeus strain KPS-C004

was able to inhabit and remain metabolically active during 45 days of a cultivation period.

However, at days 35 (lanes 17, 18 and 19) and 45 (lanes 22, 23 and 24) of a cultivation period

(Fig. 2), the intensities of 16S cDNA bands of the strain was decreased, implying that its

activity was reduced after 15 days of inoculation. This finding recommends that the

application of S. galilaeus strain KPS-C004 in crop fields requires re-inoculation after 15

days of the first inoculation.

12
3.4 Effects of S. galilaeus strain KPS-C004 on soil bacterial community

The effects of S. galilaeus strain KPS-C004 on soil bacterial community and diversity

were evaluated using PCR-DGGE and cluster analysis approach. For cluster analysis, a

dendrogram was constructed to determine the different levels of similarity shared among

samples. The UPGMA dendrogram of DNA-based analysis is shown in Fig. 4. The generated

dendrogram exhibited 2 main clusters. The first cluster consisted of only DGGE profiles of

all treatments collected at day 45. The second cluster contained DGGE profiles of all

treatments collected at days 5, 10, 15 and 35, in which those of all treatments collected at day

35 were slightly different from the others. Subclusters of all treatments collected at day 35

linked together with the other clusters at a similarity level of 0.45. Moreover, the DGGE

fingerprints of all treatments collected at days 35 and 45 consisted of a large number of

fainter bands representing less dominant ribotypes and the amounts of bands gradually

decreased along with plant age and time after planting. These results indicated the strong shift

in bacterial diversity and community during plant growth, whereas the inoculant did not

affect bacterial community structure.

In order to determine the shift in bacterial community more clearly, PCA was used to

exhibit multidimensional relationships derived from DNA-based DGGE fingerprints (Fig. 5).

The results revealed that bacterial community structures of all treatments collected at day 45

were separated from those from the other days, whereas the inoculation of S. galilaeus strain

KPS-C004 did not affect bacterial community structure. It was clearly demonstrated that

plant age and time after planting had a great influence on the bacterial community structure.

3.5 Sequence analysis of dominant ribotypes

To obtain further information about the dominant bacterial diversity and the abundant

bacterial groups that were metabolically active during 45 days of planting. Thirty-five DNA-
13
based DGGE bands and 25 RNA-based DGGE bands were excised from gels and submitted

to cloning and sequencing. Sequence analysis of these bands and their phylogenetic

affiliations are shown in Table 4. Twenty-seven DNA-based DGGE bands could be assigned

to a total of 9 distinct phylotypes. Nine of them appeared to retain as dominant populations

during 45 days of planting. These included bands B1, B8 and B35 that belonged to the genus

Fulvivirga; bands B2, B9, B14 and B18 that corresponded to the genus Pseudomonas; as well

as bands B23 and B34 that corresponded to the genus Streptomyces. This finding indicated

genera of indigenous bacteria inhabiting these soils. The intensities of bands having

equivalent mobility including bands B4, B19 and B22 that corresponded to the genus

Ramlibacter; bands B6 and B16 that corresponded to the genus Flavobacterium; band B10

that was assigned to the genus Ochrobactrum; and band B11that was assigned to the genus

Flexibacter, were decreased after 15 days of planting. Phylogenetic affiliations obtained from

bands B26 and B28, which were not detected at days 5, 10 and 15, were assigned to the

genera Idiomarina and Caulobacter, respectively. The remaining 9 bands including B5, B12,

B15, B17, B21, B24, B27, B31 and B33, which were well defined as the reference band,

occurred with equal intensities at every examined time points throughout 45 days of a

cultivation period. However, many bands of unidentified bacteria including bands B3, B7,

B13, B20, B25 and B30 also appeared at every examined time points, indicating the existence

of these common species in the soils.

RNA-based DGGE analysis was used to determine the abundance of bacteria that

contributed to the metabolic activity during 45 days of planting. Taken together, from the

results in Table 4.and Fig. 3, RNA-based DGGE profiles showed a large number of bands

whose intensities obviously decreased after 15 days of inoculation. Unidentified bands

including C1, C4, C10, C16, C21, C24 and C25, appeared to remain metabolically dominant

during 45 days of planting. 16S cDNA bands of Pedobacter (C2 and C8) and Bacillus (C6,
14
C8 and C11) disappeared after 10 days of planting. 16S cDNA bands of S. galilaeus strain

KPS-C004 at days 5, 10 and 15 (bands C3, C7, C9, C12, C14 and C15) exhibited higher

intensities than its bands at days 35 and 45 (bands C17, C19, C22 and C23), indicating the

reduction of metabolic activity of the strain after 15 days of planting. However, 16S cDNA

bands of S. galilaeus strain KPS-C004 were still detectable throughout the entire 45 days of

an experimental period.

4. Discussion

The present study shows that the inoculation of S. galilaeus strain KPS-C004 greatly

reduced the numbers of root gall, egg mass and juveniles of RKN in the root zone soils.

Inoculation of the strain in the vicinity of chili roots before RKN inoculation conferred the

highest control efficiency, with a reduction of root galls by 10%-20% as compared to

simultaneous inoculations of the strain and RKN or prior inoculation of RKN. Establishment

of the nematicidal microorganism on root systems before nematode invasion is essential for

microbial adaptation to produce nematicidal metabolites (Crawford et al., 1993; Ruanpanun

and Chamswarng, 2016). With an additional benefit, the strain could promote plant growth by

increasing dry weight as well as shoot and root length. The stimulatory effect of Streptomyces

on root growth, which could promote the absorption of nutrients and water from the soil, was

attributable to an increase of root biomass (Ma et al., 2017). The plant growth-promoting

attributes of S. galilaeus strain KPS-C004 were confirmed by the results of analysis of

quantities of elements in plant. The significant increases of all examined elements including

total N, P, K, Ca, Mg and Fe were observed in the treatment A1 (with prior inoculation of the

strain). Such an approach might promote plant health and induce immunity against pest

infection (Dowling and O’Gara, 1994; Gupta et al., 2002; Ruanpanun et al., 2010). On the

contrary, either simultaneous inoculations of the strain and RKN or prior inoculation of RKN

had less potential to impede root invasion by RKN. Streptomyces strain might require time to
15
propagate, adapt itself to new environments or produce toxic compounds before RKN can

penetrate the plant cells (Ruanpanun and Chamswarng, 2016). Cells of plant roots might

possibly be destroyed before achieving protection by the nematicidal strain of Streptomyces,

resulting in an impaired control potential of the strain when it was inoculated after RKN

infection. Noticeably, organic matter content and amounts of all examined elements in the

RKN infested soils from at least one treatment with strain inoculation significantly increased

from those of both controls without strain inoculation. This information suggested that the

strain had a great potential in increasing quantities of all examined elements in the soils,

together with controlling root knot disease, therefore it can be used as an efficient biocontrol

agent against RKN under a suitable period of time in the field.

Up-to-date, the agricultural production is currently focused on the use of biofertilizers

in the expectation of moving towards environmentally sustainable development. The

application of biofertilizers is of considerable interest since it would substantially reduce the

use of chemical fertilizers and pesticides and an increasing number of inoculants for various

crops are currently commercially available (Trabelsi and Mhamdi, 2013). Nimnoi et al.

(2014) reported the success of using Actinomadura, Nocardia, Nonomuraea and

Streptomyces as inoculants to enhance plant growth and nodulation of soybean by

Bradyrhizobium japonicum. Inoculation of actinomycetes had been reported to enhance the

growth of many legumes (El-Tarabily et al., 2008). Actinomadura, Micromonospora,

Nocardia and Streptomyces had been used as inoculants and biocontrol agents to promote

plant growth as well as control the expansion of plant pathogens (Ruanpanun et al., 2010; de

Oliveira Mendes et al., 2014; Hata et al., 2015). Nevertheless, research on the activity and

establishment of actinomycete inocula after being introduced into the competitive soil

environment is still inadvertently neglected.

16
 The results of this study exhibited that S. galilaeus strain KPS-C004 was able to

inhabit the RKN infested soils and remained metabolically active throughout a cultivation

period. Usually, the maintenance of cell survival and activity in soil is an important

consideration for the success of any inoculation procedure. Soils are heterogeneous in

composition, causing the difficulty for the inoculated bacteria to establish niches for their

survival amongst the indigenous bacteria and predators (Sivakumar et al., 2014). The

establishment seems to vary over time, a common phenomenon starts from a short-term

increase in the density of the introduced inoculants, followed by a subsequent decline to

below detection limit after a period of time (Babalola and Glick, 2012). Our finding

demonstrates the potential of using this strain as a biocontrol agent to control plant-parasitic

nematode and an inoculant to promote plant growth in the competitive soil environment.

Moreover, microbial community is largely responsible for biogeochemical cycling

and soil fertility, which has a consequence for plant health and productivity (Cong et al.,

2015; Breidenbach et al., 2016). Therefore, usage of actinomycetes that are able to promote

plant growth and control plant pathogen sas well as friendly to microbial community, as

inoculants can be a good manner of moving towards environmentally sustainable

development. In this study, DGGE fingerprints displayed that S. galilaeus strain KPS-C004

did not affect bacterial community structure. However, after 15 days of planting, the DGGE

fingerprints displayed the shift in bacterial community, implying that plant age and time after

planting were the major factors influencing bacterial community structure. These results

agreed with the comment from Piromyou et al. (2011) who demonstrated the potential of

using bacterial inoculants to promote the growth of forage corn and determined the effects of

inoculants on microbial community structure by using DGGE. They found that the inoculants

did not affect microbial community structure, whereas stages of plant growth mainly caused

the shift in microbial community structure that occurred at week 5 of planting. Sequencing of
17
DGGE bands revealed that there were many uncultured bacteria inhabiting the soils. Besides

the uncultured bacteria, Fulvivirga, Pseudomonas and Streptomyces were identified as being

indigenous bacteria inhabiting the soils and found more often at examined time points than

other genera.

Based on strong evidence about a great potential of S. galilaeus strain KPS-C004 in

several aspects including nematicidal activity against RKN, M. incognita, plant growth-

promoting attributes, friendliness to ecosystem, capability to sustain bacterial community

structure and long-term survival in the soils, product formulation of this strain should be

developed as a new alternative biological agent for root knot disease in tropical agricultural

systems.

Acknowledgements

This work was supported by Biodiversity-Based Economy Development Office

(Public organization), Thailand [grant number 42/2558].

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Fig. 1 Galling on chili roots caused by Meloidogyne incognita: treatment A1: with prior

inoculation of Streptomyces galilaeus strain KPS-C004; A4: control with only RKN

inoculation.

24
Fig. 2 DGGE fingerprinting of DNA extracted from the RKN infested soils of 5 treatments.

Reference band, lane 1; Treatment A1, lanes 2, 7, 12, 17, 22; Treatment A2, lanes 3, 8, 13,

18, 23; Treatment A3, lanes 4, 9, 14, 19, 24; Treatment A4, lanes 5, 10, 15, 20, 25; Treatment

A5, lane 6, 11, 16, 21, 26.

Treatment A1, prior inoculation of the strain; A2, prior inoculation of RKN; A3,

simultaneous inoculations of the strain and RKN; A4, without strain inoculation; A5, without

both strain and RKN inoculations

25
Fig. 3 DGGE fingerprinting of cDNA extracted from the RKN infested soils of 5 treatments.

Reference band, lane 1; Treatment A1, lanes 2, 7, 12, 17, 22; Treatment A2, lanes 3, 8, 13,

18, 23; Treatment A3, lanes 4, 9, 14, 19, 24; Treatment A4, lanes 5, 10, 15, 20, 25;

Treatments A5, lanes 6, 11, 16, 21, 26.

Treatment A1, prior inoculation of the strain; A2, prior inoculation of RKN; A3,

simultaneous inoculations of the strain and RKN; A4, without strain inoculation; A5, without

both strain and RKN inoculations

26
Fig. 4 Dendrogram generated from DNA-based DGGE fingerprints of the RKN infested soils
of 5 treatments.
C004, S. galilaeus strain KPS-C004; /A1, soil from the treatment A1; /A2, soil from the
treatment A2; /A3, soil from the treatment A3; /A4, soil from the treatment A4; /A5, soil
from the treatment A5; -5, soil collected at day 5; -10, soil collected at day 10; -15, soil
collected at day 15; -35, soil collected at day 35; -45, soil collected at day 45.

Fig. 5 PCA analysis derived from DNA-based DGGE fingerprints of the RKN infested soils

of 5 treatments.

a-e, soils from the treatments A1-A5 collected at day 5; f-j, soils from the treatments A1-A5

collected at day 10; k-ii, soils from the treatments A1-A5 collected at day 15; p-t, soils from

the treatments A1-A5 collected at day 35; u-y, soils from the treatments A1-A5 collected at

day 45.

27
Table 1 Control potential of S. galilaeus strain KPS-C004 against root knot disease of chili and plant growth-promoting under a greenhouse
condition.

Treatmen No. of egg No. of J2/20 g Galling Control Dry weight Shoot length Root length

t mass* of soil* indexa efficiencyb (g)* (cm)* (cm)*

A1 22.67 ± 1.45c** 27.00 ± 2.52c 41.67 58.33 0.89 ± 0.12a 32.80 ± 0.85a 19.13 ± 2.07a

A2 33.67 ± 0.88b 28.67 ± 2.18c 50.00 50.00 0.53 ± 0.03ab 24.97 ± 0.94ab 15.50 ± 0.76b

A3 35.67 ± 1.20b 24.33 ± 1.73c 58.30 41.70 0.59 ± 0.04ab 26.60 ± 1.05ab 11.90 ± 0.87b

A4 49.33 ± 1.76a 71.67 ± 11.83a 100 0 0.49 ± 0.12b 22.43 ± 1.74b 9.67 ± 0.47b

A5 50.33 ± 3.18a 42.33 ± 2.72b 100 0 0.59 ± 0.06ab 27.37 ± 0.46ab 10.77 ± 0.70b

a
Roots were scored for degree of gall formation: 1 ≤25 %, 2 = 25%–50 %, 3 = 51%–75 % and 4 ≥ 75 % of total number of root systems with gall

formation. Galling index = [(number of plants in class 1 × 1) + (number of plants in class 2 ×2) + (number of plants in class 3 × 3) + (number of

plants in class 4 × 4)] ×100/(total number of plants × 4)

b
Control efficiency = (galling index of treatment) ×100/galling index of control (Barker 1985).

*Average ± SD (n=15)

** Different letters in the same column represent significant differences among treatments (Tukey’s test, P< 0.05).

Treatment A1, prior inoculation of the strain; A2, prior inoculation of RKN; A3, simultaneous inoculations of the strain and RKN; A4, without

strain inoculation; A5, without both strain and RKN inoculations

28
Table 2 Physical and chemical properties of the initial soil (Ins) and the soils after planting of chili in 5 treatments.

Treatment Ins A1 A2 A3 A4 A5

pH 7.47 8.27 8.02 8.34 8.22 8.28

Electrical conductivity (dS/m) 6.15 1.57 1.99 1.51 1.55 1.54

Organic matter (%)* 1.11 ± 0.00c** 1.57 ± 0.03b 1.99 ± 0.00a 1.51 ± 0.02b 1.51±0.06b 1.47±0.05b

Total nitrogen (mg/kg) * 21.45 ± 0.57ab 20.22 ± 0.20b 23.63 ± 1.00a 20.44 ± 0.56b 7.81 ±1.43d 10.64 ± 0.92c

Total phosphorus (mg/kg) * 376.92 ± 0.59d 680.69 ± 3.66a 476.38 ± 15.83c 512.93 ± 11.34b 479.57 ±0.39c 483.23 ± 5.55c

Total potassium (mg/kg)* 138.40 ± 3.56b 146.94 ± 3.51b 186.76 ± 7.57a 144.27 ± 5.42b 118.61 ± 4.02c 124.50 ± 0.94c

Total calcium (mg/kg)* 921.41 ± 4.15c 1569.11 ± 128.01a 1431.57 ± 75.97a 1488.89 ± 34.40a 1299.96 ± 10.89b 1231.85 ± 4.15b

Total magnesium (mg/kg) * 398.08 ± 6.02c 418.95 ± 2.32ab 443.27 ± 20.39a 398.82 ± 6.40bc 394.79 ± 4.95bc 399.26 ± 0.92bc

Sand (%) 49.96 50.12 44.92 50.07 49.19 48.61

Silt (%) 37.8 36.35 40.17 36.82 37.5 40.6

Clay (%) 12.24 13.52 14.91 13.11 13.31 10.79

Texture Loam Loam Loam Loam Loam Loam

*Average ± SD (n=15)
Different letters in the same row represent significant differences among treatments (Tukey’s test, P< 0.05)
Treatment A1, prior inoculation of the strain; A2, prior inoculation of RKN; A3, simultaneous inoculations of the strain and RKN; A4, without
strain inoculation; A5, without both strain and RKN inoculations
29
Table 3 Quantities of elements in chili obtained from 5 treatments

Treatment A1 A2 A3 A4 A5

Total nitrogen (%)* 2.83 ± 0.05a** 2.69 ± 0.13ab 2.54 ± 0.00b 2.22 ± 0.13c 2.23 ± 0.10c

Total phosphorus (mg/kg)* 4192.89 ± 19.32a 3606.79 ± 89.25b 3252.55 ± 89.24c 3291.19 ± 97.25c 3330.97 ± 56.78c

Total potassium (%)* 5.61 ± 0.05a 4.54 ± 0.12b 4.10 ± 0.08c 4.00 ± 0.12c 4.04 ± 0.18c

Total calcium (mg/kg)* 8134.23 ± 461.17a 7912.33 ± 362.30ab 8032.41 ± 616.70a 6852.81 ± 463.17b 6778.84 ± 109.22b

Total magnesium (mg/kg)* 6552.29 ± 68.90a 6118.14 ± 281.28ab 5953.27 ± 278.25ab 4775.62 ± 340.37c 5481.66 ±689.67b

Total iron (mg/kg)* 285.64 ± 18.02b 487.01 ± 30.39a 246.17 ± 27.27bc 205.46 ± 26.68c 138.83 ± 17.72d

*Average ± SD (n=15)

**Different letters in the same row represent significant differences among treatments (Tukey’s test, P< 0.05).

Treatment A1, prior inoculation of the strain; A2, prior inoculation of RKN; A3, simultaneous inoculations of the strain and RKN; A4, without

strain inoculation; A5, without both strain and RKN inoculations

30
Table 4 Closest relatives of DNA and cDNA sequences derived from DGGE bands.

DGGE band number % identity Closest relative Accession Number

B1, B8 and B35 93 Uncultured Fulvivirga LC202919
B2, B9, B14 and B18 100 Uncultured Pseudomonas LC202920
B3, B13, B25, B30 and B32 98 Uncultured bacterium LC202911
B4, B19 and B22 98 Uncultured Ramlibacter LC202921
B5, B12, B15, B17, B21, B24, B27, B31 and B33 100 S. galilaeus strain C004 LC202922
B6 and B16 99 Uncultured Flavobacterium LC202909
B7and B20 95 Uncultured bacterium LC202923
B10 100 Uncultured Ochrobactrum LC202924
B11 88 Uncultured Flexibacter LC202925
B23 and B34 100 Uncultured Streptomyces LC202917
B26 91 Uncultured Idiomarina LC202908
B28 92 Uncultured Caulobacter LC202926
B29 92 Uncultured bacterium LC202916
C1 and C10 90 Uncultured bacterium LC202931
C2 and C8 100 Uncultured Pedobacter LC202928
C3, C7, C9, C12, C14, C15, C17, C19, C22 and C23 100 S. galilaeus strain C004 LC202935
C4, C16, C21 and C24 99 Uncultured bacterium LC202936
C5 and C13 100 Uncultured bacterium LC202937
C6 andC18 100 Uncultured Bacillus LC202929
C11 100 Uncultured Bacillus LC202938
C20 100 Uncultured Alicyclobacillus LC202939
C25 98 Uncultured bacterium LC203940

31
Highlights

• Grand abilities of Streptomyces galilaeus strain KPS-C004 to suppress root knot disease and promote plant growth are proposed.
• S. galilaeus strain KPS-C004 showed its capabilities to sustain bacterial community structure and survive for long terms in the root knot
nematode infested soils.
• Monitoring of S. galilaeus strain KPS-C004 in the root-knot nematode infested soils is presented.”

32