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Microbiological Research xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Microbiological Research
journal homepage: www.elsevier.com/locate/micres

Biocontrol of Rhizoctonia solani damping-off and promotion of tomato

plant growth by endophytic actinomycetes isolated from native
plants of Algerian Sahara
Yacine Goudjal a,b , Omrane Toumatia a , Amine Yekkour a , Nasserdine Sabaou a ,
Florence Mathieu c , Abdelghani Zitouni a,∗
a
Laboratoire de Biologie des Systèmes Microbiens (LBSM), Ecole Normale Supérieure de Kouba, Alger, Algeria
b
Département d’agronomie, Faculté des Sciences, Université Amar Telidji, BP 37G, Laghouat 03000, Algeria
c
LGC UMR 5503 (CNRS/INPT/UPS), Département de Bioprocédés et Systèmes Microbiens, ENSAT-INP de Toulouse, Université de Toulouse, 1 Avenue de
l’Agrobiopôle, BP 32607, 31326 Castanet-Tolosan Cedex 1, France

a r t i c l e i n f o a b s t r a c t

Article history: Thirty-four endophytic actinomycetes were isolated from the roots of native plants of the Algerian Sahara.
Received 10 May 2013 Morphological and chemical studies showed that twenty-nine isolates belonged to the Streptomyces
Received in revised form 19 June 2013 genus and five were non-Streptomyces. All isolates were screened for their in vitro antifungal activity
Accepted 22 June 2013
against Rhizoctonia solani. The six that had the greatest pathogen inhibitory capacities were subsequently
Available online xxx
tested for their in vivo biocontrol potential on R. solani damping-off in sterilized and non-sterilized soils,
and for their plant-growth promoting activities on tomato seedlings. In both soils, coating tomato seeds
Keywords:
with antagonistic isolates significantly reduced (P < 0.05) the severity of damping-off of tomato seedlings.
Endophytic actinomycetes
Biocontrol
Among the isolates tested, the strains CA-2 and AA-2 exhibited the same disease incidence reduction as
Rhizoctonia solani thioperoxydicarbonic diamide, tetramethylthiram (TMTD) and no significant differences (P < 0.05) were
Damping-off observed. Furthermore, they resulted in a significant increase in the seedling fresh weight, the seedling
Tomato length and the root length of the seed-treated seedlings compared to the control. The taxonomic position
based on 16S rDNA sequence analysis and phylogenetic studies indicated that the strains CA-2 and AA-2
were related to Streptomyces mutabilis NBRC 12800T (100% of similarity) and Streptomyces cyaneofuscatus
JCM 4364T (100% of similarity), respectively.
© 2013 Elsevier GmbH. All rights reserved.

1. Introduction and increased contamination of the soil, but may also have adverse
high toxicity on microbial communities and a degradative effect on
Rhizoctonia solani is one of the most important soil-borne fungal the ozone layer. In addition, chemical controls are not completely
pathogens, which develops in both cultured and non-cultured soils effective, and Rhizoctonia disease remains a persistent problem
(Coa et al. 2004). It lives in the soil in the form of sclerotia and does (Gees and Coffe 1989; Huang et al. 2011).
not generate asexual spores (Huang et al. 2011). Damping-off of The success of Bacillus subtilis as a potential biocontrol agent
seedlings is the most common disease caused by R. solani (Moussa encouraged research into new microbial agents as alternatives to
2002). It has a wide host range and causes disease in a variety of chemical control compounds. In recent years, the biocontrol of
crops, such as lawn grass (Parmeter et al. 1969), tomato (Coa et al. plant diseases, particularly using antibiotic metabolites of filamen-
2004), cucumber (Coa et al. 2004) and sugar beet (Sadeghi et al. tous bacteria, has been put forward as an alternative to chemical
2006). control agents (Dhanasekaran et al. 2005; Huang et al. 2011).
Chemical fungicides are often used when losses from R. solani The role of actinomycetes in the biocontrol of soil-borne plant
are substantial. They have fungicidal activity against the pathogen pathogens has been demonstrated against various pathogens such
or they are converted into toxic derivatives by the pathogen or host as Fusarium spp. (Sabaou and Bounaga 1987; Gopalakrishnan et al.
plant tissue (Grissbuhler et al. 1982). The intensive use of chemical 2011), Phytophthora spp. (Shahidi Bonjar et al. 2006), Pythium spp.
fungicides has not only created problems of fungicide resistance (Hamdali et al. 2008), Rhizoctonia spp. (Sadeghi et al. 2006), and
Verticillium spp. (Meschke and Schrempf 2010).
Actinomycetes can occur in the plant rhizosphere soil and
∗ Corresponding author. Tel.: +213 21 29 75 11; fax: +213 21 28 20 67. exercise an antagonistic and competitive effect on the microbial
E-mail address: zitouni [email protected] (A. Zitouni). communities. They have the ability to produce active compounds,

0944-5013/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.micres.2013.06.014

Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014
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such as antifungal and antibacterial antibiotics or plant growth In order to distinguish the Streptomyces genus, the cell wall type
regulators (PGRs), that have been developed for agricultural uses was determined by a chemical study based on the occurrence of
(Suzuki et al. 2000; Ilic et al. 2007). They have also been used diaminopimelic acid isomers (Becker et al. 1964).
as commercially formulated biocontrol agents of plant diseases Identification of the highest potential isolates (CA-2 and AA-2)
such as Streptomyces griseoviridis cells used to protect crops against on biocontrol of R. solani damping-off and plant growth promot-
infections by Fusarium spp. and Alternaria spp. (Lahdenperä et al. ing activity for tomato cv. Marmande seedlings was confirmed by
1991). In addition to their ability to inhibit plant pathogens, some the 16S rRNA gene sequence analysis. Genomic DNA was prepared
actinomycetes are also known to form close associations with according to the CTAB method (Liu et al. 2000). The 16S rRNA gene
plants, colonize their internal tissues without causing disease sequence was amplified by the PCR method as used by Boubetra
symptoms, and promote their growth (Kunoh 2002). et al. (2013). The PCR products obtained were sent to the MilleGen
Several native plants have successfully adapted to the stressful Company (Toulouse, France) for sequence determination. The16S
conditions of the Algerian Sahara, especially the poor sandy soil and rRNA sequences have been deposited in the GenBank data library
the drought of the arid climate. They grow and colonize these areas and assigned the accession numbers KC414006 and KC414004 for
annually without human intervention (Pouget 1980). The success of CA2 and AA2 strains, respectively. The obtained sequences were
natural regeneration from seeds and the vigorous growth of native compared with sequences present in the public sequence databases
plants under the harsh conditions of the Sahara could suggest a and with EzTaxon tools (Chun et al. 2000).
contribution of endophytic microbes to the biological protection
of germinated seeds against soil-borne pathogens and to the pro- 2.4. In vitro antagonism assay
motion of plant growth. In recent work, we initiated a study to
highlight the role of endophytic microbes from these plant species The streak method described by Boubetra et al. (2013) was used
as potential biocontrol agents against damping-off disease caused to test the antifungal activity of the endophytic actinomycete iso-
by soil-borne pathogens and as plant growth promoters (Goudjal lates against an indigenous soil-borne pathogenic strain of R. solani
et al. 2013). LRS1 isolated from tomato fields in the Laghouat region (33◦ 46 N,
The aims of the present study were to isolate endophytic actino- 2◦ 50 E). Inhibition zones were evaluated as follows: (<5 mm) no
mycetes from healthy Saharan plants and bring out their potential inhibition, (5-9 mm) weak inhibition, (10-19 mm) moderate inhi-
role on the biocontrol of R. solani damping-off and on plant growth bition, and (≥20 mm) strong inhibition.
promoting activities for tomato seedlings. In order to select the
most interesting strains for the biocontrol of R. solani damping-off, 2.5. In vivo biocontrol trials
a commercial chemical agent was used as a control.
2.5.1. Soil properties
2. Materials and methods The sandy soil used to test the effectiveness of the antago-
nistic isolates to control damping-off of tomato cv. Marmande
2.1. Sample collection and endophytic actinomycetes isolation was collected from a tomato field in the Laghouat region (33◦ 46
N, 2◦ 50 E). Its physicochemical and biological characteristics
Healthy native plants (Aristida pungens, Cleome arabica, were: pH 7.8, total organic matter (1.8%), C/N (9.3), phosphate
Solanum nigrum, Panicum turgidum, Astragallus armatus, Peganum (0.07‰), potash (0.3‰), CaCO3 (1.1%) total aerobic bacterial count
harmala, Hammada scoparia and Euphorbia helioscopia) were col- 1.52 × 108 CFU g−1 and total fungal count 3.7 × 104 CFU g−1 . Soil
lected from the Laghouat region in the northern Algerian Sahara was sterilized three times in an autoclave at 120 ◦ C for 60 min on
(33◦ 44 N, 2◦ 47 E) in March 2011. The selection of plant species three consecutive days.
for endophyte isolation was based on their abundance and adap-
tation to the poor sandy soil and arid climatic conditions of the 2.5.2. Preparation of microbial suspensions
Algerian Sahara. Five healthy root samples were harvested from Actinomycete suspensions were prepared as described by
each plant species. No record indicates that these plants have ever Errakhi et al. (2007). Tween-80 solution (0.05%) was used to recover
been studied for endophytic actinomycetes isolation previously. the spores, which were adjusted to 106 CFU ml−1 by the same solu-
Endophytic actinomycetes were isolated according to the tion using the Thomas cell. A pathogen suspension was prepared
method of Taechowisian et al. (2003). The culture medium used by growing R. solani in PDA dishes at 25 ◦ C for 10 days. Spores
was chitin-vitamin agar (Hayakawa and Nonomura 1987) sup- were directly recovered in sterile distilled water and adjusted to
plemented with cycloheximide (80 mg l−1 ) and nalidixic acid 105 CFU ml−1 in the same way.
(15 mg l−1 ) to suppress the growth of fungi and Gram-negative
bacteria, respectively. 2.5.3. Coating of tomato seeds
Tomato seeds were surface-sterilized using the method of Coa
et al. (2004). Sterilized seeds were coated separately by soaking
2.2. Effectiveness of the surface-sterilization protocol for 30 min in the actinomycete spore suspensions and then dried
under a laminar flow hood. Actinomycete spores on the coated
To prove that epiphytic actinomycetes could not grow after the seeds yielded approximately 107 CFU g−1 .
surface-sterilization of the roots and that all the isolated actino-
mycetes were endophytic, the method described by Schulz et al.
2.5.4. Preparation of infested soils
(1993) was used.
Sterilized and non-sterilized soils were infested according to a
method similar to that used by Dhanasekaran et al. (2005). Soils
2.3. Identification of the endophytic actinomycetes were conditioned in plastic pots (12 cm high × 10 cm in diameter)
and infested with 10 ml of pathogen suspension diluted in 90 ml
Endophytic actinomycetes belonging to the Streptomyces genus sterile distilled water (100 ml sterile distilled water for uninfected
were identified based on cultural and micromorphological charac- soils). The pots were covered with plastic film and incubated for
teristics according to the methods of Shirling and Gottlieb (1966) one week at room temperature to promote pathogen growth. R.
and Goodfellow and Simpson (1987). Solani density in the infested soils was evaluated at 104 CFU g−1 .

Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014
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2.5.5. Biocontrol assays

Number of isolates
Based on their in vitro antifungal activities, six antagonistic
isolates were selected for in vivo biocontrol of damping-off of
tomato cv. Marmande seedlings caused by R. Solani. Experiments
were carried out in both sterilized and non-sterilized soils, where
four treatments were performed: (1) positive control by sowing

34
5
2

5
4

5
3

1
1

2
uncoated tomato seeds in non-infested soils; (2) negative control by
sowing uncoated tomato seeds in infested soils to evaluate the vari-
etal sensitivity; (3) sowing coated tomato seeds in infested soils to

non-Streptomyces

non-Streptomyces
evaluate the biocontrol capacity of each antagonistic isolate against
R. solani damping-off; (4) use of a chemical agent, thioperoxydi-

Streptomyces
Streptomyces

Streptomyces

Streptomyces
Streptomyces

Streptomyces

Streptomyces

Streptomyces

Streptomyces

Streptomyces
carbonic diamide, tetramethylthiram (TMTD), to control R. solani
damping-off. In the last case, sterilized seeds were treated as rec-

Genus
ommended by the manufacturer (thorough mixing, for 3 min, of
10 g of the surface-sterilized tomato seeds in 0.08 g of chemical
control agent wetted with 10 ml of distilled water and drying for
2 h under a laminar flow hood), before being cultivated in infested

DAP isomers
soils.
For each treatment, six tomato seeds were sown per pot at 5 mm
from the soil surface, with ten replicates per treatment using a

DL

DL
LL
LL

LL

LL
LL

LL

LL

LL

LL

LL
fully randomized complete block. The tomato cultures were grown
under standard conditions in a plant growth room (22-26 ◦ C, 16 h
light/8 h dark). Pots were watered daily with 10 ml tap water to
maintain a moisture level favorable for seed germination.

Spore-chain types
After 30 days, seedlings were carefully removed from the soil
and washed with tap water. The disease severity was rated using a
5-class scale as described by Altier and Thies (1995).

RF

RF
S
S

S
2.6. Internal root colonization abilities

/
The method described by Misaghi and Donndelinger (1990) was
used to study the internal root colonization abilities of endophytic
Diffusible pigments

Brownish orange
actinomycete strains that significantly decreased the incidence
of damping-off and significantly promoted the growth of tomato
seedlings.
Without
Without

Without
Without

Without

Without

Without

Without
Without

Without

Without
2.7. Statistical analysis

A one-way analysis of variance (ANOVA) was performed on the

data and significant differences between means were compared
White or Yellowish white

using Fisher’s protected LSD test at P = 0.05. Differences were con-

Grey or brownish grey

sidered significant when P < 0.05.

Light brownish grey

Light brownish grey

Light brownish grey

Morphological and chemical characteristics of isolated endophytic actinomycetes.

Yellowish white
Grayish yellow
Yellowish grey

Brownish grey
Greenish grey
Aerial hyphae

Medium grey

3. Results Medium grey

3.1. Validity of the surface-sterilization protocol

All cultures of endophytic actinomycetes treated with

the surface-sterilization protocol were unable to grow on
chitin–vitamin agar medium. This indicates that the isolates
Light yellowish brown
Strong reddish brown

S, Spira; RF, Rectus flexibilis; DAP, diaminopimelic acid.

tested were endophytic and the epiphytic ones could not grow
Light brownish grey

after the sterilization protocol.

Substrate hyphae

Moderate olive

Moderate olive

3.2. Identification of endophytic actinomycetes

Colorless

Colorless

Colorless

Colorless

Colorless
Colorless

Colorless
Color of

Thirty-four endophytic actinomycetes were isolated from the

eight native Algerian Saharan plants. The morphological character-
istics and the cell wall type (Table 1) confirmed that the majority
of isolates (29 isolates) belonged to the genus Streptomyces. Endo-
Euphorbia helioscopia

phytic Streptomyces strains were isolated from all studied native

Astragallus armatus

Hammada scoparia
Panicum turgidum

Peganum harmala

plants. They were classified in three groups according to the color

Aristida pungens

Solanum nigrum
Cleome arabica

of the aerial hyphae on ISP3 agar. The majority of them (22 iso-
Host plants

lates) belonged to the grey group. The yellow and the green groups
comprised 5 and 2 isolates, respectively. The majority of the Strep-
Table 1

Total

tomyces isolates (82.8%) produced a spiral type of spore-chain and

the remaining ones (17.2%) produced a rectiflexibiles type.

Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014
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Non-Streptomyces isolates made up 14.7% of all the endophytic A 100 e e

isolates and were obtained only from the roots of Cleome arabica

Disease incidense (%)

and Hammada scoparia. 80
d
The isolates CA-2 and AA-2, which showed the highest biocon- d d
trol capacities on R. solani damping-off and the highest growth 60 c
promoting activities on tomato seedlings, were selected for the
molecular taxonomy analysis (GenBank KC414006 and KC414004, 40
b
respectively). Taxonomically, the strains CA-2 and AA-2 were ab b b ab a
ab a ab a
20 a a
ranked in two different morphological groups and they were
related to Streptomyces mutabilis NBRC 12800T (100% similar- 0
ity) and Streptomyces cyaneofuscatus JCM 4364T (100% similarity),
respectively on the basis of the 16S rRNA gene comparison.
According to the aerial spore mass color, the strain CA-2 B 2.0
c
belonged to the Grey series of the genus Streptomyces. The spore c

Seadling fresh weight

chains produced were in the section Spirales, and contained 10-50 1.5

(g/seedling)
spores per chain. This strain was compared to the phylogenetically b ab b b
b
related type strain NBRC 12800T of S. mutabilis. As described by 1.0 b ab ab ab b
ab
a a a a
Kämpfer (2012), this species can be ranked in both the Grey and a
White series and can produces spore chains in the Spirales or Retina-
culiaperti sections. Furthermore, spore chains are poorly developed, 0.5
with shorter hooks, loops, or partial spirals, and contain only 3-10
spores per chain. 0.0
The strain AA-2 belonged to the Yellow series of the genus
Streptomyces, and the spore chains produced were in the sec-
C 14 d d
tion Rectiflexibilis. Mature spore chains contained 10-50 spores per d d
chain. The strain has the same morphological characteristics as its
Seedling length (cm) 12 cd cd
c bc bc c bc
c
phylogenetically related type strain JCM 4364T of S. cyaneofuscatus 10 bc
b
(Kämpfer, 2012). 8 ab
a a ab
The both species of Streptomyces, S. mutabilis and S. cyaneofus-
catus have never been reported for their biocontrol and growth 6
promoting activities. 4
2
3.3. In vitro antagonistic activities
0
Twenty-one isolates of the genus Streptomyces showed an anti-
fungal activity against R. solani (Table 2) but only six of them D 8
d
strongly inhibited (inhibition zone >20 mm) the mycelium growth 7 d
Seedling root length (cm)

cd e
of the pathogen. The remaining isolates failed to induce, or induced
6 b cd bc d bc cd bc d
only small, inhibition zones (<20 mm) and so were not included b
5 bc
b
in subsequent studies. The antagonistic effects appeared after two b
days of paired incubation of actinomycetes and fungi. This attribute 4 a
3 a
suggests that some of the isolates tested may be efficient in con-
trolling damping-off caused by R. solani. Based on the mean size of 2
the inhibition zones, the Streptomyces strains AP-4, AP-5, CA-2, SN- 1
5, SN-10 and AA-2 were selected for in vivo biocontrol of R. solani 0
damping-off of tomato seedlings. Positive Negative TMTD AP-4 AP-5 CA-2 SN-5 SN-10 AA-2
control control
3.4. In vivo biocontrol of R. solani damping-off of tomato Seed treatments
seedlings
Fig. 1. Effect of seed treatment with TMTD and spore suspensions of antagonistic
actinomycetes (AP-4, AP-5, CA-2, SN-5, SN-10 and AA-2) on the disease incidence
High disease incidence (90.3% and 88.5%) was observed with (A), seedling fresh weight (B), seedling length (C) and root length (D) in sterilized
untreated tomato seeds in sterilized and non-sterilized soils, (white bars) and non-sterilized (grey bars) soils. Evaluation was made 30 days after
respectively (Fig. 1A). After the seeds had germinated, the rootlets planting. Bars labeled with the same letters are not significantly different according
to Fisher’s protected LSD test at P = 0.05.
were subjected to the effects of R. solani. Considerable root rot was
observed in the newly germinated seeds and caused their death
(Fig. 2A). The root rot symptoms were also observed in seedling
roots. They evolved and led to desiccation of the roots and damping- seedling rates obtained by these treatments varied from 38.0% to
off of the seedlings (Fig. 2b and c). However, normal growth and 86.6% compared to the negative control that exhibited only 19.7%
healthy seedling roots were obtained for the tomato seeds coated of healthy seedlings. The strains CA-2 and AA-2 and the com-
with spores of the strain Streptomyces sp. CA-2 growing in the soil pound TMTD had the greatest incidence of disease reduction in both
infested by R. solani (Fig. 2d). sterilized and non-sterilized soils (86.6%, 85.3% and 85.2% healthy
For the majority of seed treatments, the protective effects seedlings, respectively) and no significant differences (P < 0.05)
were more pronounced in sterilized soil than in non-sterilized soil were observed among them. However, the biocontrol capacities of
(Fig. 1A). The compound TMTD and the biocontrol agents (CA-2, the remaining strains AP-4, AP-5, SN-5 and SN-10 were less than
AA-2, AP-4, SN-10, SN-5 and AP-5) allowed a significant reduction that of TMTD. The rates of the healthy tomato seedlings obtained by
in disease incidence (P < 0.05) in seed-treated seedlings. Healthy these treatments were 82.9%, 40.4%, 49.3% and 79.8%, respectively.

Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014
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Table 2
In vitro antagonistic activity of endophytic actinomycetes against Rhizoctonia solani LRS1.

Host plants Endophytic isolates Genus Diameter of inhibition zone (mm)a

Aristida pungens AP-1 Streptomyces 0

AP-2 Streptomyces 0
AP-3 Streptomyces 0
AP-4 Streptomyces 30 ± 1.5
AP-5 Streptomyces 28 ± 0.6
AP-6 Streptomyces 0
AP-7 Streptomyces 0

Cleome arabica CA-2 Streptomyces 35 ± 1.2

CA-3 Streptomyces 0
CA-6 Streptomyces 17 ± 0.6
CA-7 non-Streptomyces 0
CA-9 Streptomyces 19 ± 1.5
CA-10 non-Streptomyces 16 ± 1.0
CA-11 non-Streptomyces 15 ± 0.6
CA-12 Streptomyces 17 ± 1.2
CA-13 non-Streptomyces 0

Solanum nigrum SN-2 Streptomyces 16 ± 0.6

SN-3 Streptomyces 0
SN-4 Streptomyces 14 ± 0.6
SN-5 Streptomyces 28 ± 1.5
SN-7 Streptomyces 14 ± 0.6
SN-8 Streptomyces 17 ± 1.3
SN-10 Streptomyces 36 ± 0.8
SN-11 Streptomyces 16 ± 1.2

Panicum turgidum PT-1 Streptomyces 19 ± 0.9

PT-3 Streptomyces 18 ± 1.2

Astragallus armatus AA-2 Streptomyces 30 ± 1.3

AA-3 Streptomyces 14 ± 0.9

Peganum harmala PH-1 Streptomyces 0

PH-8 non-Streptomyces 0

Hammada scoparia HS-1 Streptomyces 15 ± 1.5

HS-2 Streptomyces 19 ± 0.8

Euphorbia helioscopia EH-3 Streptomyces 0

EH-4 Streptomyces 0
a
Average ± standard deviation from three replicates.

In both soils, seed treatment with the strain Streptomyces to 1.67 g/seedling (Figs. 1B and 3), the seedling length from 9.88 cm
sp. CA-2 exhibited the greatest growth promoting activities on to 12.01 cm (Fig. 1C) and the root length from 4.86 cm to 6.92 cm
tomato seedlings. Compared to the positive control, it significantly (Fig. 1D). Nevertheless, smaller growth promoting activities were
increased (P < 0.05) the seedling fresh weight from 0.92 g/seedling observed on tomato seedlings that received the treatments with
spores of the Streptomyces spp. AP-4, AP-5, SN-5 and SN-10. Com-
pared to the positive control, no important changes were exhibited
in the promoting of seedling fresh weight (0.89 g/seedling to

Fig. 2. Tomato (cv. Marmande) seedlings grown in the R. solani infested soil without Fig. 3. Tomato cv. Marmande seedlings obtained by sowing uncoated tomato seeds
seed treatment (a)–(c) and by treatment of seeds with spores of the strain Strepto- in sterilized soil infested with R. solani as negative control (A), uncoated seeds sowed
myces sp. CA-2 (d). Arrows indicate the root rot symptoms caused by R. solani on in sterilized soil as positive control (B), treated seeds with the chemical TMTD sowed
primary rootlet (a) and on seedling roots (b) and (c) compared to a healthy seedling in infested soil (C), and coated seeds with spores of the strain CA-2 in infested soil
root (d). Pictures were taken after 10 days in plant growth room under standard (D). The picture was taken after 14 days under standard growth conditions in plant
growth conditions. growth room.

Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014
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1.04 g/seedling), seedling length (9.58 cm to 10.47 cm) and seedling isolates active in vitro were also active in soil conditions. Several
root length (4.30–5.02 cm). mechanisms have been proposed for the antibiosis activity, which
could be mediated by the secretion of specific or non-specific
metabolites of microbial origin, by lytic agents, enzymes, volatile
3.5. Internal root colonization abilities
compounds or other toxic substances (Fravel 1988). Hajlaou et al.
(1994) reported that such behavior was particularly interesting for
Results for the root colonization abilities of the six strains tested
biocontrol agents, because the compounds mediating the antibiosis
(AP-4, AP-5, CA-2, SN-5, SN-10 and AA-2) showed no visible growth
could diffuse in the soil, and direct contact between antagonistic
of endophytic actinomycetes from the control seedlings. However,
actinomycete and soil-borne pathogen was not necessary.
all strains were successfully isolated from the surface-sterilized
In addition, the treatment of tomato seeds with TMTD signifi-
roots of inoculated tomato seedlings. This indicates that these
cantly reduced (P < 0.05) the disease incidence by over 75% and the
strains were endophytic and their endophytic colonizing abilities
rate of healthy seedlings reached 85.3% (Fig. 1A). However, beside
were demonstrated in both their indigenous host plants and inoc-
this efficacy, the widespread use of chemical fungicides against R.
ulated tomato roots.
solani disease raises persistent problems such as resistance to the
fungicide and environment contamination (Grissbuhler et al. 1982).
4. Discussion Among the isolates tested, the Streptomyces spp. CA-2 and AA-
2 presented the same, non-significant differences at P < 0.05 and
Several studies have reported the use of actinomycete strains the same effect in reducing R. solani damping-off incidence as the
for biocontrol of R. solani damping-off (Moussa 2002; Coa et al. chemical control agent in both sterilized and non-sterilized soils.
2004; Chung et al. 2005; Sadeghi et al. 2006; Patil et al. 2010). Coating of tomato seeds by spores of these two isolates allowed
In addition, commercial products to control crop damping-off, the highest rates of healthy seedlings (86.6% and 85.3%, respec-
such as Mycostop® (Streptomyces griseoviridis strain K61) and tively). Furthermore, they significantly increased the seedling fresh
Actinovate® (Streptomyces lydicus strain WYEC108), have been reg- weight, seedling length and root length compared to the positive
istered (Lahdenperä et al. 1991). control. Several mechanisms could be involved in plant growth
In this study, six endophytic actinomycete strains (AP-4, AP- promotion by endophytic actinomycetes. The production of plant
5, CA-2, SN-5, SN-10 and AA-2) isolated from roots of native growth regulators, such as indol-3-acetic acid and gibberellic acid,
plants from the Algerian Sahara showed in vitro production of anti- the inorganic phosphate solubilization, and the nitrogen fixation
fungal compounds with a large inhibition zone (≥28 mm) for R. could be the most important ones. Several studies have reported
solani mycelium growth. Their abilities to reduce the incidence of plant growth promoting activities of endophytic actinomycetes on
damping-off and to promote the growth of tomato seedlings in both tomato seedlings (Coa et al. 2005; El-Tarabily et al. 2008; Goudjal
sterilized and non-sterilized soils were noted. et al. 2013). The enhancement of plant growth by the strains Strep-
The biocontrol of R. solani damping-off in both soils showed that tomyces sp. CA-2 and AA-2 could contribute to the protection of the
all tested strains significantly (P < 0.05) reduced the disease inci- plant against pathogenic fungi as previously reported with other
dence compared to the negative control (Figs. 1 and 3). The strains Streptomyces spp. by Xiao et al. (2002).
CA-2, AA-2, AP-4 and SN-10 reduced the disease incidence by over The transposition of the ability of the strains CA-2 and AA-2
75%. However, the disease reduction reached by the remaining to colonize the tomato roots demonstrated that their endophytic
tested strains (AP-5 and SN-5) was less than 33%. property was maintained. As defined by Wilson (1995), endophytic
Endophytic actinomycete strains selected on the basis of actinomycetes, for all or part of their life cycle, invade the tissue
in vitro antagonistic activities were effective in controlling R. solani of living plants and cause unapparent, asymptomatic infections
damping-off in both soils. Their biocontrol activities allowed 40.4% entirely within plant tissue, but cause no symptoms of disease.
to 86.6% healthy seedlings compared to the negative control that A correlation has been shown between the efficacy of biocontrol
exhibited only 9.7%. The plant protection could be resulted by the microorganisms against soil-borne pathogens and their ability to
action of antagonistic actinomycete strains on the growth of R. colonize the root system of the plant to be protected, especially
solani. Several mechanisms can be involved, especially the produc- when the mode of action used by these bacterial strains is antibio-
tion of antifungal compounds, the chitinolytic activities and the sis or competition for niches and nutrients (Chin-A-Woeng et al.
nutrient competition by the production of siderophores. However, 1998).
many factors are likely to affect biocontrol of damping-off in vivo, The growth promoting effect of the two isolates of actino-
including environmental conditions such as soil temperature, soil mycetes seemed to be correlated with root enhancement and shoot
morphology, pH value, water status, nutrient availability and inter- production. In the same cases, where the strains were strictly endo-
actions with indigenous soil microbes (Dhanasekaran et al. 2005). phytic, such effects were generally attributed to PGRs production
In the in vivo conditions, the rhizospheric microorganisms could (Shi et al. 2009). El-Tarabily et al. (2008) reported that the involve-
also interact with inoculated antagonists, which influenced their ment of PGRs could not only help the seedlings to grow better
biocontrol capacities. Our study showed that all antagonistic strains but could also help the host to compensate for tissue damage
of Streptomyces tested reduced the disease incidence more in non- caused by the pathogen agent. Several endophytic bacteria have
sterilized soil than in sterilized soil. The positive effect of interaction been reported to produce PGRs in vitro and to promote the growth
with indigenous soil microorganisms on the control of R. solani of seedlings (Kuklinsky-Sobral et al. 2004; Goudjal et al. 2013).
damping-off was also confirmed by Errakhi et al. (2007) in Scle- Furthermore, the growth promoting effect is widely attributed to
rotium rolfsii damping-off of sugar beet with Streptomyces spp. other factors, such as siderophore production and phosphate solu-
Furthermore, as reported by Shen (1997) and Kennedy (1999), bilization (Hamdali et al. 2008) and nitrogen fixation (Ribbe et al.
these findings suggest that the rhizospheric indigenous flora con- 1997).
tributes to the control of R. solani disease and could have a synergic The strains CA-2 and AA-2 were related to S. mutabilis and
effect with the antagonistic actinomycetes. S. cyaneofuscatus, respectively. Several studies have reported the
This would suggest that several mechanisms are involved in role of Streptomyces strains as potential agents in the biocontrol
controlling plant pathogens by endophytic actinomycetes in soil of soil-borne pathogenic fungi and in promoting plant growth
conditions. Trejo-Estrada et al. (1998) reported that antibiosis (Lahdenperä et al. 1991; Coa et al. 2004; Dhanasekaran et al. 2005;
is likely to be the important mechanism as the majority of the El-Tarabily et al. 2008). However, this is the first report showing the

Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014
 ARTICLE IN PRESS
G Model
MICRES-25588; No. of Pages 7

Y. Goudjal et al. / Microbiological Research xxx (2013) xxx–xxx 7

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Please cite this article in press as: Goudjal Y, et al. Biocontrol of Rhizoctonia solani damping-off and promotion of tomato plant growth by
endophytic actinomycetes isolated from native plants of Algerian Sahara. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.06.014