Evidence stage (Yekkour et al., 2012). Various chemical fungicides

Evidence of biocontrol and plant-growth-promoting
capacities of Streptosporangium becharense strain SG1: an antagonistic
actinobacterium from the Algerian SaharaABSTRACT

Sixteen actinobacterial strains isolated from various ecological niches
in the Algerian Sahara ‘were investigated for their growth
promotion effect on durum wheat plants and for their biocontrol potential of Fusarium
culmorum root rot. All actinobacteria were characterized for in vitro
antagonistic activity and plant-growth-promotion traits, for the production
of cyanhydric acid, siderophores, chitinases and indole-3-acetic acid, and for
inorganic phosphate solubilization. Strongly antagonistic actinobacteria were selected and investigated for the biocontrol of F.
culmorum in vivo and for growth
promotion of durum wheat plants in autoclaved and non-autoclaved soils. The Streptosporangium becharense strain SG1 isolate exhibited
remarkable positive results in all trials. Compared to untreated wheat seeds,
the root rot severity index was decreased significantly (P ? 0.05) by
all seed bacterization treatments. However, the highest protective effect was
obtained by the strain SG1, which reduced the disease severity index from 77.8%
to 16%, whereas it was only
reduced to 24.2% by chemical seed treatment with Dividend®. Moreover, strain SG1 led to significant increases in the shoot length,
root length and dry weight of plants. ”This is the first study
that has showed the interesting potential of biocontrol and growth improvement
of wheat plants by S. becharense SG1, it has proved to be a
powerful approach to exploit actinobacterial communities in crop enhancement.

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Actinobacteria; Streptosporangium becharense strain SG1; Biocontrol; Fusarium culmorum; Plant-growth-promotion; Durum wheat







Chemical products are commonly used as pesticides or fertilizers to
improve crop production. However, the abusive use of agrochemical compounds often causes
problems such as contamination of the soil, high toxicity on native microbial
communities, pesticide resistance and other adverse effects on the environment (Huang, Zhang, Yong, Yang, &
Shen, 2011).

Root rot and
damping-off of seedlings is a common disease caused by Fusarium species in
a variety of crop cereals, such as corn, rice, barley and wheat. In Algeria, Fusarium
culmorum is considered to be a serious problematic for the cereal crops,
which causes considerable losses especially at the seedling stage (Yekkour et al., 2012). Various chemical fungicides are frequently used to
manage the Fusarium diseases and to prevent crop losses. Nevertheless,
the majority of them are not ideally effective to eradicate these
phytopathogenic fungi (Huang et al., 2011). Du to these preoccupations, there is an increasing demand for
developing biocontrol methods
for sustainable agriculture aiming to protect the environment by reducing
chemical pesticide uses (Shimizu, 2011).

Actinobacteria are
considered as potential biocontrol agents of plant diseases. Actinobacteria can also
colonize the plant rhizosphere soil and produce adverse compounds such as cyanhydric acid, siderophores and hydrolytic
enzymes (De-Oliveira, Da Silva, & Van Der Sand, 2010; Passari et al., 2015). They can solubilize inorganic
phosphate and potash and enhance their uptake by the plant (Hamdali, Hafidi, Virolle,
& Ouhdouch, 2008). Some actinobacteria are also known to develop
symbiotic associations with crop plants, colonizing their internal tissues
without causing disease symptoms and producing plant growth regulators such as
gibberellic acid and indole-3-acetic acid (IAA) (Goudjal et al., 2013). In addition, several researchers
reported the potential of plant-associated actinobacteria as agents to manage
various soil-borne phytopathogenic fungi and/or to stimulate plant growth (Ramadan, AbdelHafez , Hassan & Saber, 2016).

In this context, we aimed to evaluate the potential
of some actinobacterial strains isolated from sandy soils or native plants that
successfully adapted to the harsh edaphoclimatic conditions of the Algerian
Sahara, as agents for biocontrol of F. culmorum root rot disease in vivo and for promoting the growth of
durum wheat plants.


Materials and methods


Actinobacterial isolates


Sixteen rhizospheric or endophytic
actinobacteria (Table 1), isolated by our research team in the Laboratory of
Biology of Microbial Systems (LBSM), ENS – Kouba, Algiers, Algeria, were
selected to investigate their efficacy in the in vivo biocontrol of Fusarium
culmorum root rot disease and in the growth promotion of wheat plants. Actinobacteria were selected based on their
efficacy in the biocontrol of soil-borne phytopathogenic fungi such as Rhizoctonia
solani (Goudjal et al., 2014) and F. oxysporum f. sp. radicis-lycopersici (Zamoum
et al., 2015; Zamoum, Goudjal, Sabaou, Mathieu & Zitouni, 2017), on their
growth promotion effect on cropped plants (Goudjal et al., 2013; 2015) and on
the fact that they were classified as novel species of actinobacteria (Chaabane
Chaouch et al., 2016a, b; Lahoum et al., 2016).


Antagonistic activity of endophytic actinobacteria


The streak method was adopted to estimate the antagonistic activities of actinobacteria
against five soil-borne phytopathogenic fungi (Fusarium culmorum (LF18),
F. graminearum (LF21), F. oxysporum f sp. radicis-lycopersici
(LF30), Rhizoctonia solani (LAG3), and Bipolaris sorokiniana
(LB12)) from the microbial collection of our laboratory. The actinobacterial
isolates were cultivated separately in straight lines on International Streptomyces
Project (ISP) 2 medium (Shirling and Gottlieb 1966) plates which are incubated for 8 days at 30ºC. After that,
target fungi were seeded in streaks perpendicular to those of actinobacteria
cultivation. After incubation at 25ºC for 5 days, the distance of inhibition
between target fungus and actinobacteria colony margins was measured (Toumatia
et al., 2015).


Determination of biocontrol and plant-growth-promotion


Hydrogen cyanide (HCN) production


Actinobacteria were grown in Bennett agar amended with 4.4 g l?1 of
glycine for studying their ability to produce HCN. Whatman paper
soaked in 0.5% picric acid (in 2% sodium carbonate) for a minute and stuck
under the Petri dish lid. The
plates were then sealed air-tight with Para film and incubated at 30 °C for 7
days”. Positive production of HCN is
indicated by an orange color on the filter paper (Passari et al., 2015).


Siderophore production


The method described by Sadeghi et al. (2012) was used to evaluate the production of
siderophores by isolates. Six millimetre disks from actinobacteria cultures
were placed on chrome azurol S plates and incubated at 30°C for 7 days.
Apparition of orange haloes around colonies was indicative to positive
siderophore production.


Chitinolytic activity


The actinobacteria were spot inoculated on colloidal chitin agar medium
to tested chitinolytic activity (Zamoum et al., 2017). Cultures were incubated at 30°C for 5
days. Measurement of the hydrolytic halo diameter surrounding the
actinobacterial colonies allows estimating chitinolytic activity.


Indole-3-acetic acid production (IAA)


For assessment of IAA synthesis, actinobacterial
isolates were inoculated in 250 mL Erlenmeyer flasks containing 50 mL of yeast extract-tryptone (YT) broth,
supplemented with 5 mg ml?1 of L-tryptophan, and kept in an incubated shaker (200 rpm, 30
ºC, 5 days). The flasks containing the culture broth were then centrifuged at
4,000 rpm for 30 min. Equimolar concentration of Salkowski reagent (1 mL 0.5 M
FeCl3 dissolved in 50 mL 35% HClO4) was added to 2 mL of
supernatant. The mixture was incubated in the dark for 30 min and the appearance of pink colour indicated the
IAA production witch was confirmed by confirmed by thin layer chromatography (TLC)
as used by Ahmad, Ahmad & Khan, (2009). Ethyl acetate fractions (10–20 )
were spotted on TLC plates (silica gel GF254, thickness 0.25 mm, Merck,
Germany) and developed in ethyl acetate: chloroform: formic acid (55:35:10, by
vol.). Spots with Rf values identical to authentic IAA were
identified under UV light (254 nm) after spraying the plates with Ehmann’s reagent. The absorbance was measured in a
spectrophotometer at 530 nm and the IAA concentration was estimated using a pure
IAA standard graph (Goudjal et al., 2013).


Phosphate solubilization


The trials were achieved in 500 ml Erlenmeyer flasks containing 100 ml
of liquid Pikovskaya medium supplemented with 5 g l?1 of Ca3(PO4)2,
AlPO4 or FePO4 as insoluble phosphate sources. Isolates were inoculated in the flasks aseptically and
kept in an incubated shaker (200 rpm, 30 ºC, 7 days). The
liquid cultures were centrifuged at 10,000 rpm for 10 min and the supernatant cultures were collected then used to
determine the amount of dissolved phosphorus using the molybdenum blue
colorimetric method (Liu et al., 2014).


In vivo biocontrol of Fusarium culmorum


The potential of the strong antagonistic actinobacteria in the in
vivo biocontrol of F. culmorum (LF18) and their ability to
promote the growth of durum wheat (cv. vitron) seedlings were tested in an
infested soil sampled cereal field in the Algerian Sahara (33°62’N, 2°91’E).
Trials were performed both in autoclaved and non-autoclaved soils.

Surface-sterilization of seeds was performed by sequential dipping in ethanol solution (70% v/v; 3
min), NaClO solution (0.9% w/v; 4 min) followed by washing three times in
sterile distilled water. After that, surface-sterilized seeds were
separately bacterized by dipping in the suspensions of antagonistic
actinobacteria strains (? 106 CFU ml?1) for 30 min and were dried under a laminar flow hood before being sown
the same day. Actinobacteria spores on the bacterized seeds were enumerated by
the plate dilution method on ISP2 medium. They yielded ? 4 × 106 CFU g?1 bacterized seeds.

Autoclaved and non-autoclaved soils were infested with the F. culmorum spore suspension
(? 103 CFU
ml?1). For this,
plastic pots (10 cm in diameter ×12 cm high) filled with soil were irrigated with 100 ml of the F. culmorum spore suspension. The density of F.
culmorum in the infested soil was evaluated at ? 1.11 × 104 CFU g?1.

Four treatments were conducted in the
biocontrol assay: (1) untreated seeds were sown in non-infested pots (negative
control); (2) untreated seeds were sown in infested soils to highlight the
virulence of F. culmorum (LF18) (positive control); (3) bacterized seeds
sown in pots with infested soil to evaluate the biocontrol potential of each
antagonistic actinobacteria strain; (4) surface-sterilized seeds were treated with a
marketed chemical fungicide Dividend® 030 FS
(Difenoconazole)by dipping for 3 min in the fungicide solution and drying for 2h under a laminar flow
hood, before being cultivated in infested soils.

Five seeds were sown per pot with 10
replicates for each treatment. In vivo biocontrol trials were conducted twice to ensure
reproducibility. Pots were then placed in a fully randomized
complete block design in a greenhouse (24?28°C, 14 h light/10 h dark). Cultures were watered daily with tap water
(10 ml per pot) for 6 weeks.

As used by Dhanasekaran et al. (2005), the F. culmorum root
rot symptoms were evaluated using the following scale:0 = no symptom, 1 = 0?25% of root browning, 2 = 26?50% of root browning, 3 = 51?75% of root browning, 4 = 76?100% of root browning and 5 = plant death. For each seed treatment, the disease severity index (DSI) was
calculated using the following formula:

R = the disease rating, N = number of plants with this rating, H =
highest rating category, T = total number of counted plants.

The effect of each seed treatment on the growth of
wheat plants was also evaluated by measuring the shoot and root lengths, and
the dry weight of healthy plants.


Statistical analysis


Three replications were performed for each experiment (10 replicates for
in vivo trials) and values represent the mean ± standard deviation. Data were subjected to one-way
analysis of variance (ANOVA). When the F-statistic was significant, Tukey’s
post hoc test (P = 0.05) was used to separate means.




Antagonistic activities


Of the 16 actinobacterial isolates, seven
(43.8%) showed positive antagonistic activities against all the fungi. Ten
isolates (62.5%) revealed antagonistic activity against at least three of the
five soil-borne phytopathogenic fungi tested (Table 1), with the most striking
antagonistic activity against Fusarium oxysporum f. sp. radicis-lycopersici
and Rhizoctonia solani. However, mycelial growth of F. culmorum was inhibited by only 25% of the isolates. It was noted that strong antagonistic activities
(inhibition zone >20 mm) were shown in four isolates and the largest zone of inhibition
was obtained by Streptosporangium becharense SG1.


Hydrogen cyanide, siderophore production
and chitinolytic activity


The results of HCN and siderophore
production, and chitinolytic activity by the four selected antagonistic
actinobacteria (strains CAR2, SG1, ZLT2 and MB29) are given in Table 2. All
isolates can produce HCN. Siderophores were produced by three isolates. All
targeted actinobacteria showed positive results for chitinolytic activity.

Indole-3-acetic acid production and
phosphate solubilization ability


Three of the four isolates tested produced IAA in YT broth, with the
isolate S. becharense SG1 showing the best production (Table 2). Our results demonstrated that all four
actinobacteria tested dissolved phosphorus from tricalcium phosphate and aluminium phosphate sources (Table 2), and only the isolate Saccharothrix
longispora MB29 was incapable of dissolving iron phosphate.


In vivo biocontrol of Fusarium culmorum


Untreated seeds sown in infested soils
(positive control) showed the highest disease severity indexes (DSI) of F.
culmorum root rot in wheat seedlings, both in autoclaved and
non-autoclaved soils (Figure
1(A), Figure 2(B),(C)). This proves the virulence of the pathogen and the
high sensitivity of durum wheat cv. vitron.

wheat seeds with spores of antagonistic actinobacteria and chemical treatment
with Dividend® significantly (P