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

Evidence of biocontrol and plant-growth-promotingcapacities of Streptosporangium becharense strain SG1: an antagonisticactinobacterium from the Algerian SaharaABSTRACTSixteen actinobacterial strains isolated from various ecological nichesin the Algerian Sahara ‘were investigated for their growthpromotion effect on durum wheat plants and for their biocontrol potential of Fusariumculmorum root rot.

All actinobacteria were characterized for in vitroantagonistic activity and plant-growth-promotion traits, for the productionof cyanhydric acid, siderophores, chitinases and indole-3-acetic acid, and forinorganic phosphate solubilization. Strongly antagonistic actinobacteria were selected and investigated for the biocontrol of F.culmorum in vivo and for growthpromotion of durum wheat plants in autoclaved and non-autoclaved soils. The Streptosporangium becharense strain SG1 isolate exhibitedremarkable positive results in all trials. Compared to untreated wheat seeds,the root rot severity index was decreased significantly (P ? 0.05) byall seed bacterization treatments. However, the highest protective effect wasobtained by the strain SG1, which reduced the disease severity index from 77.8%to 16%, whereas it was onlyreduced to 24.

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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 studythat has showed the interesting potential of biocontrol and growth improvementof wheat plants by S.

becharense SG1, it has proved to be apowerful approach to exploit actinobacterial communities in crop enhancement. KEYWORDSActinobacteria; Streptosporangium becharense strain SG1; Biocontrol; Fusarium culmorum; Plant-growth-promotion; Durum wheat     IntroductionChemical products are commonly used as pesticides or fertilizers toimprove crop production. However, the abusive use of agrochemical compounds often causesproblems such as contamination of the soil, high toxicity on native microbialcommunities, pesticide resistance and other adverse effects on the environment (Huang, Zhang, Yong, Yang, , 2011).Root rot anddamping-off of seedlings is a common disease caused by Fusarium species ina variety of crop cereals, such as corn, rice, barley and wheat.

In Algeria, Fusariumculmorum 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 tomanage the Fusarium diseases and to prevent crop losses. Nevertheless,the majority of them are not ideally effective to eradicate thesephytopathogenic fungi (Huang et al., 2011). Du to these preoccupations, there is an increasing demand fordeveloping biocontrol methodsfor sustainable agriculture aiming to protect the environment by reducingchemical pesticide uses (Shimizu, 2011).Actinobacteria areconsidered as potential biocontrol agents of plant diseases. Actinobacteria can alsocolonize the plant rhizosphere soil and produce adverse compounds such as cyanhydric acid, siderophores and hydrolyticenzymes (De-Oliveira, Da Silva, & Van Der Sand, 2010; Passari et al.

, 2015). They can solubilize inorganicphosphate and potash and enhance their uptake by the plant (Hamdali, Hafidi, Virolle,& Ouhdouch, 2008). Some actinobacteria are also known to developsymbiotic associations with crop plants, colonizing their internal tissueswithout causing disease symptoms and producing plant growth regulators such asgibberellic acid and indole-3-acetic acid (IAA) (Goudjal et al., 2013). In addition, several researchersreported the potential of plant-associated actinobacteria as agents to managevarious soil-borne phytopathogenic fungi and/or to stimulate plant growth (Ramadan, AbdelHafez , Hassan & Saber, 2016).In this context, we aimed to evaluate the potentialof some actinobacterial strains isolated from sandy soils or native plants thathadsuccessfully adapted to the harsh edaphoclimatic conditions of the AlgerianSahara, as agents for biocontrol of F. culmorum root rot disease in vivo and for promoting the growth ofdurum wheat plants.

 Materials and methods Actinobacterial isolates Sixteen rhizospheric or endophyticactinobacteria (Table 1), isolated by our research team in the Laboratory ofBiology of Microbial Systems (LBSM), ENS – Kouba, Algiers, Algeria, wereselected to investigate their efficacy in the in vivo biocontrol of Fusariumculmorum root rot disease and in the growth promotion of wheat plants. Actinobacteria were selected based on theirefficacy in the biocontrol of soil-borne phytopathogenic fungi such as Rhizoctoniasolani (Goudjal et al., 2014) and F. oxysporum f. sp.

radicis-lycopersici (Zamoumet al., 2015; Zamoum, Goudjal, Sabaou, Mathieu & Zitouni, 2017), on theirgrowth promotion effect on cropped plants (Goudjal et al., 2013; 2015) and onthe fact that they were classified as novel species of actinobacteria (ChaabaneChaouch et al., 2016a, b; Lahoum et al.

, 2016). Antagonistic activity of endophytic actinobacteria The streak method was adopted to estimate the antagonistic activities of actinobacteriaagainst 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 actinobacterialisolates were cultivated separately in straight lines on International StreptomycesProject (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 actinobacteriacultivation. After incubation at 25ºC for 5 days, the distance of inhibitionbetween target fungus and actinobacteria colony margins was measured (Toumatiaet al., 2015). Determination of biocontrol and plant-growth-promotiontraits Hydrogen cyanide (HCN) production Actinobacteria were grown in Bennett agar amended with 4.

4 g l?1 ofglycine for studying their ability to produce HCN. Whatman papersoaked in 0.5% picric acid (in 2% sodium carbonate) for a minute and stuckunder the Petri dish lid. Theplates were then sealed air-tight with Para film and incubated at 30 °C for 7days”.

Positive production of HCN isindicated 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 ofsiderophores by isolates. Six millimetre disks from actinobacteria cultureswere placed on chrome azurol S plates and incubated at 30°C for 7 days.Apparition of orange haloes around colonies was indicative to positivesiderophore production. Chitinolytic activity The actinobacteria were spot inoculated on colloidal chitin agar mediumto tested chitinolytic activity (Zamoum et al.

, 2017). Cultures were incubated at 30°C for 5days. Measurement of the hydrolytic halo diameter surrounding theactinobacterial colonies allows estimating chitinolytic activity. Indole-3-acetic acid production (IAA) For assessment of IAA synthesis, actinobacterialisolates 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, 30ºC, 5 days). The flasks containing the culture broth were then centrifuged at4,000 rpm for 30 min. Equimolar concentration of Salkowski reagent (1 mL 0.5 MFeCl3 dissolved in 50 mL 35% HClO4) was added to 2 mL ofsupernatant.

The mixture was incubated in the dark for 30 min and the appearance of pink colour indicated theIAA 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, byvol.

). Spots with Rf values identical to authentic IAA wereidentified under UV light (254 nm) after spraying the plates with Ehmann’s reagent. The absorbance was measured in aspectrophotometer at 530 nm and the IAA concentration was estimated using a pureIAA standard graph (Goudjal et al., 2013). Phosphate solubilization The trials were achieved in 500 ml Erlenmeyer flasks containing 100 mlof 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 andkept in an incubated shaker (200 rpm, 30 ºC, 7 days).

Theliquid cultures were centrifuged at 10,000 rpm for 10 min and the supernatant cultures were collected then used todetermine the amount of dissolved phosphorus using the molybdenum bluecolorimetric method (Liu et al., 2014). In vivo biocontrol of Fusarium culmorum The potential of the strong antagonistic actinobacteria in the invivo biocontrol of F. culmorum (LF18) and their ability topromote the growth of durum wheat (cv.

vitron) seedlings were tested in aninfested 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; 3min), NaClO solution (0.9% w/v; 4 min) followed by washing three times insterile distilled water. After that, surface-sterilized seeds wereseparately bacterized by dipping in the suspensions of antagonisticactinobacteria strains (? 106 CFU ml?1) for 30 min and were dried under a laminar flow hood before being sownthe same day. Actinobacteria spores on the bacterized seeds were enumerated bythe 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 CFUml?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 thebiocontrol assay: (1) untreated seeds were sown in non-infested pots (negativecontrol); (2) untreated seeds were sown in infested soils to highlight thevirulence of F.

culmorum (LF18) (positive control); (3) bacterized seedssown in pots with infested soil to evaluate the biocontrol potential of eachantagonistic actinobacteria strain; (4) surface-sterilized seeds were treated with amarketed chemical fungicide Dividend® 030 FS(Difenoconazole)by dipping for 3 min in the fungicide solution and drying for 2h under a laminar flowhood, before being cultivated in infested soils.Five seeds were sown per pot with 10replicates for each treatment. In vivo biocontrol trials were conducted twice to ensurereproducibility. Pots were then placed in a fully randomizedcomplete 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 rootrot 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) wascalculated 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 ofwheat plants was also evaluated by measuring the shoot and root lengths, andthe dry weight of healthy plants. Statistical analysis Three replications were performed for each experiment (10 replicates forin vivo trials) and values represent the mean ± standard deviation. Data were subjected to one-wayanalysis of variance (ANOVA).

When the F-statistic was significant, Tukey’spost hoc test (P = 0.05) was used to separate means. Results Antagonistic activities Of the 16 actinobacterial isolates, seven(43.8%) showed positive antagonistic activities against all the fungi. Tenisolates (62.

5%) revealed antagonistic activity against at least three of thefive soil-borne phytopathogenic fungi tested (Table 1), with the most strikingantagonistic activity against Fusarium oxysporum f. sp. radicis-lycopersiciand 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 inhibitionwas obtained by Streptosporangium becharense SG1. Hydrogen cyanide, siderophore productionand chitinolytic activity The results of HCN and siderophoreproduction, and chitinolytic activity by the four selected antagonisticactinobacteria (strains CAR2, SG1, ZLT2 and MB29) are given in Table 2. Allisolates can produce HCN. Siderophores were produced by three isolates.

Alltargeted actinobacteria showed positive results for chitinolytic activity.Indole-3-acetic acid production andphosphate solubilization ability Three of the four isolates tested produced IAA in YT broth, with theisolate S. becharense SG1 showing the best production (Table 2). Our results demonstrated that all fouractinobacteria tested dissolved phosphorus from tricalcium phosphate and aluminium phosphate sources (Table 2), and only the isolate Saccharothrixlongispora 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 andnon-autoclaved soils (Figure1(A), Figure 2(B),(C)). This proves the virulence of the pathogen and thehigh sensitivity of durum wheat cv. vitron.

Coatingwheat seeds with spores of antagonistic actinobacteria and chemical treatmentwith Dividend® significantly (P < 0.05) decreased thedisease incidence, which was more noticeable in non-autoclaved soil than inautoclaved soil (Figure 1(A)). Compared to the positive control and with reference totheir antagonistic activities, the four actinobacteria selected showedbiocontrol effects on F.

culmorum root rot in vivo. Bacterization of wheat seeds significantly (P <0.05) reduced the disease severity index.

Compared to untreatedwheat seeds in non-infected soils (negative control), the isolate S. becharenseSG1 achieved the highest effect in promoting growth of wheat seedlings. Itsignificantly (P < 0.05) increased the shoot length from 15.

88 cm to21.58 cm (Figure 1(B)), root length from 7.11 cm to 12.

62 cm (Figure 1(C)) anddry weight from 0.26 g to 0.7 g (Figure 1(D)). Discussion Several studies have already reported that the antagonist Streptomycesspecies can be considered as active against numerous phytopathogenic fungi,such as F.

culmorum,F. oxysporum f. sp. radicis-lycopersici, R. solani,and Bipolaris sorokiniana (Yekkour et al.,2012; Goudjal et al., 2014; Zamoum et al., 2015), and have been suggested for use as biocontrolagents, or involved in the in vivo biocontrolof the wheat root rot caused by F.

culmorum (Toumatia et al., 2015). The study by El-Tarabily, Hardy, Sivasithamparam, Hussein & Kurtboöke (1997) was the first to provide evidenceof S. albidum in the biocontrol of Pythium coloratum. Theyreported that the mechanism involved in disease reduction appeared to beantibiosis by production of non-volatile antifungal compounds.

Antifungalcompounds from actinobacteria may facilitate the biocontrol of plant diseasesbut this does not mean that it is the only mechanism by which biocontrol occurs(El-Tarabily et al. 1997; Franco-Correaet al. 2010).Our results showed that all isolates can produce HCN. This volatileantifungal compound can inhibit growth of F. culmorum and reduceroot rot disease as noted by Aydi-Benabdallah, Jabnoun-Khireddine, Nefzi,Mokni-Tlili, & Daami-Remadi (2016). Our findings showed that S.becharense SG1 isolated from Saharan soil (Chaabane Chaouch et al.

, 2016b)produced HCN and significantly reduced the F. culmorum root rotof durum wheat (Fig. 1A). Furthermore, Defago et al. (1990) suggested that HCNproduction worked by inducing resistance in host plants. Nevertheless, resultsby El-Tarabily et al.

(1997) showed that Streptosporangium albidumfailed to produce volatile antifungal compounds. However, to the best of our knowledge, this is the first report showingHCN production by a Streptosporangium species.Siderophores were produced by three isolates (Table 2). These low molecular weight compounds can solubilize and sequester iron fromthe soil (Sadeghi et al., 2012).Siderophores are secreted by manyactinobacterial genera, such as Streptomyces (Zamoum et al., 2015), andpermit the acquisition of ferric ion, thus inhibiting phytopathogen growth bycompetition for iron (Ramadan et al.

, 2016). The possible association of siderophore production with the biocontrolability of actinobacteria has been reported by Cao, Qiu, You, Tan & Zhou (2005). Our findings showing siderophore productionby S. becharense SG1 are consistent with those of Sudisha et al.(2016), who reported siderophore production by Streptosporangium roseum SJ_UOM?18?09.All isolates showed positive results for chitinolytic activity, which can be involved in the cell wall degradation of several phytopathogenicfungi. However, many authors have reported the potential of actinobacteria producing chitinase for the biocontrolof F.

oxysporum f. sp. radicis-lycopersici, F.oxysporum f. sp. lini, F.

culmorum and Botrytis cinerea in situ (Goudjal, Zamoum, Sabaou, Mathieu , 2016; Das, Kumar, Kumar, Solanki, Kapur, 2017).Recording to our results, the isolate S. becharense SG1show the best production of IAA.

This phytohormone improves the growth of plants by increasing seed dry weight, seedling elongation, and germinationrate (Goudjal et al., 2014). Several actinobacterial species havealready been reported to produce IAA but this is the first report highlightingIAA production by a species from the genus Streptosporangium.

Another mechanism by which actinobacteria play an important role in the improvementof plant growth is the solubilization of inorganic phosphate as reported by Hamdali et al. (2008). The overall growth of plants is affected by theavailability of essential plant nutrients such as phosphorus (P) (Hamdali etal., 2008).

Several bacterial, fungal and actinobacteria strains have beenrevealed to be phosphate solubilizing organisms (Khan, Almas & Ees, 2014). They convert insoluble forms ofphosphate, such as tricalcium phosphate (Ca3(PO4)2),aluminium phosphate (AlPO4) and iron phosphate (FePO4),to soluble phosphorus forms (Khan et al., 2014).The isolate S. becharense SG1 obtained the highest amount ofdissolved phosphorus from tricalcium phosphate.

These Franco-Correa etal. (2010) demonstrated high activities of actinobacteria in thesolubilization of tricalcium phosphate. Furthermore, Mba (1997) reported similarresults for the solubilization of inorganic phosphate by Streptosporangium species.Accordingto our results, surface treatment of seeds with the control chemical agentshowed a marked protective effect against F. culmorum root rot.

Toumatia et al. (2015) obtained similar results, indicating that chemical treatment of wheat seedswith Tebuconazole is effective in controlling F. culmorumdisease. However, the massive use of these chemical pesticides can lead toenvironmental pollution, which is a major worry in agricultural production (Shimizu, 2011).The strongest biocontrol potential in vivo was obtained by S.

becharense SG1 (Figure 2(A)). This suggests that antibiosis is a factorthat can be involved in biocontrol insitu, and that the production of HCN, siderophoresand chitinases may also be effective mechanisms for controlling F. culmorum root rot (Franco-Correa et al., 2010). Additionally, biocontrol of root rot in vivo may be affected bymany factors besides nutrient availability, water status, soil morphology, soiltemperature, pH value, and interactions with indigenous soil microbes (Dhanasekaran et al., 2005).

Our findings show that the biocontrol effect of F.culmorum is more marked in non-autoclaved soil, which suggests the presence of a synergic effect between the antagonisticactinobacteria and soil indigenous microflora. Similarresults have been reported by Errakhi, Bouteau, Lebrihi & Barakate (2007), who highlighted the effect of soil microflora in controlling Fusariumroot rot of sugar beet.Biocontrol of plantdiseases is often associated with promotion of plant growth (Shimizu, 2011).The isolate S.

becharense SG1 presented the highest effect inpromoting growth of wheat seedlings. It increased the shoot length, root lengthand dry weight. Our results are consistent with those of Zamoum etal. (2015), who reported thatseveral actinobacteria may have a suppressive effect of Fusarium rootrot disease.  Furthermore, the efficacyof Streptosporangium species in the biocontrol of” Pythium coloratum and Sclerospora graminicola has been reportedby El-Tarabily et al.

(1997) and Sudisha et al. (2016).However, as far as we know, this is the first work reporting the efficacy of S.becharense SG1 in the biocontrol of F. culmorum root rotdisease. S.

becharense is a new species of Streptosporangiumdiscovered very recently by Chaabane Chaouch et al. at our laboratory (2016b)and no study of its efficacy in biocontrol has yet been carried out.The strain SG1 showedthe best results for all attributesdetermined in our study.

Thus, it showed the greatest effect in the biocontrol of F. culmorum in vivo and the highestplant-growth-promoting activities on durum wheat (cv. vitron).

This is the firstreport highlighting such properties for the rhizospheric actinobacterium S.becharense SG1, it has proved to be a powerful approach to exploit actinobacterialcommunities in crop enhancement. AcknowledgementsWe thank Susan Becker,translator and native English speaker, for her kind contribution in revisingthe English of this article. Disclosure statementNo potential conflict of interest wasreported by the authors.