The potential of antagonistic moroccan Streptomyces isolates for the biological control of damping-off disease of pea (Pisum sativum L.) caused by Aphanomyces euteiches

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Legumes are very important to agriculture as they fix nitrogen in association with rhizobia, thereby reducing the need for fertilizer inputs. Economically, they are an important source of protein and other nutrients for human needs (Iqbal, Khalil, Ateeq, & Sayyar, 2006; Maphosa & Jideani, 2017). However, many pathogens cause major yield losses in legumes. Soilborne oomycete pathogens attack roots of legumes, limit nutrient uptake and cause the damping-off and/or root rot disease complex resulting in the death of plants (Attard et al., 2014; Black, 2000). The soilborne pathogenic oomy-cete, Aphanomyces euteiches, has been reported in most pea growing

areas of North America, northern Europe, Australia, New Zealand and Japan (Heyman, 2008; Wakelin, McCarthy, Stewart, & Walter, 1998).

Damping-off and root rot are caused in saturated soil conditions when A. euteiches infects seeds or the root of pea plants via zoospores. After an early attack, the plants often collapse and die. The intensity of symptoms of root rot varies with the environmental conditions (Gaulin et al., 2008; Wakelin, Walter, Jaspers, & Stewart, 2002). There are currently no efficient fungicides or other treatments available for the control of this pathogen. Some chemicals effectively suppress A. euteiches under controlled conditions but have limited protective effects in field trials (Wu et al., 2018). Moreover, the oospores can

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sativumĺő1-v;70Aphanomyces euteiches

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1_{Laboratory of Biology and Biotechnology}

of Microorganisms, Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech, Morocco

2_{Laboratory of Interaction}

Plant-Microorganisms, Department of Biology, University of Fribourg, Fribourg, Switzerland

ƒ_{Agrobiosciences & Fertilizers}

Program, University Mohammed IV Polytechnic (UM6P), Benguerir, Morocco

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Mustapha Barakate, Laboratory of Biology and Biotechnology of Microorganisms (LBBM), Department of Biology, Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech, Morocco.

Emails: mbarakate@uca.ma; mbarakate@ hotmail.com

m7bm]bm=oul-|bom

Swiss Government Excellence Scholarships - the Federal Commission for Scholarships for Foreign Students FCS, Grant/Award Number: 2016.0822; the Ministry of National Education, Professional Training, Higher education and Scientific Research of Morocco, Grant/Award Number: Ƒƕ&ƑƏƐƔ

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Three hundred and fifty-nine isolates of actinobacteria collected from different Moroccan soils were evaluated for their in vitro antimicrobial activity against the oomycete pathogen Aphanomyces euteiches, the causal agent of damping-off of pea and other legumes. Eighty-seven isolates (24%) had an inhibitory in vitro effect against A. euteiches. Fourteen bioactive isolates with the greatest inhibitory effect against A. euteiches and no inhibitory effect on plant beneficial rhizobia were tested for their ability to protect pea seeds and seedlings against the damping-off disease using culture supernatants or spore suspensions as treatments. The two most ruo|;1|b; bvoѴ-|;vķ ƑƐ -m7 ƐƔķ vb]mb=b1-m|Ѵ u;71;7ķ 1olr-u;7 |o m|u;-|;7 1om|uoѴrѴ-m|vķ7-lrbm]Ŋo==0ƒƒѷ-m7Ɠƕѷķu;vr;1|b;Ѵĺ$_;|o0bo-1|b;bvoѴ-|;v were classified as species of the genus Streptomyces based on 16S rDNA analysis and morphological and chemical characteristics.

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16S rDNA, actinobacteria, Aphanomyces euteiches, biocontrol, damping-off, pea, Streptomyces

http://doc.rero.ch

Published in "Journal of Phytopathology 167(2): 82–90, 2019"

which should be cited to refer to this work.

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persist in soil for 10 years excluding crop rotation as mean of control (Sauvage et al., 2007). Alternative strategies have been developed to fight against this pathogen, but only the biological control strategies are promising for sustainable protection. Bacillus, Paenibacillus and Pseudomonas strains (P. fluorescens and P. cepacia) were reported to control A. euteiches (Parke, Joy, & King, 1991; Wakelin et al., 2002). However, no previous study had tested the potential of actinobacteria to control diseases caused by A. euteiches.

Actinobacteria are well known to produce several kinds of bio-active secondary metabolites that are used in biological control of several plant pathogens (Elbendary et al., 2018; Ganesan et al., ƑƏƐƕĸ + ;| -Ѵĺķ ƑƏƐƔőĺ rruobl-|;Ѵ ѶƏѷ o= |_; hmom 1oll;u-cial antibiotics used in medicine and agriculture have initially been isolated from actinobacteria, and especially from strains of genus Streptomyces (Sanglier, Haag, Huck, & Fehr, 1996), that proved to be a rich source of antibiotics. Furthermore, Streptomyces have been reported in several studies as biological control and showed poten-tial to control a wide range of plant pathogenic bacteria, fungi and oomycetes by producing diverse bioactive substances (Baz, Lahbabi, et al., 2012; Baz, Tran, et al., 2012; Errakhi, Lebrihi, & Barakate, 2009; Goudjal, Zamoum, Sabaou, Mathieu, & Zitouni, 2016; Samri ;| -Ѵĺķ ƑƏƐƔőĺ $_;v; 0-1|;ub- Ѵbh; l-m o|_;u lb1uoou]-mbvlv 1-m also promote plant growth by producing phytohormones (Goudjal ;|-ѴĺķƑƏƐƒőķ0blruo;7r_ovr_-|;lo0bѴb-|bomŐ-l7-Ѵbķ-=b7bķ (buoѴѴ;ķş_7o1_ķƑƏƏѶő-m70ruo71|bomo=vb7;uor_ou;v=ou iron acquisition (Macagnan, Romeiro, Pomella, & Souza, 2008). The aim of the present study was to analyse the potential of actinobac-teria isolates to protect legumes against A. euteiches. To this end, a Moroccan collection of actinobacteria was screened for antagonistic properties against A. euteiches and no inhibitory effect on rhizobial bacteria. The ability of selected antagonistic strains to reduce damp-ing-off symptoms was further analysed on pea seeds, and their tax-onomic affiliation was determined.

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Morocco) were used in the present study in order to avoid selected bioactive actinobacteria that could negatively affect the symbiotic relationship between legumes and rhizobia.

ƑĺƒՊ|ՊAphanomyces euteiches

The A. euteiches strain ATCC 201684 was kindly provided by Prof. Elodie Gaulin (Laboratoire de recherche en sciences végétales, University of Toulouse III Paul Sabtier, France). The pathogen was v|ou;7bm1ouml;-Ѵ-]-u-|ƓŦĺ

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-1|bb|-]-bmv|A. euteiches

ƑĺƔՊ|Պ"1u;;mbm]o=v;Ѵ;1|;70bo-1|b;-1|bmo0-1|;ub-

bvoѴ-|;v=oubm_b0b|bomo=u_bo0b-To avoid that selected bioactive actinobacteria isolates would negatively affect the symbiotic relationship between legumes and rhizobia, the active isolates were screened against four different strains of rhizobia, using the method of agar disc-diffusion technique as described by Bauer, Kirby, Sherris, and Turck (1966). Agar plugs of active actinobacteria isolates (9 mm in diameter) were placed on solid yeast extract mannitol medium (K₂₄ƏĺƔ]ņĸ]"₄ 7H₂ 0.2 g/L; NaCl 0.1 g/L; yeast extract 0.6 g/L; mannitol 10 g/L; agar ƐƔ]ņőru;bovѴv;;7;7b|_-ƐƏ &ņlѴvvr;mvbomo=bm7bb7-Ѵ u_bo0b-v|u-bmvĺѴ-|;v;u;h;r|bm-u;=ub];u-|ouŐƓŦő=ou-|Ѵ;-v| Ɠ_u-m7;u;|_;mbm10-|;7-|ƑѶŦbm|_;7-uh=ouƒŋƓ7-vĺ

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Actinobacteria isolates that inhibited mycelia growth of A. euteiches and did not have any activity against rhizobia strains were tested for their ability to control damping-off of pea seeds caused by A. euteiches using spore suspensions or culture filtrates. For spore production, -1|bmo0-1|;ub-bvoѴ-|;v;u;]uomom;mm;||-]-ul;7bl-|ƑѶŦķ and after 14 days of incubation, spores were harvested in distilled water and counted with Malassez haemocytometer and the density was adjusted to 108_{spores/ml. Culture filtrates were obtained by}

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culturing agar plug of purified selected actinobacteria isolates in ƔƏlѴ o= ;mm;|| Ѵbtb7 l;7bl -m7 -=|;u bm10-|bom -| ƑѶŦ =ou 7 days. The supernatants were harvested after centrifugation ŐƐƔlbm-|ƑƔķƑƏƏgő-m7h;r|-|ƓŦm|bѴv;ĺ

ƑĺƕՊ|Պ,oovrou;ruo71|bomo=A. euteiches

Zoospores of A. euteiches ATCC 201684 were produced by the modified method of Deacon and Saxena (1998). Agar discs of A. euteiches ]uom om l;7bl ;u; 1Ѵ|u;7 bm ƒƏlѴ  0uo|_=ouƐƏ7-v-|ƑƓŦbm|_;7-uhĺ$_;l1;Ѵbl-v_-u;v|;7 and washed three times with autoclaved Volvic water (VW) and bm10-|;7 =ou Ƒ_u -| ƑƓŦ bm ƑƏlѴ o= -|o1Ѵ-;7 () -=|;u ;-1_ wash. The production of zoospores was initiated by incubation of |_; l1;Ѵbl -v_;7 bm ƑƏlѴ () =ou ƐƖ_u -| ƑƓŦ bm |_; 7-uhĺ Zoospores were harvested in autoclaved VW, and the density was adjusted to 10Ɣ_{zoospores/ml using a Malassez haemocytometer.}

ƑĺѶՊ|Պ==;1|o=v;Ѵ;1|;7-1|bmo0-1|;ub-bvoѴ-|;vom

A. euteiches7-lrbm]Ŋo==o=r;-v;;7v-m7v;;7Ѵbm]v

Seeds of Pisum sativum L. were surface-sterilized with 4% sodium _ro1_Ѵoub|; =ou Ɣlbmķ -v_;7 |_u;; |bl;v b|_ v|;ubѴ; 7bv|bѴѴ;7 -|;u-m7-buŊ7ub;7Ő;mb7bu;;|-ѴĺķƑƏƐƔőĺboѴo]b1-Ѵ1om|uoѴv|7b;v were performed as described by Sadeghipour and Abbasi (2012) with some modifications. Disinfected pea seeds were immersed in flasks containing culture supernatant or spore suspension of each selected -1|bmo0-1|;ub- bvoѴ-|; =ou ƒƏlbm b|_ - vѴo v_-hbm]ĺ ;|ub 7bv_;v were covered by Whatman paper Number 1, and pea seeds were v0l;u];7bmƔlѴo=ƐƏƔ_{zoospore/ml of A. euteiches=ouƔlbmĺ$;m}

v;;7vr;u7bv_;u;bm10-|;7-|ƑƓŦ-m7ƕƔѷo=_lb7b|bm|_; dark. Seed germination and seedling stand were determined after 1 week (inhibition of germination, disease severity index, seedling fresh weight and seedling root length). Tests were repeated three times and included four replicate dishes for each treatment.

ƑĺƖՊ|Պour_oѴo]b1-Ѵķr_vboѴo]b1-Ѵ-m7

1_;lo|-omolb11_-u-1|;ub-|bomvo=v;Ѵ;1|;7

-m|-]ombv|b1-1|bmo0-1|;ub-bvoѴ-|;v

The taxonomic classification of the two selected antagonistic actinobacteria isolates was evaluated as described in the International Streptomyces Project (ISP) (Shirling & Gottlieb, ƐƖѵѶőĺ ѴѴ 1Ѵ|u;v ;u; bm10-|;7 -| ƑѶŦ =ou ƕ7-vķ -m7 |_; colour of the aerial spore mass of colonies and the colour of the growth medium were observed according to the scale adopted by Nonomura (1974). The production of melanoid pigments was tested on peptone yeast extract iron agar (ISP6) and tyrosine agar (ISP7) (Shirling & Gottlieb, 1968). The assimilation of carbohydrates as sole carbon sources at concentration 1% (w/v) was studied using the ISP9 medium (Shirling & Gottlieb, 1968). The chemical analyses of the diaminopimelic acid isomer were performed as adopted by Becker, Lechevalier, Gordon, and Lechevalier (1964).

ƑĺƐƏՊ|Պ ru;r-u-|bomķ-lrѴb=b1-|bom-m7

v;t;m1bm]o=|_;Ɛѵ"u

The DNA of the two selected actinobacteria isolates was extracted -v 7;v1ub0;7 0 oroo7 ;| -Ѵĺ ŐƐƖѶƔőĺ $_; Ɛѵ" u -v !Ŋ -lrѴb=b;7vbm]|_;rubl;uvƑƕ=ŐƔனŊ$$$$$$Ŋƒனő -m7ƐƓƖƑuŐƔனŊ$$$$$$$$ŊƒனőŐ-m;ķƐƖƖƐőĺ!;-1|bom conditions were as follows: initial denaturation of the target DNA at ƖƕŦ=ouƓlbmķ=oѴѴo;70ƒƔ11Ѵ;vŐƖƓŦ=ouѵƏvķƔƑŦѵƏv-m7 ƕƑ=ouƔlbmőĺlrѴb=b1-|bomruo71|v;u;v;r-u-|;7om-ƏĺѶѷŐņő agarose gel and stained with ethidium bromide. The primers used for sequencing were the same as mentioned above. Sequences were compared by blast search for similarity with sequences present in genomic database at NCBI (http://www.ncbi.nih.gov/). A phylogenetic tree based on 16S rDNA sequences was constructed with the neighbour-joining algorithm in MEGA version 6.0. The nucleotide v;t;m1;o=|_;|obvoѴ-|;vƑƐ-m7ƐƔ_-vruob7;7;m-mh 11;vvbom ml0;uv -v "&ƓƒƐƖƕƕƏ Streptomyces MH820126 and "&ƓƓѶѶƒƔƓStreptomyces MH828291, respectively.

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All collected quantitative data were analysed statistically by variance -m-Ѵvbv|;v|Ő(őķ-m7-ѴѴml;ub17b==;u;m1;vbm|_;7-|-;u; considered significantly different at the probability level of pƽƏĺƏƔĺ

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bm_b0b|bomo=A. euteiches0|mo-1|bb||o-u7v

ƒĺƑՊ|Պbo1om|uoѴo=A. euteiches7-lrbm]Ŋo==om

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resulted in a germination rate of about 92% (culture supernatant) and 88% (spore treatment) as compared to 6% in the untreated con-trol (Table 1). The results of the diseases severity index (DSI) show |_-| ƑƐ -v lou; ;==b1b;m| |_-m ƐƔ bm ;b|_;u |r; o= |u;-|-l;m|Ő b]u;ƒ-őĺ$_; "o=m|u;-|;71om|uoѴv;;7Ѵbm]v-vƖƑĺƔѷĺ $_; bmo1Ѵ-|bom o= r;- v;;7v b|_ 1Ѵ|u; vr;um-|-m| o= ƑƐ 7;1u;-v;7 vb]mb=b1-m|Ѵ |_; " |o ƑƔĺƓѷķ _;u;-v |u;-|l;m| o= v;;7vb|_ƑƐvrou;vѴ;7|o- "o=ƒƕĺƖѷĺ"blbѴ-uѴķ|_;1Ѵ|u; vr;um-|-m|o=|_;ƐƔ-vlou;;==b1b;m|bm7;1u;-vbm]|_; " ŐƒƕĺѶѷő|_-m|_;-rrѴb1-|bomo=ƐƔvrou;vŐ "Ɣƒĺѵѷőĺ$_;-rrѴb-cation of different treatments of the two tested actinobacteria iso-lates had a significant effect on the seedling fresh weight compared |o |_; bmo1Ѵ-|;7 1om|uoѴ Ő b]u; ƒ0őĺ $_; -;u-]; =u;v_ ;b]_| o= ƑƐŊ|u;-|;7 v;;7Ѵbm]v -v ƕĺƕ] Ő1Ѵ|u; vr;um-|-m| |u;-|l;m|ő and 6.9 g (spore treatment) compared to 4.0 g of untreated control v;;7Ѵbm]vĺ$_;ƐƔbvoѴ-|;-vѴ;vv;==b1b;m|b|_-;u-];v;;7Ѵbm] ;b]_|vo=ѵĺƔ-m7Ɣĺƕ]ķu;vr;1|b;Ѵĺvv_ombm b]u;ƒ1ķ|u;-|-l;m|vb|_ƑƐouƐƔ]u;-|Ѵblruo;7|_;v;;7Ѵbm]uoo|Ѵ;m]|_ compared to controls infected by A. euteiches alone. Treatment with

1Ѵ|u; =bѴ|u-|;v o= ƑƐ -m7 ƐƔ Ѵ;7 |o -m -;u-]; uoo| Ѵ;m]|_ o=ѵĺƒ-m7ƔĺƐ1lķu;vr;1|b;Ѵķ1olr-u;7|o-Ѵ;m]|_o=ƏĺƔ1lbm untreated inoculated controls. Together, the results presented in b]u;ƒvrrou||_;1om1Ѵvbom|_-|0o|_ķ|_;ƑƐ-m7|_;ƐƔ isolates, protect pea seedlings against A. euteiches.

ƒĺƒՊ|Պ$-omolb11_-u-1|;ub-|bomo=|_;

v;Ѵ;1|;7bvoѴ-|;v

$_;|o-m|-]ombv|b1ruolbvbm]bvoѴ-|;vƑƐ-m7ƐƔ;u;|;v|;7 for taxonomical identification using morphological, cultural, physi-ological and biochemical criteria as well as other features (Table 2). The morphology of colonies and the mycelium colour (aerial and substrate) were determined on ISP media. The analysis showed that |_; |o v|u-bmvķ ƑƐ -m7 ƐƔķ _-; 7b==;u;m| ruo=bѴ;v bm 7b==;u-;m||;v|l;7b-ĺm"ƒl;7blķ|_;1oѴouo=|_;-;ub-Ѵl1;Ѵblo= ƑƐ-v]u;;mbv_ķ0||_;ƐƔ1oѴouo=|_;-;ub-Ѵl1;Ѵbl-v _b|;ĺ$_;v0v|u-|;l1;Ѵbl1oѴou=ouƑƐbm"Ƒ-v0uomķ _;u;-vb|-v;ѴѴo=ouƐƔŐ$-0Ѵ;Ƒőĺ$_;u;vѴ|vv_o;7-Ѵvo  & ! Ɛ Պ (a) Percentage of actinobacteria isolates able to inhibit the growth of Aphanomyces euteiches mycelium in Petri dish test, (b) percentage of active actinobacteria isolates against A. euteiches that not able to cause any damage to rhizobia in Petri dish test by using the confrontation method

& ! Ƒ Պ Screening method to isolate: (a) isolates able to inhibit the growth of Aphanomyces euteiches in PGA medium, (b) actinobacteria isolates with an in vitro inhibitory activity against A. euteiches but without any activity against rhizobia

(a) (b)

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|_-|ƑƐ-m7ƐƔ7omo|ruo71;l;Ѵ-mbmrb]l;m|om"ѵ-m7 ISP7 media. Both selected strains were able to use sucrose, galac-tose, lacgalac-tose, mannose, mannitol, arabinose, frucgalac-tose, dextrin and

maltose as sole carbon sources. However, they were not able to use inositol, raffinose and rhamnose (Table 2).

& ! ƒ Պ uo|;1|b;;==;1|o=|_;-rrѴb1-|bomo=vr;um-|-m|Ѵbtb71Ѵ|u;-m7vrou;vvr;mvbomvo=-m|-]ombv|b1-1|bmol1;|;vƑƐ -m7ƐƔomr;-v;;7vrubou|obm=;1|bomb|_ƐƏƔ_{spores/ml of Aphanomyces euteiches zoospores. (a) Effect of the treatment on disease}

v;;ub|bm7;Ő "őķŐ0ő;==;1|o=|_;|u;-|l;m|omv;;7Ѵbm]v=u;v_;b]_|ķŐ1ő;==;1|o=|u;-|l;m|omv;;7Ѵbm]uoo|Ѵ;m]|_ĺƑƐ"rĹ vr;um-|-m|Ѵbtb71Ѵ|u;o=ƑƐĸƑƐ"vĹvrou;vvr;mvbomo=ƑƐĸƐƔ"rĹvr;um-|-m|Ѵbtb71Ѵ|u;o=ƐƔĸƐƔ"vĹvrou; vvr;mvbomo=ƐƔ

$u;-|l;m||r; "r;um-|-m|Ѵbtb71Ѵ|u; "rou;|u;-|l;m| om|uoѴ

Isolate

ƑƐ ƖƑĺƔѷƼƒĺƒ ѶƕĺƔѷƼƒĺƑ ѵĺƒѷƼƑĺѵ

ƐƔ ƖƑĺƔѷƼƐĺѶ ѶƕĺƔѷƼƑĺƔ

$ Ɛ Պ The protective effect of bmo1Ѵ-|bomb|_ƑƐ-m7ƐƔ treatment on the germination rate of pea seeds infected with 10Ɣ_{spore/ml of} Aphanomyces euteiches

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$_; 1;ѴѴ -ѴѴ r;r|b7o]Ѵ1-m o= 0o|_ bvoѴ-|;v ƑƐ -m7 ƐƔ contained LL-diaminopimelic acid and glycine, indicating that they had a chemotype I cell wall (Lechevalier & Lechevalier, 1970), which is characteristic of the genus Streptomyces. The amplification and sequence analysis of 16S rDNA confirmed the assignment of both isolates to the genus StreptomycesŐ b]u;vƓ-m7ƔőĺƐƔ-m7ƑƐ bvoѴ-|;v ;_b0b|;7 ƖƖĺƕƖѷ -m7 ƖƖĺƒƔѷ v;t;m1; b7;m|b|ķ u;vr;1-tively, to Streptomyces hydrogenans NBRC 13475T_{. The comparison of}

cultural, morphological and biochemical criteria, however, showed several differences. Streptomyces hydrogenans NBRC 13475T_in

bm|_;v-l;l;7blŐ$-0Ѵ;ƒőĺ

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There is a growing interest in endophytic and soil microorganisms that can protect plants against pathogens and thus may contribute to sustainable agriculture. The protective effect of such biocontrol microorganisms is often based on the production of compounds that directly interfere with spore germination and pathogen growth Őolr-m|ķ ==ķ o-hķ Ѵ;ķ ş -uh-ķ ƑƏƏƔĸ u;b|-vķ ;ulb7-ķ ş De, 1991). Biocontrol strains can also have positive indirect effects on disease resistance by stimulation of plant defence mechanisms (Lehr, Schrey, Hampp, & Tarkka, 2008; Prévost, Couture, Shipley, Brzezinski, & Beaulieu, 2006). Actinobacteria are widespread soil microorganisms known to produce a large number of bioactive compounds including antibiotics used in medicine. Bioactive actino-bacteria from indigenous Moroccan habitats were reported before $ Ƒ Պ

uo|_r;u=oul-m1;-m7vol;r_;mo|rb11_-u-1|;ubv|b1vo=v|u-bmƑƐ-m7ƐƔom7b==;u;m|l;7b-;7bl

uo|_ ;ub-Ѵl1;Ѵbl "0v|u-|;_r_-; "oѴ0Ѵ;rb]l;m|

ƑƐ ƐƔ ƑƐ ƐƔ ƑƐ ƐƔ ƑƐ ƐƔ

ISP 2 +++ +++ White/brown White Brown Yellow None None

"ƒ +++ +++ Greenish White Brown Brown None None

ISP 4 +++ +++ White White Greenish Yellow None None

ISP 6 +++ +++ White/brown White/yellow Brown Brown None Brown

ISP 7 +++ +++ Greenish White/pink Brown Yellow/brown None Brown

BENNETT +++ +++ White/yellow White White White-grey None None

GYEA +++ +++ White/greenish White Brown White Brown Brown

Note. (+++) Good growth.

& ! Ɠ Պ ;b]_0ouŊfobmbm]r_Ѵo];m;|b1|u;;0-v;7omƐѵ"u v;t;m1;vv_obm]|_;u;Ѵ-|bomv_brv0;|;;mv|u-bmvƐƔ-m7 related bacteria from the GenBank database. The numbers in parentheses represent the sequence accession numbers in GenBank. The scale bar represents 0.02 substitutions/nucleotide position

BA15

Streptomyces hydrogenans

NBRC 13475

T

(AB184868)

Streptomyces koyangensi

s VK-A60

T

(AY079156)

Streptomyces intermediu

s NBRC 13049

T

(AB184277)

Streptomyces prasinosporus

NRRL B-12431

T

(DQ026655)

Streptomyces cinereospinus

NBRC 15397

T

(AB184648)

Streptomyces poonensis

NBRC 134805

T

(AB184437)

Streptomyces purpurascens

NBRC 13077

T

(AB184859)

Streptomyces luteogriseus

NBRC 13402

T

(AB184379)

Streptomyces geysiriensis

NBRC 15413

T

(AB184661)

Streptomyces pilosus

NBRC 12807

T

(AB184161)

Streptomyces flavoviridis

NBRC 12772

T

(AB184842)

100

99

59

76

57

74

71

50

36

0.005

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(Barakate et al., 2002; Baz, Lahbabi, et al., 2012; Baz, Tran, et al., ƑƏƐƑĸ-l7-Ѵb;|-ѴĺķƑƏƏѶĸ"-lub;|-ѴĺķƑƏƐƔőĺ-mv|7b;v=o1v;7 on the role of actinobacteria as biocontrol agents against a large ml0;u o= r_|or-|_o];mv Őov|-ķ ,11_bķ ş ;Ѵoķ ƑƏƐƒĸ o7f-Ѵ et al., 2016; Shrivastava, Kumar, & Yandigeri, 2017). However, no previous studies reported the use of actinobacteria against legume diseases caused by A. euteiches. Therefore, we investigated in this study the potential of actinobacteria isolated from Moroccan eco-systems for biological control of the damping-off disease of pea caused by A. euteichesĺ mѴ ƒĺѶѷ o= |_; v1u;;m;7 -1|bmo0-1|;ub- isolates were antagonistic towards A. euteiches without causing

damage to rhizobia. Isolates with specific antioomycete activity ;u; u;rou|;7 ru;bovѴ Ő;; ;| -Ѵĺķ ƑƏƏƔőĺ $_bv =bm7bm] 1om=bulv that actinobacteria, especially the genus of Streptomyces, have an important potential to produce bioactive compounds such as ami-noglycosides, which are selectively active against several oomycete pathogens. Seed treatment with supernatants of liquid cultures and spore suspensions used in the present study dramatically increased pea seed germination rates in the presence of A. euteiches from 6% bm|_;m|u;-|;71om|uoѴ|oƖƑĺƔѷ-m7ѶƕĺƔѷķu;vr;1|b;Ѵĺ"blbѴ-u;=-=;1|v;u;u;rou|;7bmru;bovv|7b;vŐѶƏĺƔѷķ-m7;Ѵvl-mķ!-==;Ѵķ ;v|;uķ)m7;uѴb1_ķşu-ķƐƖƖƏĸƖƑѷķ)-h;Ѵbm;|-ѴĺķƑƏƏƑĸƑƔѷķ "-7;]_bķ;vv-mķvh-ubķ]_b]_bķş"_-_b7bomf-uķƑƏƏѵőĺuu;vѴ|v v_o;7|_-||_;-rrѴb1-|bomo=ƑƐouƐƔ1-v;7-1omvb7;u-0Ѵ; decrease in the diseases severity index (DSI) compared to untreated 1om|uoѴvĺ";;7|u;-|l;m|7;1u;-v;7|_; "|o-Ѵ;v0;|;;mƑƔѷ -m7 ƔƓѷ 7;r;m7bm]om |_; |r;o= |u;-|l;m|ĺ olr-u-0Ѵ; u;vѴ|v ;u; u;rou|;7 0 -lb7ķ _-|ķ "o=bķ _-|ķ -m7 vb= ŐƑƏƐƒő _o showed that Trichoderma and Pseudomonas fluorescens strains can 7;1u;-v;|_; "|oƐƑѷ-m7ƐƔѷķu;vr;1|b;Ѵĺ|u;l-bmvmhmom whether the decrease in DSI is only caused by the production of antimicrobial compounds or by an additional effect on the accumu-Ѵ-|bom o= rѴ-m|Ŋ7;ub;7 7;=;m1; 1olrom7vĺ $u;-|l;m| b|_ ƑƐ -m7ƐƔu;vѴ|;7bm;m_-m1;7]uo|_o=r;-v;;7Ѵbm]v1olr-u;7|o unprotected controls. Treatment with the two selected actinobacte-ria clearly increased both the pea seedling weight and root length. Wakelin et al. (2002) reported a significant promotion of plant and root weight by inoculation of pea seed with spore-forming bacte-ria after infection with A. euteiches. Sadeghi et al. (2006) reported enhanced plant weight by using Streptomyces strains as biocontrol agents against damping-off and root rot caused by Rhizoctonia solani. & ! Ɣ Պ ;b]_0ouŊfobmbm]r_Ѵo];m;|b1|u;;0-v;7omƐѵ"u v;t;m1;vv_obm]|_;u;Ѵ-|bomv_brv0;|;;mv|u-bmvƑƐ-m7 related bacteria from GenBank database. The numbers in parentheses represent the sequence accession numbers in GenBank. The scale bar represents 0.02 substitutions/nucleotide position

OB21

Streptomyces hydrogenans

NBRC

T

(13475 AB184868)

Streptomyces koyangensis

VK-A60

T

(AY079156)

Streptomyces tacrolimicus

ATCC 55098

T

(FN429653)

Streptomyces luteogriseus

NBRC 13402

T

(AB184379)

Streptomyces purpurascens

NBRC 13077

T

(AB184859)

Streptomyces janthinus

ISP 5206

T

(AJ399478)

Streptomyces flavoviridis

NBRC 12772

T

(AB184842)

Streptomyces geysiriensis

NBRC 15413

T

(AB184661)

Streptomyces prasinosporus

NRRL B-12431

T

(DQ026655)

Streptomyces naganishii

NBRC 12892

T

(AB184224)

Streptomyces cinereospinus

NBRC 15397

T

(AB184648)

99

74

70

90

35

46

40

0.005

$ ƒ Պ _;mo|rb1ruor;u|b;v|_-|v;r-u-|;v|u-bmƑƐ-m7 ƐƔ=uolu;Ѵ-|;7Streptomyces species _-u-1|;ubv|b1v ƑƐ ƐƔ Streptomyces hydrogenans NBRC 13475T "ƒl;7bl Colour of aerial mycelium

Greenish White White

Colour of soluble pigment Brown Brown Use of Sucrose + + ƴ D-Fructose + + ƴ Mannitol + + ƴ Rhamnose ƴ ƴ + NoteĺŐƳőovb|b;u;-1|bomĸŐƴőm;]-|b;u;-1|bomĺ

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|_;u v|7b;v 7;lomv|u-|;7 |_; -0bѴb| o= Streptomyces species to be effective agents for biocontrol of soilborne pathogens such as Rhizoctonia solaniŐ_m]ķ-m]ķş-m]ķƑƏƏƔőķSclerotium rolfsii (Errakhi et al., 2009), Pectobacterium carotovorum and P. atrosepti-cum (Baz, Lahbabi, et al., 2012; Baz, Tran, et al., 2012). Streptomyces species were also reported to control diseases caused by oomy-cete pathogens (Bibi, Yasir, Song, Lee, & Chung, 2012; Rodríguez-Villarreal et al., 2014).

The taxonomical results proved that both selected actinobac-teria isolates belong to the genus Streptomyces. Sequencing of 16S rDNA revealed highest sequence identity (99%) with S. hydrogenans NBRC 13475T _{Őbm7m;uķ mhķ ;vl-mmķ ş "1_lb7|Ŋ$_ol࣐ķ ƐƖƔѶőĺ}

However, cultural, morphological and biochemical studies revealed v;;u-Ѵ 7b==;u;m1;v bm7b1-|bm] |_-| ƑƐ -m7 ƐƔ u;ru;v;m| m; strains of Streptomyces.

The present study demonstrates that the two selected Streptomyces isolates isolated from Moroccan soils showed a good potential to protect pea seedling against the damping-off disease caused by A. euteiches. It remains to be tested how effective this protection is under field conditions.

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This work was financially supported by the excellence grant ŦƑƕ&ƑƏƐƔ --u7;7 0 |_; bmbv|u o= -|bom-Ѵ 71-|bomķ Professional Training, Higher education and Scientific Research of Morocco and a Swiss Government Excellence Scholarships awarded by the Federal Commission for Scholarships for Foreign Students "ņŦƑƏƐѵĺƏѶƑƑĺ

 ! 

Mustapha Barakate _||rvĹņņou1b7ĺou]ņƏƏƏƏŊƏƏƏƐŊƕƖƓƖŊƔƔƖƑ

! ! "

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eng.2018.07.006

+ķĺķ,_-m]ķĺķbķ ĺķ bķ*ĺķbķ+ĺķşblķĺ"ĺŐƑƏƐƔőĺvoѴ-|bom-m7 characterization of actinobacteria from Yalujiang coastal wetland, North China. Asian Pacific Journal of Tropical Biomedicine, 5ķƔƔƔŋƔѵƏĺ _||rvĹņņ7obĺou]ņƐƏĺƐƏƐѵņfĺ-rf|0ĺƑƏƐƔĺƏƓĺƏƏƕ

The potential of antagonistic moroccan Streptomyces isolates for the biological control of damping-off disease of pea (Pisum sativum L.) caused by Aphanomyces euteiches

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http://doc.rero.ch

Published in "Journal of Phytopathology 167(2): 82–90, 2019"

which should be cited to refer to this work.

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BA15

Streptomyces hydrogenans

NBRC 13475

(AB184868)

Streptomyces koyangensi

s VK-A60

(AY079156)

Streptomyces intermediu

s NBRC 13049

(AB184277)

Streptomyces prasinosporus

NRRL B-12431

(DQ026655)

Streptomyces cinereospinus

NBRC 15397

(AB184648)

Streptomyces poonensis

NBRC 134805

(AB184437)

Streptomyces purpurascens

NBRC 13077

(AB184859)

Streptomyces luteogriseus

NBRC 13402

(AB184379)

Streptomyces geysiriensis

NBRC 15413

(AB184661)

Streptomyces pilosus

NBRC 12807

(AB184161)

Streptomyces flavoviridis

NBRC 12772

(AB184842)

100

99

59

76

57

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