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Isolation and evaluation of endophytic Streptomyces endus OsiSh-2

with potential application for biocontrol of rice blast disease
Accepted Article
Ting Xu1†

Email: seesaw0105@outlook.com

Yan Li1†

Email: liyanshine@hnu.edu.cn

Xiadong Zeng1†

Email: zengxiadong@hun.edu.cn

Xiaolu Yang1

Email: yxl_gongzuo@126.com

Yuanzhu Yang2

Email: yzhuyah@163.com

Shanshan Yuan1

Email: yss0911@163.com

Xiaochun Hu2

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/jsfa.7841

This article is protected by copyright. All rights reserved.

Email: hxc@lpht.com.cn
Accepted Article
Jiarui Zeng1

Email: zengjiarui@hotmail.com

Zhenzhen Wang1

Email: wzz@hnu.edu.cn

Qian Liu1

Email: lq1420@hnu.edu.cn

Yuqing Liu1

Email: belindaYiYi@163.com

Hongdong Liao1

Email: liaohongdong@126.com

Chunyi Tong1

Email: freeradical00@163.com

Xuanming Liu1*

*
Corresponding author

This article is protected by copyright. All rights reserved.

Phone: 08673188821721
Accepted Article
Email: xml05@hnu.edu.cn

Yonghua Zhu1*

*
Corresponding author

Phone: 08673188664048

Email: yonghuaz@outlook.com


Equal contributors

1
Hunan Province Key Laboratory of Plant Functional Genomics and

Developmental Regulation, College of Biology, Hunan University, Changsha

410082, Hunan, PR China

2
Yahua Seeds Science Research Institute, Longping High-tech, Changsha

410119, Hunan, PR China

Abstract

BACKGROUND: Biocontrol is a promising strategy in the control of rice blast disease.

In this study, we isolated and characterized a novel antagonist to the pathogen

Magnaporthe oryzae from rice endophytic actinomycetes.

RESULTS: Out of 482 endophytic actinomycetes isolated from rice blast infected and

healthy rice, Streptomyces endus OsiSh-2, exhibited a remarkable in vitro antagonistic

This article is protected by copyright. All rights reserved.

Chemical fungicides treatment and development of resistant rice varieties can effectively control this disease. IAA and ACC deaminase.64%. . partially due to the highly variable of M.oryzae.2 In the context of seeking promising This article is protected by copyright. Rice blast. All rights reserved. Preliminary separation indicated that the methanol extract of OsiSh-2 could suppress growth of pathogen. In addition. rice yield is greatly reduced due to the rice blast disease caused by pathogenic fungi Magnaporthe oryzae. Magnaporthe oryzae. Scanning electron microscopy observation of M. activity.1 Plant resistance (often based on a single gene) is normally not durable in the field.) is the most important food source for over half of the world’s population. CONCLUSION: Endophytic Streptomyces endus OsiSh-2 has potential as a biocontrol agent against rice blast in agriculture. Antagonistic agents. gelatinase. Streptomyces endus OsiSh-2 INTRODUCTION Rice (Oryza sativa L. both fermentation broth of OsiSh-2 and its cell-free filtrates could inhibit growth of M. siderophore. In two-year’s field tests. spraying OsiSh-2 spore solution (107 spores mL-1) is capable of reducing rice blast disease severity by 59. suggesting the presence of active enzymes and secondary metabolites. its major active component was identified as nigericin.oryzae. However. OsiSh-2 is tested positive for PKS-I and NRPS genes and can produce cellulase.oryzae treated by OsiSh-2 Accepted Article revealed significant morphological alterations in hyphae. chemical agents often negatively impacts the environment and even the consumers. protease.1 However. Keywords: Rice endophytes.

researchers have started to isolate untapped microbes from special environments. Plant beneficial microorganisms [biological control agents (BCAs)] have been chosen as such an approach due to their lower toxicity and lack of pathogen resistance. by the secretion of bioactive secondary metabolites to kill the pathogens directly. Several species of actinomycetes. Now. rice endophyte Harpophora oryzae exhibited a unique protection for rice blast disease. and have been proven to be a potential source for biocontrol agents over the last years. amyloliquefaciens FZB24 displayed a significantly lower disease incidence of bacterial leaf blight under This article is protected by copyright. for example.9 Numerous studies have indicated that endophytes are capable of improving the resistance of host plants to adversities. .3 Actinomycetes.11 Endophytic Bacillus subtilis var. such as pathogen challenges. or induce systemic resistance of host plant indirectly.6 and also rice blast. most of these antibiosis actinomycetes are from traditional sources. such as rhizosphere and soil.5 sheath blight disease. All rights reserved. endophytes inside plants exhibit complex interactions with their hosts. well known for their capacity to synthesize many extracellular enzymes and various antibiotics. new approaches for sustainable rice production. Endophytes are microorganisms that can colonize the inner parts of plants for all or part of their lifetime. the exploration of alternative methods Accepted Article has received growing interest in the last decade.7 By far.8 Analog to the gut microflora in humans.4 are one of these potential BCAs.10 In the case of rice disease. have been isolated and selected to control rice diseases such as rice blight disease. especially Streptomyces. such as plant endophytic environments.

15 tap water yeast extract agar (TWYE)15 and water agar medium (WA). 16. grown on fields used for rice blast control test in Liuyang. there are very Accepted Article few reports concerning endophytic actinomycetes as BCAs for rice blast disease. along with a higher rice grain yield. by far. stem. We evaluate the pathogen control efficacy of the antagonistic isolate in vitro and especially in vivo.13 In an effort to discover novel biocontrol candidates against M.12 However. MATERIALS AND METHODS Isolation of rice endophytic actinomycetes Two rice varieties Xiang’aizao 7 and Gumei 4 (indica). 19 weeks old. we preliminary analyzed its secondary metabolites with potential value for biocontrol agents. China (22. All rights reserved.15 mannitol soybean agar (MS). The actinomycete or actinomycete-derived components could then be used as fungal suppressors.41′44′′E). Isolation plates were This article is protected by copyright.16 Each medium was supplemented with 20 mg L-1 nalidixic acid and 50 mg L-1 benomyl as antibacterial and antifungal agents respectively. oryzae. The plants were washed thoroughly and dried overnight. In addition.14 The dried plant segments were cut into pieces (10 mm in length) and transferred onto four isolation media: humic acid vitamin B agar (HV). then divided into root. then lifted into paper bags to the laboratory. . sheath and leaf tissues and subjected to a surface sterilization procedure as described by Xiong. Hunan. 11. 7. were used as the plant materials. 103. the objective of this study is to isolate and characterize a rice endophytic actinomycete with capacity to suppress rice blast pathogen effectively. Blast infected and healthy rice were harvested respectively when they were 2.54′30′′N. 4. field conditions.

14 Screening of antagonists against Magnaporthe oryzae Rice blast pathogenic fungi Magnaporthe oryzae RB3.6 Evaluation of antifungal activity for the selected antagonist in vitro To detect the effect of the antagonists on the morphology of M. was collected every day and inoculated in the PDA plates mixed with spore suspension of M. China). After incubated at 28 °C for 7 days. Guy1118 was provided by Hunan Academy of Agricultural Sciences (Changsha.oryzae obtained from 10-day-old PDA plates (200 μL.20 The growth inhibition of M. oryzae.19 Dual-culture assay was performed to screen the endophytic actinomycetes with antagonistic activity. All rights reserved. China). The emerged actinomycetes Accepted Article colonies were transferred to half-strength potato dextrose agar (half-PDA) for purification. M.oryzae This article is protected by copyright.oryzae towards the actinomycete colony on dual-culture assay plates were collected and its ultrastructure was visualized using scanning electron microscopy (SEM). mycelia of M. The efficacy of the surface sterilization was tested as described by Xiong.oryzae colony radius was measured to assess its growth inhibition by actinomycete. which was cultured in International Streptomyces Project (ISP)-221 liquid medium at 28 °C. The inhibition percentage was calculated as described by Boukaew. .oryzae by fermentation broth of actinomycete was tested as follows: fermentation broth (100 μL) of actinomycete.17 62 and S07 were provided by Yahua Seeds Science Research Institute (Changsha. Spore suspension of M. 106 spores mL-1) was inoculated in the center of PDA plate and actinomycetes were one-line streaked 30 mm away from the center. incubated at 27 °C and 37 °C and checked regularly. All the strains were confirmed by ITS sequence analysis as described by Wang.

and 50 cm between plots. (106 spores mL-1) at 1:25 (v/v). The antifungal volatile compounds production was also tested as described by Spence.oryzae was transferred to a new PDA plate and incubated at 28 °C to test their viability. and the mycelia of M. Accepted Article The fermentation broth of actinomycetes at 7 days was also collected to determine the antifungal activity of its cell free filtrate. Each treatment was done with three replicates in different fields with 100 rice plants and chosen in a randomized block arrangement. The spore suspension of actinomycete (107 spores mL-1) containing 2 mL L-1 Tween 20 was sprayed onto the leaves 4 times in 2 days. All rights reserved.000 g for 20 min and supernatant was filtered through a 0.oryzae in vitro was selected to evaluate its ability to reduce rice blast in rice cultivar Xiang’aizao 7 (susceptible to M.oryzae) in fields during the cropping period of 2014 and 2015. then incubated at 28 °C and observed. The fungal spore suspension was inoculated in the center of the medium and incubated at 28 °C. the rice seedlings were surrounded by infected seedlings to let the airborne spores serve as inoculum. Disease index = ∑ (Number of diseased leaves at all levels × The relevant disease level)/(Total number of research This article is protected by copyright.22 μm membrane filter. Disease incidence was evaluated in 30th day by collecting 30 rice leaves randomly.oryzae was measured. To mimic the natural condition. The cells were removed by centrifugation at 4. . the semidiameter of M. Plants were grown in plots with each plot covered an area of 1 m2. After 7 days.22 Field trial of the most potent antagonist against rice blast The isolate that evidenced the greatest suppression activity against M. Then the filtrate was mixed with PDA (1:10. v/v) and poured into Petri plates.

All rights reserved. MS.8 was used to align the sequences retrieved from databases and Neighbor-joining phylogenetic tree was constructed using MEGA5. The isolation frequency = number of endophytic antagonist / total number of endophytic actinomycetes ×100%. Nutrient agar (NA) and a variety of ISP media. Actinomycetes inside the rice were isolated from these rice samples as the isolation procedure described above. Shanghai Generay Biotech Co. leaves × Highest disease level) × 100%. Clustalx 1. LTD) following the manufacturer’s protocol. Growth affection factors for the most potent antagonist Growth curve of actinomycete was conducted as follows: the actinomycete grew in This article is protected by copyright. The sequence was blasted in EzTaxon server (http://www.0 software. The colonies were identified based on their morphological characteristics or further confirmed by their 16S rRNA sequence analysis.21 Biochemical characterization tests were carried out according to Bergey’s Manual of Systematic Bacteriology. Accepted Article Estimation of internal rice colonization of the most potent antagonist The 30-day-old rice seedlings treated or not treated by antagonist in field test were also taken for estimation of internal rice colonization of antagonist. Identification and characterization of the most potent antagonist The cultural characteristics of actinomycete were determined on PDA.15 The purified PCR product was sequenced in Sangong Biotech (Shanghai. The 16S rRNA gene was amplified by PCR with universal primers 27f and 1492r.ezbiocloud.net/eztaxon). Genomic DNA was extracted using a commercial DNA extraction kit (GeneRay Bacterial Genomic DNA Extraction Kit. China). ..

All rights reserved.30 Extraction and characterization of antifungal metabolites produced by the most potent antagonist The actinomycete was inoculated on cellophane placed PDA to separate the mycelia from agar media. Accepted Article The temperature sensitivity was determined from 25 °C to 45 °C with the interval of 5 °C. The PCR reaction was programmed as described by Qin.28 II29 and nonribosomal peptide synthetases (NRPS)28 from actinomycete were used. The NaCl concentration sensitivity was conducted in medium amended with NaCl at the concentrations of 10 to 50 g L-1 with the interval of 10 g L-1. The pH sensitivity was detected from pH 5-13 with the interval of pH 1. .24 Gelatinase production was tested as described by Bose. After 20 days. Hydrocyanic acid (HCN).27 All tests were incubated at 28 °C for 7 days. and the cell pellets were dried at 60 °C and weighed every day. the cellophanes were removed and the agar was cut into This article is protected by copyright. Amplification of PKS and NRPS sequences from the most potent antagonist Three sets of primers for the amplification of genes encoding polyketide synthases (PKS) I.26 Mineral phosphate solubilization was tested as described by Nautiyal. protease.23 Cellulase. Growth on sole carbon sources was studied as described by Gottlieb.25 1-amino-cyclopropane-1-carboxylate (ACC) deaminase production was performed as described by Penrose. siderophore and IAA production was done as described by Gopalakrishnan. ISP2 broth up to 8 days.21 Determination of enzymes and secondary metabolites produced by the most potent antagonist Chitinase production was tested as described by Kotasthane.

oryzae RB3 by This article is protected by copyright. 5 strains showed antagonism against M. oryzae was determined as described above. we focused on the 171 isolates from 4-week-old rice. All rights reserved. . The effect of extract on growth of M. One-way ANOVA analysis and Duncan’s multiple range test were performed to detect statistical significance. USA). the samples of two rice varieties Xiang’aizao 7 and Gumei 4 which were susceptible and resistant to rice blast respectively were collected.05.oryzae To isolate endophytic actinomycetes with antagonistic activities.31 The nigericin standard used for identification is from InvivoGen (CA. and the differences were considered significant when P <0. From 16840 rice segments. Out of these isolates. RESULTS Isolation and screening of endophytic actinomycetes antagonistic to M. The operating parameters of the instrument were as described in Silva. and the extract with best inhibition activity was conducted to characterize the antifungal metabolites on a Waters ACQUITY UPLC I-Class system coupled to the Xevo TQ-S triple quadrupole mass spectrometer with positive electrospray ionization (ESI+) mode and multiple reaction monitoring (MRM). pieces to extract with different organic solvents (methanol. Statistical analysis Statistical analysis of the data was performed with SPSS. The extract was filtrated through three-layer gauze to remove the solid agar and was evaporated to obtain crude metabolites. ethyl acetate and petroleum Accepted Article ether) and shaken for 2 h. we isolated 482 endophytic actinomycete strains (Table S1). Because seedling blast usually breaks out within 30 days during the cropping period.

After secondary screening against 4 physiological races of M. the fermentation broth of OsiSh-2 revealed inhibition effect on the growth of M. Compared with the control. Its cell-free filtrates also showed strong suppress effect against M. one isolate appeared the strongest antifungal activity with mycelia growth inhibition of 75.oryzae hyphal structure conformed this. S07 and 62). dual-culture assay. All rights reserved. This strain was isolated from sheath of healthy Gumei 4 on MS medium in 27 °C. and its antifungal activity increased with the OsiSh-2 culture time extending (Fig.oryzae Accepted Article (RB3. and the result is negative.oryzae. Biocontrol efficacy of OsiSh-2 against rice blast in field Biocontrol efficiency of OsiSh-2 against rice blast under field conditions was evaluated in 2014 and 2015.oryzae exposed to OsiSh-2 showed the cellular changes in hyphal morphology including hyphal swelling and cytoplasm aggregation (Fig.3% at maximum. The inhibition effect and inhibition percentage are shown in Fig. indicating that OsiSh-2 could inhibit the growth of M. oryzae. We also examined whether OsiSh-2 might produce volatile antifungal metabolites to hinder pathogen growth. 2A). M. The mycelia of these growth suppressed M. 1A and 1C. oryzae. SEM observations of M. In vitro antagonistic activities of OsiSh-2 Dual-culture assay indicated that OsiSh-2 could inhibit the growth of M. As expected. The colony semidiameter of pathogen grown on filtrates-mixed PDA plate was significantly less than control (Fig. In 2014. 2B and C). 1B).oryzae resumed growth again when transferred to a new PDA plate without filtrates. two treatments of OsiSh-2 were proceeded: 1) spraying 5 This article is protected by copyright.oryzae but not kill it. Guy11. . and we named it OsiSh-2.

83% of untreated rice. Meanwhile. while OsiSh-2 twice-sprayed treatment reduced the disease symptoms with the disease index as lowest as 16. and black when matured. .48%. 3).74% came from the twice sprayed rice shoot. obviously lower than the 58. in 2015. Untreated rice was seriously damaged by rice blast with disease index of 41. days before and 5 days after rice blast outbreak. then was 12.25%) was a little worse than the untreated rice (Fig. The aerial spore mass is white or gray. The physiological and biochemical properties of OsiSh-2 are summarized in Table 1.19% of twice sprayed rice. However. Concerning that the effect of spraying OsiSh-2 before and after rice blast occurrence was better than the only after treatment (Fig. The color of substrate mycelium is brown or yellow and the aerial mycelium is white or gray. 3B).75 cm of untreated rice. 2) spraying 5 days after rice blast Accepted Article outbreak. its aerial hypha was differentiated into spiral spore morphology (Fig.12% from once sprayed rice shoot. 4A).3%. when OsiSh-2 was introduced to rice. effect of this before treatment was the best with the disease index of 30.67% (Fig. Isolation of internal rice colonized OsiSh-2 showed that the highest isolation frequency of 67. In fact. All rights reserved.2 cm. Identification and characteristics of OsiSh-2 OsiSh-2 can grow on a range of agar media (Table S3). even lower than the 36. This article is protected by copyright. the disease severity of spraying after the rice blast (59. The SEM observation showed after grew on PDA for 10 days. and no OsiSh-2 from untreated rice (Table S2). the length of above-ground part was taller than untreated rice. The length of the best disease-controlled rice reached 57. significantly higher than the 35.3B). we added a treatment of once before rice blast outbreak.

16S rRNA sequence analysis indicated that OsiSh-2 clustered within the genus Accepted Article Streptomyces and has the highest similarity to Streptomyces endus strain NRRL 2339 (NR_043379. It can grow at pH ranging from 6 to 12 and showed NaCl tolerance up to 30 g L-1 (Table 1).32 OsiSh-2 produced gelatinase with a clear zone surround the colony on gelatin medium (Fig. . OsiSh-2 was designated as Streptomyces endus (Genbank Accession number KU324469. S1A) and cellulose (Fig. revealing the production of iron chelator siderophore. indicating OsiSh-2 can secrete CWDEs protease and cellulose. Enzymes and secondary metabolites production by OsiSh-2 Production of active secondary metabolites and fungal cell-wall degrading enzymes (CWDEs) is a prominent character of many biocontrol agents. morphological. S1C). 4B). physiological and biochemical characteristics. we detected the enzymatic activities and secondary metabolites produced by OsiSh-2 (Table 2). The OsiSh-2 strain was deposited in the China General Microbiological Collection Center (CGMCC) under accession number CGMCC-8716. It enters stationary phase on the 4th day and death phase on the 7th day at 28 °C in ISP2 lipid culture (Data not shown). OsiSh-2 can decolor the blue colored ferric CAS complex into orange (Fig. OsiSh-2 does not grow greater than 45 °C with an optimal growth temperature of 30-40 °C. S1B) media with a clear zone surround the colonies due to the substrate hydrolysis. which might lead to phytopathogen death due to lack of ions. Combined with the cultural. Thus. All rights reserved.1) (99. OsiSh-2 can grow well on casein (Fig.1).93%) (Fig. In addition. S1D). Gelatinase might suppress disease occurrence by inhibiting fungal spore adhesion to the This article is protected by copyright.

were chosen to extract the solid culture of OsiSh-2. identifying antifungal metabolites of OsiSh-2 can be helpful to its biocontrol application. 5). methanol. S2B). . S2A). methanol extract showed maximum antifungal activity followed by ethyl acetate whereas petroleum ether had no inhibitory effects. The amplification products from OsiSh-2 with PKS-I and NRPS primers were positive. Three organic solvents. Further. OsiSh-2 can produce plant hormone IAA and ACC deaminase which can promote plant growth by reducing harmful effects of ethylene. Preliminary extraction and characterization of the active metabolites of OsiSh-2 PKS and NRPS represent two groups of natural products with remarkable biological activities. cellulose substrate. All rights reserved. however. indicating OsiSh-2 was likely to synthesize bioactive compounds including antibiotics. thus improve the host plant’s resistance against pathogen.34 The fact that few endophytic This article is protected by copyright. and the antifungal activity of methanol extract exhibited a dose-dependent effect (Fig.84 min (Fig. DISCUSSION Endophytic actinomycetes have been confirmed as promising resources for the biocontrol of fungi-induced plant diseases. Among all.33 Chitinase is an important CWDE and HCN also can suppress Accepted Article disease. UPLC-MS/MS of the methanol extract produced one major active peak at a retention time 4. and the peak was identified as nigericin by comparison with nigericin standard (Fig. Some microbial enzymes and secondary metabolites may promote plant growth. ethyl acetate and petroleum ether with different polarity. OsiSh-2 was unable to produce them in our detection.

For example. Encouragingly. The disease incidence varied in two years. reports on rice blast biocontrol using endophytic actinomycetes are seldom based on variable. 3B).oryzae in vitro. even better than spraying simultaneously before and after disease occurrence (Fig. This article is protected by copyright.36 Bacillus methylotrophicus BC79 culture filtrate showed 84.19 It should be noted that in this study we only used spore suspension of OsiSh-2. We propose that timing of OsiSh-2 treatment is very critical to rice blast disease control efficiency. All rights reserved.64% in 2014 and 48. . dry flowable formulations of Bacillus subtilis strain T429 were effective in controlling rice blast with efficiency up to 78. We are currently optimizing the formulation of OsiSh-2 which will improve the effect. The impressive reports were come from other species microorganisms. field-realistic conditions. almost all OsiSh-2 treatments showed a suppression of rice blast.35 we carried out field tests for two years to confirm the biocontrol effects of OsiSh-2. The disease severity was reduced by 59.5%.07%. Spraying OsiSh-2 before rice blast occurrence has the best performance. 3). Concerning the experiments in laboratory do not necessarily reflect their inhibitory ability in field condition. actinomycetes has been reported to control rice blast reveals that this microbe might be Accepted Article an under explored reservoir of novel potential agents in this field.8% biocontrol effect for M.19% in 2015 (Fig. and it was more serious in 2015. By far.37 Culture filtrate of Chaetomium aureum MF-91 reduced the disease index of rice panicle blast by 66. we isolated 171 endophytic actinomycetes from 4-week-old rice and obtained a Streptomyces endus strain OsiSh-2. In this study. which exhibited a remarkable antagonism effect against M.oryzae.

Correspondingly. the inoculation of pathogen in rice cost energy to induce defense response. BCAs should be used as early as possible to improve plant resistance. the high isolation frequency of OsiSh-2 from inner part of rice confirmed that it could colonize in rice tissues efficiently and dominantly. Further field tests with prolonged investigation on the biocontrol effects of OsiSh-2 are underway to confirm our inference. To our knowledge. subtilis SB24 declined on soybean leaves. While rice blast is severely outbroken (e. it was not surprising to find that spraying OsiSh-2 after rice blast occurrence showed lowest performance in 2015. it was found that plant-endophyte interactions in healthy plants were different from those of host plants under stressful conditions. assimilation OsiSh-2 might decrease the host rice immunity to pathogen for a period of time.8 Hence. The result of OsiSh-2 shows similar pattern. All rights reserved. the observed reduction of disease symptoms was associated with the infection level of This article is protected by copyright. . Xue38 indicated that biocontrol efficiency was frequently correlated with the colonization ability of BCAs.37 Additionally. even a little worse than control (Fig. in year 2015).g. The effect of OsiSh-2 treatment is also consistent with the report that in field tests. In our study. instead of curing rice blast. Under this station. present commercial chemical fungicides are only effective for Accepted Article prevention. We reason that pretreated OsiSh-2 can effectively contribute to the plant resistance of disease by producing antifungal metabolites and plant growth promoters (Table 2). and Babalola39 further highlighted that BCAs must dominate the ecological niche within plant to effectively attenuate disease symptom. 3B). Zhang40 reported that the suppression of sclerotinia stem rot was affected when the population of antagonist B. On the other hand.

As compared to actinomycetes in rhizosphere. SEM observations showed that OsiSh-2 caused the cellular changes of M. MJM4426 showed biocontrol potential on rice bacterial blight.oryzae. We consider OsiSh-2 may facilitate its distribution through the plant and have better implications for plant protection compared to unrelated antagonists due to its beneficial actions. .4 Staurosporine from Streptomyces sp. CWDES such as chitinase and protease secreted by antagonists might cause abnormal hyphal morphology of pathogen. padanus JAU4234 suppressed rice blast pathogen. 1B).43 Resistomycin This article is protected by copyright. OsiSh-2 shows IAA and ACC deaminase activity and the rice pretreated with OsiSh-2 were indeed taller than untreated in field (Table 2). endophytic actinomycetes can colonize plants well and have symbiotic relationship with host plant.oryzae were likely due to the presence of cellulase and protease from OsiSh-2. it also might be due to the antagonism of actinobacteria in the protection of host rice against pathogens thus promoting growth of rice. Streptomyces are prolific producers for antibiotics. OsiSh-2 in plants: the highest isolation frequency of OsiSh-2 67. 3). a new polyene macrolide antibiotic from S. which can protect plants from pathogen attack.38 The fact that OsiSh-2 can produce extracellular enzymes and metabolites indicates it has the capacity to demonstrate multiple mechanisms against M.64% (Fig.42 Antifungalmycin 702.74% (Table S2) was Accepted Article correlated with the highest disease control efficiency 59. Firstly. These plant growth promoters might indirectly improve plant disease resistance.41 So the hyphal deformation and growth suppression of M. All rights reserved. It was also reported that the biocontrol efficacy of BCAs had a positive correlation with their biocontrol capabilities. Secondly.oryzae in hyphal morphology (Fig. Of course.

such as polyether46 and 1H-pyrrole-2-carboxylic acid. sindeneusis 26348 strongly inhibited M. This article is protected by copyright. 50 To the best of our knowledge. indicating the active compound in methanol is valuable. endus strain exhibited strong antagonistic activity against M.7 In Accepted Article our study. S.7 S. Nigericin was among the first polyether ionophores to be discovered.oryzae. S.49. methanol extract of OsiSh-2 showed strong antagonism. The characterization of methanol extract of OsiSh-2 by UPLC-MS/MS identified the presence of nigericin. possessed significant preventive efficacy against rice blast. canus BYB02.oryzae. from S. padanus JAU4234. endus was only reported in potato and Zinnia with low efficiency. canus BYB02.43 S.44 and it showed strong activity against Gram positive bacteria. The genus Streptomyces strains have antagonistic activity against wide range of phytopathogen. All together. . this study is the first report about an endophytic S.28 Further research to verify which compounds are responsible for the antifungal activity of OsiSh-2 (nigericin or other antibiotics) are still under investigation in our lab. All rights reserved.oryzae both in vitro and in vivo. rarely researches reported nigericin possessing the capacity to inhibit M. Given that numerous studies have discovered many antifungal antibiotics in methanol extract of some BCAs.45 However. The antifungal activity of S. As for rice blast.47 and PCR amplification of PKS and NRPS confirmed OsiSh-2 had the genes involved in biosynthesize of active compounds including antibiotics.endus OsiSh-2 can be a promising candidate as biocontrol agent for rice blast in agriculture application.

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This article is protected by copyright. L) using some biocontrol agents and plant extracts. Altalhi A. Elhariry H. Molecular screening of Streptomyces isolates for antifungal activity and family 19 chitinase enzymes. J Agri Sci 2: 221-30 Accepted Article (2010). elegans. . Appl Microbiol 4: 243-50 (1956). 50 Gherbawy Y. El-Deeb B and Khiralla G. 51 Backus EJ and Tresner HD. J Microbiol 50: 459-68 (2012). All rights reserved. A broadened concept of the characteristics of Streptomyces hygroscopicus.

All rights reserved. .Accepted Article This article is protected by copyright.

respectively. (B) Growth inhibition of M. M. The antagonism of OsiSh-2 against M.05) according to the Duncan’s multiple range test. .oryzae by OsiSh-2 fermentation broth (A). Figure 1. Bars represent standard errors of three replicates. oryzae by OsiSh-2. Different letter indicates significant differences (P <0. Bars represent standard errors of three replicates. Different This article is protected by copyright.The corresponding hyphae structure observed by scanning electron microscopy are (B1) and (B2).oryzae by OsiSh-2 cell free filtrate from 7-day-old fermentation broth. The fermentation time of OsiSh-2 are from 1 to 7 days.oryzae was cultured not Accepted Article exposed (A1) or exposed to OsiSh-2 (A2). and the corresponding colony semidiameter (C). Figure 2. Growth inhibition of M. (C) Inhibition percentage of four different physiological races of M. All rights reserved.oryzae.

Different letter indicates significant differences (P <0.5 days before rice blast outbreak (once before). (A) Representative images of 30 days’ rice in year 2015. Bars represent standard errors of three replicates. . Control: without any treatment. OsiSh-2: spraying OsiSh-2 spores suspension 5 days before the rice blast outbreak. (B) Disease index of 30 days’ rice treated by OsiSh-2 in year 2014 and 2015.05) according to the Duncan’s multiple Accepted Article range test.05) according to the Duncan’s multiple range test. Figure 3. letter indicates significant differences (P <0. Biocontrol efficiency of OsiSh-2 against rice blast in field at 30 days. 5 days after rice blast outbreak (once after). All rights reserved. This article is protected by copyright. Treatments include: spraying OsiSh-2 spores suspension 5 days before and 5 days after rice blast outbreak (twice).

Accepted Article Figure 4. Antifungal activities of methanol extract of OsiSh-2. or meanwhile add methanol (up) and methanol extract of OsiSh-2 (down) 30 mm away from the fungal (B). The plates were incubated for 10 days. Spores of M. This article is protected by copyright. control was 200 μL methanol. All rights reserved. . Scanning electron micrograph of spiral spore chains of OsiSh-2 grown on PDA agar for 10 days (A).oryzae RB3 was inoculated in the center of PDA plates (A). or add different methanol extract volume of OsiSh-2 (C). Figure 5. Neighbour-joining phylogenetic tree of OsiSh-2 based on 16S rRNA sequences analysis (B).

- Decomposition of hydrogen peroxide + ND Starch + + Casein + + Gelatin + + Growth at pH 5 + ND pH 6-12 ++ ND pH 13 + ND 25 °C + ND 30-40 °C ++ ND 45 °C . +. utilization doubtful. E.endus NRRL 2339 Test OsiSh-2 S. . ++. ±.195651. All rights reserved. +. -. - Maltose ± + NaAc . J. no growth. endus strain NRRL 2339* Growth on sole carbon sources(10 g L-1) D-Glucose + + D-Xylose ± + L-Arabinose + + L-Rhamnose + + D-Fructose + + D-Galactose . ++. Comparison of physiological and biochemical properties of OsiSh-2 and S. endus strain NRRL 2339* are from Backus. - Lactose . positive utilization. -. poor growth. - Raffinose . - Mannitol ± + Inositol ± - Salicin ± + Sucrose . Tables Accepted Article Table 1. good growth. ND Growth in the presence of Sodium chloride 30 g L-1 ++ ND -1 Sodium chloride 40 g L + ND ND. strong positive utilization. utilization negative. This article is protected by copyright. *Data for S. not determined.

This article is protected by copyright. Table 2. Production of enzymes and secondary metabolites by OsiSh-2 Accepted Article Enzymes and secondary OsiSh-2 metabolites Chitinase - Cellulase + Protease + Gelatinase + ACC deaminase + Siderophore + IAA + HCN - Phosphate solubilization - +: positive. . -: negative. All rights reserved.