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

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 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

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 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

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 activity. Scanning electron microscopy observation of M.oryzae treated by OsiSh-2
Accepted Article
revealed significant morphological alterations in hyphae. In two-year’s field tests,

spraying OsiSh-2 spore solution (107 spores mL-1) is capable of reducing rice blast

disease severity by 59.64%. In addition, both fermentation broth of OsiSh-2 and its

cell-free filtrates could inhibit growth of M.oryzae, suggesting the presence of active

enzymes and secondary metabolites. OsiSh-2 is tested positive for PKS-I and NRPS

genes and can produce cellulase, protease, gelatinase, siderophore, IAA and ACC

deaminase. Preliminary separation indicated that the methanol extract of OsiSh-2 could

suppress growth of pathogen; its major active component was identified as nigericin.

CONCLUSION: Endophytic Streptomyces endus OsiSh-2 has potential as a biocontrol

agent against rice blast in agriculture.

Keywords: Rice endophytes; Antagonistic agents; Magnaporthe oryzae; Rice blast;

Streptomyces endus OsiSh-2

INTRODUCTION

Rice (Oryza sativa L.) is the most important food source for over half of the world’s

population. However, rice yield is greatly reduced due to the rice blast disease caused

by pathogenic fungi Magnaporthe oryzae. Chemical fungicides treatment and

development of resistant rice varieties can effectively control this disease.1 However,

chemical agents often negatively impacts the environment and even the consumers.1

Plant resistance (often based on a single gene) is normally not durable in the field,

partially due to the highly variable of M.oryzae.2 In the context of seeking promising

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 new approaches for sustainable rice production, the exploration of alternative methods
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has received growing interest in the last decade. Plant beneficial microorganisms

[biological control agents (BCAs)] have been chosen as such an approach due to their

lower toxicity and lack of pathogen resistance.3 Actinomycetes, well known for their

capacity to synthesize many extracellular enzymes and various antibiotics,4 are one of

these potential BCAs. Several species of actinomycetes, especially Streptomyces, have

been isolated and selected to control rice diseases such as rice blight disease,5 sheath

blight disease,6 and also rice blast.7 By far, most of these antibiosis actinomycetes are

from traditional sources, such as rhizosphere and soil. Now, researchers have started to

isolate untapped microbes from special environments, such as plant endophytic

environments.

Endophytes are microorganisms that can colonize the inner parts of plants for all or part

of their lifetime.8 Analog to the gut microflora in humans, endophytes inside plants

exhibit complex interactions with their hosts, and have been proven to be a potential

source for biocontrol agents over the last years.9 Numerous studies have indicated that

endophytes are capable of improving the resistance of host plants to adversities, such as

pathogen challenges, by the secretion of bioactive secondary metabolites to kill the

pathogens directly, or induce systemic resistance of host plant indirectly.10 In the case of

rice disease, for example, rice endophyte Harpophora oryzae exhibited a unique

protection for rice blast disease.11 Endophytic Bacillus subtilis var. amyloliquefaciens

FZB24 displayed a significantly lower disease incidence of bacterial leaf blight under

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 field conditions, along with a higher rice grain yield.12 However, by far, there are very
Accepted Article
few reports concerning endophytic actinomycetes as BCAs for rice blast disease.13

In an effort to discover novel biocontrol candidates against M. oryzae, the objective of

this study is to isolate and characterize a rice endophytic actinomycete with capacity to

suppress rice blast pathogen effectively. The actinomycete or actinomycete-derived

components could then be used as fungal suppressors. We evaluate the pathogen control

efficacy of the antagonistic isolate in vitro and especially in vivo. In addition, we

preliminary analyzed its secondary metabolites with potential value for biocontrol

agents.

MATERIALS AND METHODS

Isolation of rice endophytic actinomycetes

Two rice varieties Xiang’aizao 7 and Gumei 4 (indica), grown on fields used for rice

blast control test in Liuyang, Hunan, China (22.54′30′′N, 103.41′44′′E), were used as the

plant materials. Blast infected and healthy rice were harvested respectively when they

were 2, 4, 7, 11, 16, 19 weeks old, then lifted into paper bags to the laboratory. The

plants were washed thoroughly and dried overnight, then divided into root, stem, sheath

and leaf tissues and subjected to a surface sterilization procedure as described by

Xiong.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),15 mannitol

soybean agar (MS),15 tap water yeast extract agar (TWYE)15 and water agar medium

(WA).16 Each medium was supplemented with 20 mg L-1 nalidixic acid and 50 mg L-1

benomyl as antibacterial and antifungal agents respectively. Isolation plates were

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 incubated at 27 °C and 37 °C and checked regularly. The emerged actinomycetes
Accepted Article
colonies were transferred to half-strength potato dextrose agar (half-PDA) for

purification. The efficacy of the surface sterilization was tested as described by Xiong.14

Screening of antagonists against Magnaporthe oryzae

Rice blast pathogenic fungi Magnaporthe oryzae RB3,17 62 and S07 were provided by

Yahua Seeds Science Research Institute (Changsha, China), Guy1118 was provided by

Hunan Academy of Agricultural Sciences (Changsha, China). All the strains were

confirmed by ITS sequence analysis as described by Wang.19

Dual-culture assay was performed to screen the endophytic actinomycetes with

antagonistic activity. Spore suspension of M.oryzae obtained from 10-day-old PDA

plates (200 μL; 106 spores mL-1) was inoculated in the center of PDA plate and

actinomycetes were one-line streaked 30 mm away from the center. After incubated at

28 °C for 7 days, M.oryzae colony radius was measured to assess its growth inhibition

by actinomycete. The inhibition percentage was calculated as described by Boukaew.6

Evaluation of antifungal activity for the selected antagonist in vitro

To detect the effect of the antagonists on the morphology of M. oryzae, mycelia of

M.oryzae towards the actinomycete colony on dual-culture assay plates were collected

and its ultrastructure was visualized using scanning electron microscopy (SEM).20

The growth inhibition of M.oryzae by fermentation broth of actinomycete was tested as

follows: fermentation broth (100 μL) of actinomycete, which was cultured in

International Streptomyces Project (ISP)-221 liquid medium at 28 °C, was collected

every day and inoculated in the PDA plates mixed with spore suspension of M.oryzae

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 (106 spores mL-1) at 1:25 (v/v), then incubated at 28 °C and observed.
Accepted Article
The fermentation broth of actinomycetes at 7 days was also collected to determine the

antifungal activity of its cell free filtrate. The cells were removed by centrifugation at

4,000 g for 20 min and supernatant was filtered through a 0.22 μm membrane filter.

Then the filtrate was mixed with PDA (1:10, v/v) and poured into Petri plates. The

fungal spore suspension was inoculated in the center of the medium and incubated at

28 °C. After 7 days, the semidiameter of M.oryzae was measured, and the mycelia of

M.oryzae was transferred to a new PDA plate and incubated at 28 °C to test their

viability. The antifungal volatile compounds production was also tested as described by

Spence.22

Field trial of the most potent antagonist against rice blast

The isolate that evidenced the greatest suppression activity against M.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. Plants

were grown in plots with each plot covered an area of 1 m2, and 50 cm between plots.

Each treatment was done with three replicates in different fields with 100 rice plants and

chosen in a randomized block arrangement. To mimic the natural condition, the rice

seedlings were surrounded by infected seedlings to let the airborne spores serve as

inoculum. 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. Disease incidence was

evaluated in 30th day by collecting 30 rice leaves randomly. Disease index = ∑ (Number

of diseased leaves at all levels × The relevant disease level)/(Total number of research

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 leaves × Highest disease level) × 100%.
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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. Actinomycetes inside the

rice were isolated from these rice samples as the isolation procedure described above.

The colonies were identified based on their morphological characteristics or further

confirmed by their 16S rRNA sequence analysis. The isolation frequency = number of

endophytic antagonist / total number of endophytic actinomycetes ×100%.

Identification and characterization of the most potent antagonist

The cultural characteristics of actinomycete were determined on PDA, MS, Nutrient

agar (NA) and a variety of ISP media.21 Biochemical characterization tests were carried

out according to Bergey’s Manual of Systematic Bacteriology.

Genomic DNA was extracted using a commercial DNA extraction kit (GeneRay

Bacterial Genomic DNA Extraction Kit, Shanghai Generay Biotech Co., LTD)

following the manufacturer’s protocol. The 16S rRNA gene was amplified by PCR with

universal primers 27f and 1492r.15 The purified PCR product was sequenced in Sangong

Biotech (Shanghai, China). The sequence was blasted in EzTaxon server

(http://www.ezbiocloud.net/eztaxon). Clustalx 1.8 was used to align the sequences

retrieved from databases and Neighbor-joining phylogenetic tree was constructed using

MEGA5.0 software.

Growth affection factors for the most potent antagonist

Growth curve of actinomycete was conducted as follows: the actinomycete grew in

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 ISP2 broth up to 8 days, and the cell pellets were dried at 60 °C and weighed every day.
Accepted Article
The temperature sensitivity was determined from 25 °C to 45 °C with the interval of

5 °C. The pH sensitivity was detected from pH 5-13 with the interval of pH 1. 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. Growth on sole carbon

sources was studied as described by Gottlieb.21

Determination of enzymes and secondary metabolites produced by the most potent

antagonist

Chitinase production was tested as described by Kotasthane.23 Cellulase, protease,

Hydrocyanic acid (HCN), siderophore and IAA production was done as described by

Gopalakrishnan.24 Gelatinase production was tested as described by Bose.25

1-amino-cyclopropane-1-carboxylate (ACC) deaminase production was performed as

described by Penrose.26 Mineral phosphate solubilization was tested as described by

Nautiyal.27 All tests were incubated at 28 °C for 7 days.

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,28 II29 and nonribosomal peptide synthetases (NRPS)28 from actinomycete were

used. The PCR reaction was programmed as described by Qin.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. After 20 days, the cellophanes were removed and the agar was cut into

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 pieces to extract with different organic solvents (methanol, ethyl acetate and petroleum
Accepted Article
ether) and shaken for 2 h. The extract was filtrated through three-layer gauze to remove

the solid agar and was evaporated to obtain crude metabolites. The effect of extract on

growth of M. oryzae was determined as described above, 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). The operating parameters of the instrument were as described in

Silva.31 The nigericin standard used for identification is from InvivoGen (CA, USA).

Statistical analysis

Statistical analysis of the data was performed with SPSS. One-way ANOVA analysis

and Duncan’s multiple range test were performed to detect statistical significance, and

the differences were considered significant when P <0.05.

RESULTS

Isolation and screening of endophytic actinomycetes antagonistic to M.oryzae

To isolate endophytic actinomycetes with antagonistic activities, the samples of two rice

varieties Xiang’aizao 7 and Gumei 4 which were susceptible and resistant to rice blast

respectively were collected. From 16840 rice segments, we isolated 482 endophytic

actinomycete strains (Table S1). Because seedling blast usually breaks out within 30

days during the cropping period, we focused on the 171 isolates from 4-week-old rice.

Out of these isolates, 5 strains showed antagonism against M.oryzae RB3 by

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 dual-culture assay. After secondary screening against 4 physiological races of M.oryzae
Accepted Article
(RB3, Guy11, S07 and 62), one isolate appeared the strongest antifungal activity with

mycelia growth inhibition of 75.3% at maximum. The inhibition effect and inhibition

percentage are shown in Fig. 1A and 1C. This strain was isolated from sheath of healthy

Gumei 4 on MS medium in 27 °C, and we named it OsiSh-2.

In vitro antagonistic activities of OsiSh-2

Dual-culture assay indicated that OsiSh-2 could inhibit the growth of M.oryzae. SEM

observations of M.oryzae hyphal structure conformed this. Compared with the control,

M.oryzae exposed to OsiSh-2 showed the cellular changes in hyphal morphology

including hyphal swelling and cytoplasm aggregation (Fig. 1B).

As expected, the fermentation broth of OsiSh-2 revealed inhibition effect on the growth

of M. oryzae, and its antifungal activity increased with the OsiSh-2 culture time

extending (Fig. 2A). Its cell-free filtrates also showed strong suppress effect against M.

oryzae. The colony semidiameter of pathogen grown on filtrates-mixed PDA plate was

significantly less than control (Fig. 2B and C). The mycelia of these growth suppressed

M.oryzae resumed growth again when transferred to a new PDA plate without filtrates,

indicating that OsiSh-2 could inhibit the growth of M.oryzae but not kill it.

We also examined whether OsiSh-2 might produce volatile antifungal metabolites to

hinder pathogen growth, and the result is negative.

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. In 2014, two treatments of OsiSh-2 were proceeded: 1) spraying 5

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 days before and 5 days after rice blast outbreak; 2) spraying 5 days after rice blast
Accepted Article
outbreak. Untreated rice was seriously damaged by rice blast with disease index of

41.3%, while OsiSh-2 twice-sprayed treatment reduced the disease symptoms with the

disease index as lowest as 16.67% (Fig. 3). Meanwhile, when OsiSh-2 was introduced

to rice, the length of above-ground part was taller than untreated rice. The length of the

best disease-controlled rice reached 57.2 cm, significantly higher than the 35.75 cm of

untreated rice. Isolation of internal rice colonized OsiSh-2 showed that the highest

isolation frequency of 67.74% came from the twice sprayed rice shoot, then was

12.12% from once sprayed rice shoot, and no OsiSh-2 from untreated rice (Table S2).

Concerning that the effect of spraying OsiSh-2 before and after rice blast occurrence

was better than the only after treatment (Fig. 3B), in 2015, we added a treatment of once

before rice blast outbreak. In fact, effect of this before treatment was the best with the

disease index of 30.48%, obviously lower than the 58.83% of untreated rice, even lower

than the 36.19% of twice sprayed rice. However, the disease severity of spraying after

the rice blast (59.25%) was a little worse than the untreated rice (Fig.3B).

Identification and characteristics of OsiSh-2

OsiSh-2 can grow on a range of agar media (Table S3). The color of substrate mycelium

is brown or yellow and the aerial mycelium is white or gray. The aerial spore mass is

white or gray, and black when matured. The SEM observation showed after grew on

PDA for 10 days, its aerial hypha was differentiated into spiral spore morphology (Fig.

4A). The physiological and biochemical properties of OsiSh-2 are summarized in Table

1.

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 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_043379.1) (99.93%) (Fig. 4B). Combined with the cultural, morphological,

physiological and biochemical characteristics, OsiSh-2 was designated as Streptomyces

endus (Genbank Accession number KU324469.1). The OsiSh-2 strain was deposited in

the China General Microbiological Collection Center (CGMCC) under accession

number CGMCC-8716.

In addition, OsiSh-2 does not grow greater than 45 °C with an optimal growth

temperature of 30-40 °C. It can grow at pH ranging from 6 to 12 and showed NaCl

tolerance up to 30 g L-1 (Table 1). 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).

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. Thus, we detected the

enzymatic activities and secondary metabolites produced by OsiSh-2 (Table 2). OsiSh-2

can grow well on casein (Fig. S1A) and cellulose (Fig. S1B) media with a clear zone

surround the colonies due to the substrate hydrolysis, indicating OsiSh-2 can secrete

CWDEs protease and cellulose. OsiSh-2 can decolor the blue colored ferric CAS

complex into orange (Fig. S1C), revealing the production of iron chelator siderophore,

which might lead to phytopathogen death due to lack of ions.32 OsiSh-2 produced

gelatinase with a clear zone surround the colony on gelatin medium (Fig. S1D).

Gelatinase might suppress disease occurrence by inhibiting fungal spore adhesion to the

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 cellulose substrate.33 Chitinase is an important CWDE and HCN also can suppress
Accepted Article
disease, however, OsiSh-2 was unable to produce them in our detection.

Some microbial enzymes and secondary metabolites may promote plant growth, thus

improve the host plant’s resistance against pathogen. 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. The amplification products from OsiSh-2 with PKS-I and NRPS primers were

positive, indicating OsiSh-2 was likely to synthesize bioactive compounds including

antibiotics. Further, identifying antifungal metabolites of OsiSh-2 can be helpful to its

biocontrol application. Three organic solvents, methanol, ethyl acetate and petroleum

ether with different polarity, were chosen to extract the solid culture of OsiSh-2. Among

all, methanol extract showed maximum antifungal activity followed by ethyl acetate

whereas petroleum ether had no inhibitory effects, and the antifungal activity of

methanol extract exhibited a dose-dependent effect (Fig. 5). UPLC-MS/MS of the

methanol extract produced one major active peak at a retention time 4.84 min (Fig.

S2A), and the peak was identified as nigericin by comparison with nigericin standard

(Fig. S2B).

DISCUSSION

Endophytic actinomycetes have been confirmed as promising resources for the

biocontrol of fungi-induced plant diseases.34 The fact that few endophytic

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 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.

In this study, we isolated 171 endophytic actinomycetes from 4-week-old rice and

obtained a Streptomyces endus strain OsiSh-2, which exhibited a remarkable

antagonism effect against M.oryzae in vitro.

Concerning the experiments in laboratory do not necessarily reflect their inhibitory

ability in field condition,35 we carried out field tests for two years to confirm the

biocontrol effects of OsiSh-2. The disease incidence varied in two years, and it was

more serious in 2015. Encouragingly, almost all OsiSh-2 treatments showed a

suppression of rice blast. The disease severity was reduced by 59.64% in 2014 and

48.19% in 2015 (Fig. 3). By far, reports on rice blast biocontrol using endophytic

actinomycetes are seldom based on variable, field-realistic conditions. The impressive

reports were come from other species microorganisms. For example, dry flowable

formulations of Bacillus subtilis strain T429 were effective in controlling rice blast with

efficiency up to 78.5%.36 Bacillus methylotrophicus BC79 culture filtrate showed 84.8%

biocontrol effect for M.oryzae.37 Culture filtrate of Chaetomium aureum MF-91 reduced

the disease index of rice panicle blast by 66.07%.19 It should be noted that in this study

we only used spore suspension of OsiSh-2. We are currently optimizing the formulation

of OsiSh-2 which will improve the effect.

We propose that timing of OsiSh-2 treatment is very critical to rice blast disease control

efficiency. Spraying OsiSh-2 before rice blast occurrence has the best performance,

even better than spraying simultaneously before and after disease occurrence (Fig. 3B).

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 To our knowledge, present commercial chemical fungicides are only effective for
Accepted Article
prevention, instead of curing rice blast. The result of OsiSh-2 shows similar pattern. The

effect of OsiSh-2 treatment is also consistent with the report that in field tests, BCAs

should be used as early as possible to improve plant resistance.37 Additionally, it was

found that plant-endophyte interactions in healthy plants were different from those of

host plants under stressful conditions.8 Hence, it was not surprising to find that spraying

OsiSh-2 after rice blast occurrence showed lowest performance in 2015, even a little

worse than control (Fig. 3B). 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). While rice blast is severely outbroken (e.g. in year

2015), the inoculation of pathogen in rice cost energy to induce defense response. Under

this station, assimilation OsiSh-2 might decrease the host rice immunity to pathogen for

a period of time. Further field tests with prolonged investigation on the biocontrol

effects of OsiSh-2 are underway to confirm our inference.

Xue38 indicated that biocontrol efficiency was frequently correlated with the

colonization ability of BCAs, and Babalola39 further highlighted that BCAs must

dominate the ecological niche within plant to effectively attenuate disease symptom. On

the other hand, Zhang40 reported that the suppression of sclerotinia stem rot was

affected when the population of antagonist B. subtilis SB24 declined on soybean leaves.

In our study, the high isolation frequency of OsiSh-2 from inner part of rice confirmed

that it could colonize in rice tissues efficiently and dominantly. Correspondingly, the

observed reduction of disease symptoms was associated with the infection level of

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 OsiSh-2 in plants: the highest isolation frequency of OsiSh-2 67.74% (Table S2) was
Accepted Article
correlated with the highest disease control efficiency 59.64% (Fig. 3). As compared to

actinomycetes in rhizosphere, endophytic actinomycetes can colonize plants well and

have symbiotic relationship with host plant. 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.

It was also reported that the biocontrol efficacy of BCAs had a positive correlation with

their biocontrol capabilities.38 The fact that OsiSh-2 can produce extracellular enzymes

and metabolites indicates it has the capacity to demonstrate multiple mechanisms

against M.oryzae. Firstly, SEM observations showed that OsiSh-2 caused the cellular

changes of M.oryzae in hyphal morphology (Fig. 1B). CWDES such as chitinase and

protease secreted by antagonists might cause abnormal hyphal morphology of

pathogen.41 So the hyphal deformation and growth suppression of M.oryzae were likely

due to the presence of cellulase and protease from OsiSh-2. Secondly, OsiSh-2 shows

IAA and ACC deaminase activity and the rice pretreated with OsiSh-2 were indeed

taller than untreated in field (Table 2). These plant growth promoters might indirectly

improve plant disease resistance. Of course, 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, which can protect plants from

pathogen attack.4 Staurosporine from Streptomyces sp. MJM4426 showed biocontrol

potential on rice bacterial blight.42 Antifungalmycin 702, a new polyene macrolide

antibiotic from S. padanus JAU4234 suppressed rice blast pathogen.43 Resistomycin

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 from S. canus BYB02, possessed significant preventive efficacy against rice blast.7 In
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our study, methanol extract of OsiSh-2 showed strong antagonism, indicating the active

compound in methanol is valuable. The characterization of methanol extract of OsiSh-2

by UPLC-MS/MS identified the presence of nigericin. Nigericin was among the first

polyether ionophores to be discovered,44 and it showed strong activity against Gram

positive bacteria.45 However, rarely researches reported nigericin possessing the

capacity to inhibit M.oryzae. Given that numerous studies have discovered many

antifungal antibiotics in methanol extract of some BCAs, such as polyether46 and

1H-pyrrole-2-carboxylic acid,47 and PCR amplification of PKS and NRPS confirmed

OsiSh-2 had the genes involved in biosynthesize of active compounds including

antibiotics.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.

The genus Streptomyces strains have antagonistic activity against wide range of

phytopathogen. As for rice blast, S. canus BYB02,7 S. padanus JAU4234,43 S.

sindeneusis 26348 strongly inhibited M.oryzae. The antifungal activity of S. endus was

only reported in potato and Zinnia with low efficiency.49, 50

To the best of our

knowledge, this study is the first report about an endophytic S. endus strain exhibited

strong antagonistic activity against M.oryzae both in vitro and in vivo.

All together, S.endus OsiSh-2 can be a promising candidate as biocontrol agent for rice

blast in agriculture application.

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 Acknowledgements We are grateful to Yahua Seeds Science Research Institute,
Accepted Article
Longping High-tech for offering the fields used for rice blast control test. This research

was supported by the National Natural Science Foundation of China (51378191 and

31571635), Science and Technology Planning Project of Hunan Province, China

(2014WK2005).

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Accepted Article

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 Figure 1. The antagonism of OsiSh-2 against M.oryzae. M.oryzae was cultured not
Accepted Article
exposed (A1) or exposed to OsiSh-2 (A2).The corresponding hyphae structure observed

by scanning electron microscopy are (B1) and (B2), respectively. (C) Inhibition

percentage of four different physiological races of M. oryzae by OsiSh-2. Bars represent

standard errors of three replicates. Different letter indicates significant differences (P

<0.05) according to the Duncan’s multiple range test.

Figure 2. Growth inhibition of M.oryzae by OsiSh-2 fermentation broth (A). The

fermentation time of OsiSh-2 are from 1 to 7 days. (B) Growth inhibition of M.oryzae

by OsiSh-2 cell free filtrate from 7-day-old fermentation broth, and the corresponding

colony semidiameter (C). Bars represent standard errors of three replicates. Different

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 letter indicates significant differences (P <0.05) according to the Duncan’s multiple
Accepted Article
range test.

Figure 3. Biocontrol efficiency of OsiSh-2 against rice blast in field at 30 days. (A)

Representative images of 30 days’ rice in year 2015. 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. Treatments

include: spraying OsiSh-2 spores suspension 5 days before and 5 days after rice blast

outbreak (twice);5 days before rice blast outbreak (once before); 5 days after rice blast

outbreak (once after). Bars represent standard errors of three replicates. Different letter

indicates significant differences (P <0.05) according to the Duncan’s multiple range

test.

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Accepted Article

Figure 4. Scanning electron micrograph of spiral spore chains of OsiSh-2 grown on

PDA agar for 10 days (A). Neighbour-joining phylogenetic tree of OsiSh-2 based on

16S rRNA sequences analysis (B).

Figure 5. Antifungal activities of methanol extract of OsiSh-2. Spores of M.oryzae RB3

was inoculated in the center of PDA plates (A), or meanwhile add methanol (up) and

methanol extract of OsiSh-2 (down) 30 mm away from the fungal (B), or add different

methanol extract volume of OsiSh-2 (C), control was 200 μL methanol. The plates were

incubated for 10 days.

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 Tables
Accepted Article
Table 1. Comparison of physiological and biochemical properties of OsiSh-2 and
S.endus NRRL 2339
Test OsiSh-2 S. 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 - -
Raffinose - -
Mannitol ± +
Inositol ± -
Salicin ± +
Sucrose - -
Lactose - -
Maltose ± +
NaAc - -
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 - ND
Growth in the presence of
Sodium chloride 30 g L-1 ++ ND
-1
Sodium chloride 40 g L + ND
ND, not determined.
*Data for S. endus strain NRRL 2339* are from Backus, E. J.195651.
++, strong positive utilization; +, positive utilization; ±, utilization doubtful; -,
utilization negative.
++, good growth; +, poor growth; -, no growth.

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 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;

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