Diversity and functional characterization of bacterial ...

Diversity and functional characterization of bacterial endophytes dwelling in various rice (Oryza sativa L.) tissues, and their seed-borne dissemination into rhizosphere under gnotobiotic P-stress Asif Hameed, Meng-Wei Yeh, Yu-Ting Hsieh, Wei-Ching Chung, Chaur-Tsuen Lo, et al. Plant and Soil An International Journal on Plant-Soil Relationships ISSN 0032-079X Plant Soil DOI 10.1007/s11104-015-2506-5

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Author's personal copy Plant Soil DOI 10.1007/s11104-015-2506-5

REGULAR ARTICLE

Diversity and functional characterization of bacterial endophytes dwelling in various rice (Oryza sativa L.) tissues, and their seed-borne dissemination into rhizosphere under gnotobiotic P-stress Asif Hameed & Meng-Wei Yeh & Yu-Ting Hsieh & Wei-Ching Chung & Chaur-Tsuen Lo & Li-Sen Young Received: 25 July 2014 / Accepted: 4 May 2015 # Springer International Publishing Switzerland 2015

Abstract Aim Endophytic bacterial diversity in four rice cultivars grown in two soil-types, their plant-probiotic features and rhizospheric deployment under P-stress were investigated. Methods Oryza sativa cvs. TCN1, TCS10, TK8, and TN71 were cultivated in greenhouse using non-sterile acidic and near-neutral paddy soils for 60 days. Root, stem and leaf tissues were screened for culturable bacterial endophytes using nutrient agar. Endophytes were identified and profiled for plant-probiotic features. The richness, Shannon-Weiner diversity, evenness and Venn’s distribution in terms of endophytic strains were evaluated. Seed-borne endophytes were characterized through DGGE. The deployment of endophytes into the rhizosphere in TCN1 and TK8 under gnotobiotic P-stress was assessed.

Responsible Editor: Choong-Min Ryu. Electronic supplementary material The online version of this article (doi:10.1007/s11104-015-2506-5) contains supplementary material, which is available to authorized users. A. Hameed : Y.1300 bp each) showing Gram-negative cultured rice-endophytic bacterial strains (CREBS, highlighted in bold type) and related reference type strains. Clades of the class αproteobacteria, β-proteobacteria and γ-proteobacteria are given in red, green and blue colored lines, respectively. Bootstrap values of >70 % after 1000 replications are shown at branching nodes. Scale bar, 0.05 substitutions per nucleotide position

99

Bacilli and Actinobacteria 87 Rhizobium sp. LS-079 (KF870446) 99 Rhizobium radiobacter ATCC 19358T (AJ389904)

Rhizobium sp. LS-099 (KJ584032) Rhizobium larrymoorei 3-10T (Z30542) Rhizobium sp. LS-004 (KF870419) T 98 Rhizobium daejeonense KCTC 12121 (AY341343) Rhizobium leguminosarum ATCC 10004T (U29386) 86 Sphingomonas sp. LS-116 (KF870453) Sphingomonas koreensis JSS26T (AF131296) T 100 Sphingomonas trueperi LMG 2142 (X97776) Sphingomonas sp. LS-026 (KF870430) 100 Sphingomonas sp. LS-039 (KF870435) 100 Sphingomonas aquatilis LS-081 (KJ584031) Sphingomonas aquatilis JSS7T (AF131295) Sphingomonas sp. LS-101 (KF870449) Sphingomonas sanguinis IFO 13937T (D84529) 77 Sphingomonas paucimobilis ATCC 29837T (U37337) 82 76 Alcaligenes sp. LS-124 (KF870456) 99 Alcaligenes sp. LS-083 (KF870447) Alcaligenes faecalis VKM B-1518T (D88008) Alcaligenes aquatilis LMG 22996T (AJ937889) 99 84 Achromobacter sp. LS-032 (KF870432) Achromobacter xylosoxidans NBRC 15126T (Y14908) 100 Achromobacter sp. LS-058 (KF870441) 100 T 92 Achromobacter animicus LMG 26690 (HE613448) 100 Herbaspirillum sp. LS-111 (KF870451) Herbaspirillum seropedicae DSM 6445T (Y10146) Burkholderia sp. LS-047 (KF870439) 82 Burkholderia sp. LS-044 (KF870438) T 100 Burkholderia cepacia ATCC 25416 (U96927) Burkholderia seminalis R-24196T (AM747631) 100 Rheinheimera sp. LS-001 (KJ584022) 100 Rheinheimera tangshanensis JA3-B52T (DQ874340) Rheinheimera baltica OSBAC1T (AJ441080) Cedecea davisae DSM 4568T (AF493976) 94 Cedecea sp. LS-120 (KF870454) Cedecea lapagei GTC 346T (AB273742) 100 100 Enterobacter sp. LS-010 (KJ584024) 90 Enterobacter oryzendophyticus REICA 082T (JF795011) Enterobacter cloacae ssp. cloacae ATCC 13047T (CP001918) 76 Shigella sonnei GTC 781T (FR870445) 86 Shigella dysenteriae ATCC 13313T (X96966) Shigella sp. LS-125 (KF870457) 87 Pantoea sp. LS-013 (KF870425) Pantoea stewartii ssp. indologenes LMG 2632T (JN175332) Pantoea allii LMG 24248T (AY530795) 94 Pantoea ananatis ATCC 19321T (EAU80196) Pantoea sp. LS-123 (KJ584033) 85 Pantoea sp. LS-042 (KJ584027) 79 Pantoea agglomerans DSM 3493T (AJ233423) 100 Acinetobacter oryzae B23T (GU954428) 100 Acinetobacter sp. LS-110 (KF870450) Acinetobacter calcoaceticus NCCB 22016T (AJ888983) 99 Pseudomonas taiwanensis LS-020 (KF870428) Pseudomonas taiwanensis BCRC 17751T (EU103629) Pseudomonas sp. LS-088 (KF870448) Pseudomonas sp. LS-037 (KF870434) Pseudomonas toyotomiensis HT-3T (AB453701) 95 Pseudomonas sp. LS-041 (KF870437) 100 97 Pseudomonas sp. LS-040 (KF870436) Pseudomonas alcaligenes NBRC 14159T (D84006) Pseudomonas aeruginosa DSM 50071T (HE978271) Pseudomonas otitidis MCC10330T (AY953147) 81 96 Pseudomonas sp. LS-011 (KF870423) Stenotrophomonas maltophilia IAM 12423T (AB294553) Xanthomonas campestris ATCC 33913T (AE008922) 100 Xanthomonas sacchari LMG 471T (Y10766) Xanthomonas sp. LS-012 (KF870424) 87 99

100

99

0.05

100

Author's personal copy Plant Soil Table 2 Localization of cultured rice-endophytic bacterial strains (CREBS) in cultivars planted at the acidic and near-neutral soils Class Endophytic strain

A†

B†

C†

D¶

E¶

Rhizobium sp. LS-004 Rhizobium sp. LS-079 Rhizobium sp. LS-099 Sphingomonas aquatilis LS-081 Sphingomonas sp. LS-026 Sphingomonas sp. LS-039 Sphingomonas sp. LS-101 Sphingomonas sp. LS-116 Achromobacter sp. LS-032 Achromobacter sp. LS-058 Alcaligenes sp. LS-083 Alcaligenes sp. LS-124 Burkholderia sp. LS-044 Burkholderia sp. LS-047 Herbaspirillum sp. LS-111 Acinetobacter sp. LS-110 Cedecea sp. LS-120 Enterobacter sp. LS-010 Pantoea sp. LS-013 Pantoea sp. LS-042 Pantoea sp. LS-123 Pseudomonas sp. LS-011 Pseudomonas sp. LS-037 Pseudomonas sp. LS-040 Pseudomonas sp. LS-041 Pseudomonas sp. LS-088 Pseudomonas taiwanensis LS-020 Rheinheimera sp. LS-001 Shigella sp. LS-125 Xanthomonas sp. LS-012 Bacillus aryabhattai LS-019 Bacillus aryabhattai LS-121 Bacillus safensis LS-066 Bacillus sp. LS-003 Bacillus sp. LS-005 Bacillus sp. LS-008 Bacillus sp. LS-016 Bacillus sp. LS-018 Bacillus sp. LS-023 Bacillus sp. LS-029 Bacillus sp. LS-035 Bacillus sp. LS-036 Bacillus sp. LS-057 Bacillus sp. LS-059 Bacillus sp. LS-063 Bacillus sp. LS-071 Enterococcus faecium LS-002 Lysinibacillus sp. LS-065 Staphylococcus sp. LS-114 Staphylococcus warneri LS-054 Curtobacterium sp. LS-077 Microbacterium sp. LS-009

TCN1 Root Stem Leaf a b a b a b

TCS10 Root Stem Leaf a b a b a b

TK8 Root Stem Leaf a b a b a b

TN71 Root Stem Leaf a b a b a b

Actinobacteria.† , Gram-negative; ¶ , Gram-positive

were retrieved. The DGGE band 1 represented Pantoea sp. LS-042 as it shared 100 % sequence identity with

this organism besides having highest query coverage. In addition, this band also shared high sequence similarity

Author's personal copy Plant Soil

to rice-associated cultured Pantoea sp. LS-013 (100 %) and Pantoea sp. LS-123 (99.6 %), and uncultured Pantoea SHCB0588 (100 %) and Pantoea sp. [DGGE gel band 10] (99.7 %) while establishing a strong (99 % bootstrap) phyletic cluster associated with Pantoea. In the DGGE bands 2–6 sequences were from Bacillus as they formed a separate and distinct phylogenetic cluster closely linked with other rice-associated endophytic Bacillus. In addition, bands 2–6 showed high sequence similarities to rice-associated uncultured Bacillus sp. [DGGE gel band 15] (97.8–96.8 %) and cultured Bacillus sp. P-150 (97.6–96.8 %). The sequence of DGGE band 7 showed a high identity to Massilia niastensis 5516S-1T (99.3 %), whereas DGGE band 13 showed high sequence similarity to Noviherbaspirillum suwonense 5410S-62T (99.5 %). These two sequences (bands 7 and 13) were found to be localized in the clade that accommodated both Massilia and Noviherbaspirillum species. The sequences of DGGE bands 8–9, and 18–19 were identified to correspond to sequences from the genus Xanthomonas, whereas sequences that correspond to DGGE bands 10, 12 and 15 were found to be affiliated to the genus Stenotrophomonas. These seven bands shared high sequence similarity to rice-associated uncultured Stenotrophomonas SHCB1148 (100–99.5 %), Stenotrophomonas sp. [DGGE gel band 9] (100– 99.4 %) and Xanthomonas sp. LS-012 (99.8–98.8 %). The sequences of DGGE bands 14 and 17 were from Pseudomonas oryzihabitans IAM 1568T. In addition, these bands also shared the highest sequence similarity to rice-associated Pseudomonas sp. REICA_175 (100 %) followed by uncultured Pseudomonas sp. [DGGE gel bands 2 and 12] (99.7 % each), Pseudomonas sp. [DGGE gel band 14] (98.7–98.4 %) and Pseudomonas SHCB0777 (98.1–97.8 %), and

Fig. 4 Venn’s diagrams showing the genus-wise distribution of cultured bacterial endophytes in rice cultivars originated from the acidic (a) and near-neutral soil (b). Numbers indicate the number of bacterial genera as given Table S3–S4

cultured Pseudomonas spp. LS-037 (99.8 %), LS-040 (99.8 %), LS-011 (98.7–98.4 %) and MDR7 (98.8 %). The representative images of total rhizobacteria and P-solubilizing rhizobacteria appeared on NA and CaP agar plates, respectively, for TCN1 and TK8 cultivated for 12 days under gnotobiotic P-stress conditions are shown in Fig. 7a. The corresponding rhizobacterial counts are given in Fig. 7b. On NA, TK8 showed statistically similar rhizobacterial CFU for all three treatments. Similarly, TCN1 also exhibited almost stable rhizobacterial CFU during various treatments, irrespective of accommodating significantly lower rhizobacteria as compared to TK8. The P-solubilizing rhizobacterial CFU in TK8 in the presence of soluble (1P) and insoluble P (XP) was slightly but not significantly higher than control without P (0P). On the contrary, abundance of P-solubilizing rhizobacteria was significantly high in TCN1 particularly in XP as compared to 0P and 1P. The 0P treatments showed predominance of P-solubilizing Paenibacillus sp., and Xanthomonas sp. in TK8 and TCN1 rhizospheres, respectively. On the other hand, in XP and 1P treatments, TK8 and TCN1 consistently deployed Burkholderia sp. and Acidovorax sp., respectively, as predominant P-solubilizing rhizobacteria.

Discussion The number of bacterial endophytes in plant tissues is believed to be affected by various factors including the tissue-type, developmental stage, macerating method and isolation media (Kumar et al. 2012). Rice endophytic bacteria that have been isolated through universal fullstrength and/or diluted NA (Kaga et al. 2009; Mano et al. 2006, 2007; Okunishi et al. 2005; Ruiza et al. 2011), full-strength or diluted tryptic soy agar/broth

Author's personal copy Plant Soil Table 3 Differential plant-probiotic characteristics of cultured rice-endophytic bacterial strains (CREBS) Class Endophytic strain A†

B†

C†

D¶

N2fixation

Siderophore- Chitin Antagonism vs production hydrolysis Sr/Rs

–

+

–

–

–

+

–

+

–

–

Ecozone

CaPIAAdissolution production

Rhizobium sp. LS-004

R

+


S

–


L

–

+

+

–

–

–

Sphingomonas aquatilis LS-081

S

+

+

+

–

–

–

Sphingomonas sp. LS-026

S

+

–

+

–

–

–


S

+

+

+

–

–

–


L

+

+

+

–

–

–


S

–

–

+

–

–

–

Achromobacter sp. LS-032

L

+

+

–

–

–

–

Achromobacter sp. LS-058

R

–

–

+

–

–

–

Alcaligenes sp. LS-083

L

+

–

–

–

–

–

Alcaligenes sp. LS-124

L

+

+

–

–

–

–

Burkholderia sp. LS-044

R

–

+

+

–

–

+

Burkholderia sp. LS-047

R

–

+

–

–

–

–

Herbaspirillum sp. LS-111

R

–

–

–

–

–

–

Acinetobacter sp. LS-110

R

–

–

+

–

–

–

Cedecea sp. LS-120

S

–

–

+

–

–

– –

Enterobacter sp. LS-010

S

–

+

+

–

–

Pantoea sp. LS-013

S, L

+

–

+

–

–

–

Pantoea sp. LS-042

R, S, L

–

+

+

+

–

–

Pantoea sp. LS-123

L

–

+

+

–

–

–

Pseudomonas sp. LS-011

R, S

–

+

+

+

–

–


S

–

+

+

+

–

–


S

–

+

–

–

–

–


S

–

–

–

+

–

–


R

+

+

+

+

–

–

Pseudomonas taiwanensis LS-020 R

–

+

+

+

–

–

Rheinheimera sp. LS-001

R, S

–

+

+

–

–

–

Shigella sp. LS-125

L

+

–

–

–

–

–

Xanthomonas sp. LS-012

L

–

–

+

+

+

–

Bacillus aryabhattai LS-019

R, L

–

+

+

–

–

–

Bacillus aryabhattai LS-121

R, L

+

–

–

–

–

–

Bacillus safensis LS-066

S

–

–

–

–

–

–

Bacillus sp. LS-003

R, S, L

–

+

–

–

–

–

Bacillus sp. LS-005

R

–

+

+

–

–

–

Bacillus sp. LS-008

R

+

–

+

–

–

–

Bacillus sp. LS-016

R

+

+

+

+

–

–

Bacillus sp. LS-018

R

–

+

+

–

–

+

Bacillus sp. LS-023

R

+

+

+

–

–

–

Bacillus sp. LS-029

S

–

+

–

–

–

–

Bacillus sp. LS-035

R

–

+

–

–

–

–

Bacillus sp. LS-036

R, S

–

+

+

+

–

+

Bacillus sp. LS-057

R

+

+

+

–

–

–

Bacillus sp. LS-059

R

–

+

–

–

–

–

Author's personal copy Plant Soil Table 3 (continued) Class Endophytic strain

E¶

Ecozone

CaPIAAdissolution production

N2fixation

Siderophore- Chitin Antagonism vs production hydrolysis Sr/Rs

Bacillus sp. LS-063

S

–

–

+

+

–

–

Bacillus sp. LS-071

R

–

+

+

–

–

–

Enterococcus faecium LS-002

R

–

+

+

–

+

–

Lysinibacillus sp. LS-065

S

–

+

+

–

–

–

Staphylococcus sp. LS-114

S

–

–

+

–

–

–

Staphylococcus warneri LS-054

L

–

+

–

+

–

–

Curtobacterium sp. LS-077

S

+

–

+

–

–

–

Microbacterium sp. LS-009

S

–

+

+

+

–

–

A, α-proteobacteria; B, β-proteobacteria; C, γ-proteobacteria; D, Bacilli; E, Actinobacteria; +, positive; −, negative; Gram-negative; , Gram-positive; IAA, indole acetic acid; CaP, tricalcium phosphate; R, root; S, shoot; L, leaf; Sr, Sclerotium rolfsii; Rs, Rhizoctonia solani

¶

(TSA/TSB; Ji et al. 2014; Muthukumarasamy et al. 2007), R2A agar (Hardoim et al. 2012; Loaces et al. 2011), Luria–Bertani agar (Feng et al. 2006), isolatespecific media (Muthukumarasamy et al. 2007; Tian et al. 2007) or probiotic-property-specific media (Barraquio et al. 1997; Chaudhary et al. 2012; Ji et al. 2014; Loaces et al. 2011) showed significant variation in their number. On NA, in the present work, four rice cultivars showed ~1.4×106 to 1.0×108 endophytic bacterial CFU g−1 seed endosphere. These values were found to be slightly higher than the data (5 × 106 CFU g−1 tissue) reported earlier by Ruiza et al. (2011) for rice seed bacterial endophytes isolated on NA. This discrepancy in numbers suggests possible variation in endophytic bacterial abundance in the seed endosphere of different rice genotypes. The gnotobiotic study revealed relatively higher rhizobacterial count in TK8 as compared to TCN1, which is in line with the

Fig. 5 Venn’s diagrams showing the distribution of plant-probiotic functionality (bacterial endophyte-derived) in rice cultivars originated from the acidic (a) and near-neutral soil (b). Numbers indicate the number of plant-probiotic features

seed endosphere CFU data obtained for these two cultivars. Interestingly, endophytic bacterial count obtained in this study for various tissues after 60 days of cultivation under non-sterile paddy soil was lower (~3.5×10 to ~6.0×104 CFU g−1 tissue) as compared to that of seed endosphere. This might be due to active dispersal of endophytes into various tissues as well as into the rhizoshpere. In addition, the surface disinfection and moisture content were believed to affect negatively the bacterial count in the wet tissues. The rice root tissues were identified to possess higher endophytic bacteria as compared to stem and leaf tissues in this study. Rice plants that are cultivated under aseptic soil were reported to accommodate large number of bacterial endophytes in the shoot than the root tissues (Hardoim et al. 2012). In contrast, plant roots originated from the open fields (non-sterile soil) contained higher endophytic bacteria as compared

Author's personal copy Plant Soil Noviherbaspirillum suwonense 5410S-62T (JX275858) Noviherbaspirillum malthae CC-AFH3T (DQ490985) DGGE band 13 (KM065400) 99 DGGE band 7 (KM065395) Massilia niastensis 5516S-1T (EU808005) Massilia timonae UR/MT95T (U54470) 79 Massilia haematophila CCUG 38318T (AM774589) 90 Uncultured Pantoea sp. [DGGE gel band 10] (JN110455) Pantoea agglomerans DSM 3493T (AJ233423) DGGE band 1 (KM065389) Pantoea sp. LS-042 (KJ584027) Pantoea sp. LS-123 (KJ584033) T 99 Pantoea ananatis ATCC 19321 (U80209) Pantoea septica LMG 5345T (EU216734) Uncultured Pantoea SHCB0588 (JN697822) Pantoea sp. LS-013 (KF870425) Uncultured Pseudomonas sp. [DGGE gel band 14] (JN110459) 85 Pseudomonas sp. LS-020 (KF870428) Uncultured Pseudomonas SHCB0777 (JN697940) Pseudomonas sp. LS-088 (KF870448) Pseudomonas sp. LS-041 (KF870437) Pseudomonas sp. MDR7 (AM911672) Uncultured Pseudomonas sp. [DGGE gel band 12] (JN110457) Pseudomonas sp. LS-011 (KF870423) T 83 94 Pseudomonas aeruginosa DSM 50071 (HE978271) Uncultured Pseudomonas sp. [DGGE gel band 2] (JN110447) Pseudomonas sp. REICA_175 (JN697674) Pseudomonas oleovorans ssp. oleovorans IAM 1508T (D84018) Pseudomonas sp. LS-040 (KF870436) Pseudomonas sp. LS-037 (KF870434) DGGE band 14 (KM065401) DGGE band 17 (KM065403) Pseudomonas psychrotolerans C36T (AJ575816) Pseudomonas oryzihabitans IAM 1568T (AM262973) Xanthomonas cynarae CFBP 4188T (AF208315) Xanthomonas campestris ATCC 33913T (AE008922) Xanthomonas sacchari LMG 471T (Y10766) DGGE band 9 (KM065397) DGGE band 18 (KM065404) 85 DGGE band 19 (KM065405) DGGE band 8 (KM065396) Xanthomonas sp. LS-012 (KF870424) 87 DGGE band 10 (KM065398) DGGE band 15 (KM065402) DGGE band 12 (KM065399) Stenotrophomonas maltophilia ATCC 13637T (AB008509) Uncultured Stenotrophomonas SHCB1148 (JN698214) Stenotrophomonas pavanii ICB 89T (FJ748683) Uncultured Stenotrophomonas sp. [DGGE gel band 9] (JN110454) Stenotrophomonas humi R-32729T (AM403587) DGGE band 5 (KM065393) Bacillus muralis LMG 20238T (AJ628748) 93 DGGE band 3 (KM065391) DGGE band 6 (KM065394) DGGE band 4 (KM065392) DGGE band 2 (KM065390) Bacillus simplex NBRC 15720T (AB363738) Uncultured Bacillus sp. [DGGE gel band 15] (JN110460) Bacillus sp. P-150 (AM412171) 91 Bacillus sp. LS-035 (KF870433) 98 Bacillus sp. LS-059 (KJ584029) Bacillus sp. LS-063 (KF870442) Bacillus sp. LS-003 (KJ584023) Bacillus sp. LS-019 (KF870427) Bacillus sp. LS-071 (KJ584030) Bacillus sp. LS-121 (KF870455) Bacillus sp. LS-023 (KF870429) Bacillus sp. LS-066 (KF870444) Bacillus sp. LS-057 (KJ584028) Bacillus sp. LS-008 (KF870421) 82 Bacillus alt LS-016 (KJ584025) Bacillus sp. LS-005 (KF870420) Bacillus subtilis DSM10T (AJ276351) Bacillus sp. LS-036 (KJ584026) 89 Bacillus sp. LS-018 (KF870426) Bacillus sp. LS-029 (KF870431) Nitrososphaera viennensis EN76T (FR773157) 0.05


Fig. 6

Maximum likelihood tree based on 16S rRNA gene sequences (~350 bp each) showing phylogenetic relationship of cultured and uncultured rice endophytes. Present PCR-DGGE data highlighted in bold-type. Data given in blue-colored font indicates previously described rice-associated taxa. Clades given in red, green and blue color indicates representatives of class βproteobacteria, γ-proteobacteria and Bacilli. Bootstrap values of >70 % after 1000 replications are shown at branching nodes. Scale bar, 0.05 substitutions per nucleotide position. Inset shows PCRDGGE profiles of seed-endosphere of four different cultivars of rice. Soil archaeon Nitrososphaera viennensis EN76T (FR773157) was used as an outgroup. The band identification data are summarized in Table S5

to other tissues (Hallmann et al. 1997; Sun et al. 2008). Increased root endophytes in the non-sterile

soil could be due to active invasion and colonization of soil-borne bacteria (Botta et al. 2013; Ji et al. 2010). A higher endophytic bacterial count in the rice plants cultivated in the near-neutral soil suggested pH sensitivity and possibly the importance of plant physiology in response to their environment, and/or differences in root secretion profiles that may affect plant-microbe interaction and community establishment in plants. Both culture-dependent and -independent analyses revealed significant diversity of rice endophytic bacteria (Chaudhary et al. 2012; Mano et al. 2008; Sun et al. 2008). In particular, Bacillus strains have been found

(a)

(b)

Fig. 7 Deployment of rhizobacteria in TCN1 and TK8 under gnotobiotic P-stress. Total and P-solubilizing rhizobacteria (a) and their quantitative analysis (b) are shown. Data obtained after 12 days of rice cultivation without P (OP), with 0.5 mM tricalcium phosphate (XP) and with 0.5 mM KH2PO4 (1P) followed by

plating the aliquots of rhizozphereic fluid on NA and CaP agar. Different letters (uppercase, NA; lowercase, CaP) denote significant differences (P≤0.05) according to Duncan’s multiple range test


frequently associated with rice (Kaga et al. 2009; Hallmann and Berg 2006; Mano and Morisaki 2008; Okunishi et al. 2005; Yang et al. 2008). Although, the PCR-DGGE profile of seed endospheres revealed predominance of Bacillus particularly restricted to TCN1 and TCS10, our culture-dependent analysis clearly showed prevalence of Bacillus strains in the rice cultivars planted at two contrasting soil-types. Interestingly, several endophytic Bacillus strains isolated in this study resembled the air-borne strains that were collected at high altitudes (Shivaji et al. 2006), whereas others were related to plant-probiotic strains (Niu et al. 2011; Jeong et al. 2012; Lee et al. 2012) and heavy metal resistant species (Suresh et al. 2004). In addition, almost all (except for Bacillus safensis LS-066) endophytic Bacillus showed single or several plant-probiotic features. The motility, spore-forming ability and possible tolerance to high osmotic pressure have been presumed to favor endophytic colonization of Bacillus in rice (Okunishi et al. 2005). In addition, Bacillus strains were identified to secrete lipopeptides that prevent plant disease (Ongena and Jacques 2008). The root-originated Bacillus spp. LS-018 and LS-036 were detected to be antifungal through this study. In addition, we observed peculiar nucleotide insertions/deletions particularly at the upstream end of 16S rRNA gene sequences (data not shown), which reflects unique genetic diversity existing in Bacillus. Endophytic colonization of Enterococcus, Staphylococcus and Lysinibacillus representatives in rice has never been reported. However, Actinobacteria such as Curtobacterium and Microbacterium were earlier documented to be rice endophytes (Cottyn et al. 2009; Mano et al. 2007; 2006). Members of the Gram-negative genera Pantoea, Pseudomonas and Stenotrophomonas, have been reported to be rice-endophytes (Hardoim et al. 2012). PCR-DGGE signatures correspond to Pantoea, Pseudomonas, Stenotrophomonas, Xanthomonas and Noviherbaspirillum/Massilia in rice endosphere were detected in this study. In addition, Pantoea (n=3), Pseudomonas (n=6) and Xanthomonas (n=1) representatives were isolated from various tissues of four different cultivars. Unfortunately, members of Stenotrophomonas and Noviherbaspirillum/Massilia were not recovered in this study. In line with Bacillus, Pantoea strains were also reportedly associated with rice tissues

irrespective of growth stages (Kaga et al. 2009; Okunishi et al. 2005; Mano et al. 2006). All four rice genotypes consistently accommodated the endophytic representatives of the genus Pantoea in the near-neutral soil and all Pantoea strains isolated in this study showed ≥2 plant-probiotic features. Pantoea sp. LS-042 believed to be one of the most competent endophytes particularly in TCN1. Similarly, all four rice cultivars constantly possessed endophytic Pseudomonas strains. All Pseudomonas were identified to be plant-probiotic in this study, in which LS-088 possessed a maximum of four and LS-020 exhibited at least three plant growth-promoting features. Furthermore, these two Pseudomonas strains were genetically related to the insecticidal toxin-producing biocontrol strain Pseudomonas taiwanensis BCRC 17751T (Wang et al. 2010; Liu et al. 2010). Many strains of Stenotrophomonas maltophilia have been identified to be plant-associated (Hardoim et al. 2012; Hayward et al. 2010), which substantiates our detection of Stenotrophomonas in the rice seed endosphere. Some Xanthomonas strains are responsible for bacterial blight and leaf streak diseases in rice (Swings et al. 1990). We have isolated Xanthomonas sp. LS-012 from the leaf tissue of TCS10 in near-neutral soil. The detection of Noviherbaspirillum or Massilia through PCRDGGE in the seed endospheres of TCS 10 and TK8 is considered to be significant as these two taxa have never been reported to be rice-associated. Five N2-fixing Sphingomonas strains have been detected in TCN1 and TN71 grown in near-neutral soil, in which LS-026 and LS-039 were related to Sphingomonas trueperi, a reclassified diazotroph previously known as ‘Pseudomonas azotocolligans’ (Kämpfer et al. 1997). PCR-DGGE analysis of rice cultivars by Hardoim et al. (2011a) revealed ubiquitous occurrence of Rhizobium across ten different rice cultivars, whereas Burkholderia and Enterobacter representatives were found to be heterogeneous in distribution. No evidence for the presence of Rhizobium, Burkholderia and Enterobacter in the seeds through PCR-DGGE was obtained in this study. However, we were able to isolate strains of Burkholderia (n= 2), Rhizobium (n=5) and Enterobacter (n=1) with plant-probiotic features from various tissues of different rice cultivars. In line with the biocontrol


attributes of Burkholderia reported earlier (Cuong et al. 2011), we found that the root-originated Burkholderia sp. LS-044 with multiple plantprobiotic traits, was antagonistic to Sclerotium rolfsii and Rhizoctonia solani. Predominance of Gram-positive strains in the root tissues, and Gram-negative strains in the stem and leaf tissues was recorded in this study. Similar observation was also made earlier on the tissues of mature rice plants by Mano et al. (2006). Among 52 CREBS affiliated to 20 different genera identified in this study, ~60 % genera have been reported to be seed-borne (Hardoim et al. 2012). In particular, Rheinheimera sp. LS-001, Acinetobacter sp. LS-110, and Enterobacter sp. LS-010 were genetically related to rice root-associated bacterium Rheinheimera tangshanensis (Zhang et al. 2008), diazotrophic rice endophyte ‘Acinetobacter oryzae’ (Chaudhary et al. 2012) and rice seed endophyte ‘Enterobacter oryzendophyticus’ (Hardoim et al. 2013), respectively. However, endophytic colonization of strains such as Alcaligenes, Cedecea and Shigella in rice has never been reported. Genetically related isolates of the genera Achromobacter, Bacillus, Burkholderia, Pseudomonas, Cedecea, Shigella, Massilia and Staphylococcus reported to exhibit clinical/pathogenic attributes (Veitch et al. 2014; Vandamme et al. 2013; Holt et al. 2012; Vanlaere et al. 2008; Clark et al. 2006; La Scola et al. 1998; Dan et al. 1984; Grimont et al. 1981). Taken together, it seems that rice cultivars served not only as hosts for diverse plant-probiotic bacterial stains, but also for putative opportunistic pathogenic bacteria. Reports suggest that rice seed itself can acts as a source of endophytic bacteria (Hardoim et al. 2012; Kaga et al. 2009; Mano et al. 2006). In addition, Hardoim et al. (2012) demonstrated an active dissemination of seed-borne endophytes in to shoot and root tissues of rice using aseptic soil. Rice endophytic bacterial taxa have been reported to fluctuate considerably according to the seed generation, plant growth stage, tissue type and host genotype (Hardoim et al. 2012; Kaga et al. 2009; Mano et al. 2006, 2007; Okunishi et al. 2005; Elbeltagy et al. 2000; Engelhard et al. 2000). In addition, soil-effect has also been reported to be the major contributing factor in shaping endophytic bacterial community (Hallmann and Berg 2006; van Overbeek and van Elsas 2008). Diversity indices and

Venn’s plot presented in this study substantiated the fluctuation in terms of endophytic bacterial taxa according to the soil-type and rice genotype. Endophytic bacteria might actively influence the physiology of host by producing phytohormones and/ or the modulation of host ethylene levels. Many other plant-probiotic functions, such as N2fixation, P-solubilization, provision of micronutrients, promotion of photosynthetic activity, induction of the plant defense system, production of antibiotics, biotransformation of heavy metals and biodegradation of organic pollutants, might also enhance host fitness (Compant et al. 2010). Our results showed that the rice cultivars steadily maintained genetically diverse endophytic bacteria with at least three to four plant-probiotic functions irrespective of the soil type tested. The presence of diazotrophic strains/bacterial N2-fixing genes has been reported from diverse varieties and cultivars of rice (Barraquio et al. 1997; Chaudhary et al. 2012; Knauth et al. 2005; Muthukumarasamy et al. 2012). Rice plant genotype has been identified to influence the association with N2-fixing bacteria in its rhizosphere (Hirota et al. 1978). Genetic diversity of endophytic diazotrophs in rice appears to be far more complex as 36 distinct strains affiliated to 17 discrete genera were isolated in this study. Endophytic siderophore-producing bacteria are reported to be ubiquitous in grains, roots and leaves of the rice (Loaces et al. 2011). In this study, diverse siderophore-producing strains of the genera Pseudomonas (n=5), Bacillus (n=3), Microbacterium (n = 1), Pantoea (n = 1), Rhizobium (n = 1), Staphylococcus (n=1) and Xanthomonas (n=1) were isolated. Iron deficiencies have been usually encountered in alkaline pH soils, which would explain enhanced occurrence of endophytic siderophoreproducers in the cultivars originated from slightly alkaline soil. Considerable amounts of endophytic Psolubilizers and IAA-producers were also detected in rice. The high proportion of P-solubilizing endophytes particularly observed in the rice cultivars grown in acidic soil suggested possible preference for P-solubilizers in P-limited soil. Plant-probiotic endophytes previously presumed to disseminate and colonize rhizosphere to accelerate specific plant growth-promoting or biocontrol effects, which could be of great advantage for the host (Hartmann et al. 2009). A short-term gnotobiotic Pstress experiment under acidic condition was performed by using TCN1 and TK8 to investigate the possible


transformation of endophytes into rhizobacteria in rice. Our results suggest that rice cultivars consistently deployed a steady rhizobacterial community that includes P-solubilizers, irrespective of the presence or absence of P. TCN1 found to deploy predominantly P-solubilizing Acidovorax sp., whereas TK8 established P-solubilizing Burkholderia sp. in 1P and XP treatments. TCN1 mainly recruited P-solubilizing Xanthomonas sp., whereas TK8 deployed P-solubilizing Paenibacillus sp. during 0P treatments. Inoculation of P-solubilizing bacteria has been reported to increase P-solubilization as well as rice growth (Panhwar et al. 2011, 2014), which may partially explain the rhizospheric deployment of P-solubilizers by rice. The P-solubilizing rhizobacterial strains presently isolated have been found to be unique (as determined by 16S rRNA gene sequencing, data not shown) when compared to 52 shortlisted endophytic strains, which further corroborated both genetic complexity and diversity of endophytic taxa associated with rice. Analysis of plant growth-promoting effects of selfdeployed P-solubilizing rhizobacteria by rice would be an interesting field of further research. The interactions between plant and microbes are quite complex, and the mechanisms involved in host selection and specificity would require additional molecular approaches to elucidate.

Conclusions Present culture-dependent and -independent analyses corroborated significant genetic diversity of bacterial endophytes existing in the rice. In addition, this study provided baseline information regarding the richness, and diversity in terms of cultivable bacterial endophytes and their differential plant-probiotic functions in four rice genotypes grown in two different soils. Rice plants found to harbor both plant-probiotic and putative pathogenic bacterial strains as endophytes. We provided direct evidence that shows transforming ability of endophytes into rhizobacteria in rice. The distribution within plant, and rhizospheric deployment of bacterial endophytes in rice found to be influenced by host genotype, edaphic factors and nutrient stress. Present study will be helpful in designing and testing multifunctional endophytic bacterial consortia that could assist plants to withstand a variety of biotic- and abiotic-stresses.

Acknowledgments Authors would like to thank the editor and anonymous reviewers for their constructive comments on this manuscript. Authors acknowledge Dr. Wei-An Lai for technical support. This research was funded by grants (Grant No. 100-2313B-150-001-MY3 and 102-2321-B-150-001) from Ministry of Science and Technology, Taiwan.

Conflict of interest The authors declare that they have no conflict of interest.

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