Acidophilic actinomycetes from rhizosphere soil: diversity and properties beneficial to plants

ABSTRACT Three hundred and fifty-one isolates of actinomycetes were recovered from 21 rhizospheric soil samples using acidified media of pH 5.5. They were evaluated for their antifungal,

siderophore production and phosphate solubilization activities. The total count of actinomycetes growing on acidified starch casein agar and Gause no. 1 agar were below 2.48 × 104 CFU g−1

soil. Two hundred and twelve isolates were assigned to acidophiles and the remaining 139 isolates were neutrophiles. Of these actinomycetes, 57.8, 32.5 and 50.4%, showed antagonistic

activity against three rice pathogenic fungi; _Fusarium moniliforme, Helminthosporium oryzae_ and _Rhizoctonia solani_, respectively. More than half of the isolates (68.1%) inhibited at

least one tested pathogenic fungus, whereas 25.9% exhibited antifungal activities against all tested fungi. Three hundred and thirty-eight isolates (96.3%) produced siderophore and 266

isolates (75.8%) solubilized phosphate. A greater proportion of the acidophilic actinomycetes exhibited antifungal, siderophore production and phosphate solubilization activity compared with

the neutrophiles. Three hundred and twenty-five isolates (92.6%) were classified as streptomycetes based on their morphological characteristics and the presence of the _LL_-isomeric form of

diaminopimelic acid in whole-cell hydrolysates. The 16S ribosomal RNA (rRNA) gene analysis of representative non-streptomycete strains showed that the isolates belonged to seven genera,

that is, _Allokutzneria_, _Amycolatopsis_, _Mycobacterium_, _Nocardia_, _Nonomuraea_, _Saccharopolyspora_ and _Verrucosispora_. The potential antifungal acidophilic isolates, R9-4, R14-1,

R14-5 and R20-5, showed close similarity to _Streptomyces misionensis_ NBRC 13063T (AB184285) in terms of morphological characteristics and 16S rRNA gene sequences. SIMILAR CONTENT BEING

VIEWED BY OTHERS ISOLATION AND EVALUATION OF QATARI SOIL RHIZOBACTERIA FOR ANTAGONISTIC POTENTIAL AGAINST PHYTOPATHOGENS AND GROWTH PROMOTION IN TOMATO PLANTS Article Open access 12 December

2023 IDENTIFICATION, FERMENTATION OPTIMIZATION, AND BIOCONTROL EFFICACY OF ACTINOMYCETE YG-5 FOR THE PREVENTION OF _ALTERNARIA_ LEAF SPOT DISEASE IN STAR ANISE Article Open access 10 August

2024 EXPLORING THE RHIZOSPHERE OF PERENNIAL WHEAT: POTENTIAL FOR PLANT GROWTH PROMOTION AND BIOCONTROL APPLICATIONS Article Open access 01 October 2024 INTRODUCTION Actinomycetes are a

diverse group of Gram-positive bacteria with high GC content, which are common in soil and widely distributed in various environments. They represent the most economically and biologically

valuable bacteria among prokaryote, producing various biologically active substances such as antibiotics, antitumors and enzymes.1, 2, 3 Although actinomycetes are commonly known as

neutrophiles, which grow well in neutral or slightly alkaline conditions, a few actinomycetes such as _Streptomyces acidiphilus_4 and members of the genus _Streptacidiphilus_5 have been

reported to require acidic conditions (pH 2.6–5.5) for growth. In addition, the members of the genera _Actinospica_6 and _Catenulispora_7 are also known as acidophilic actinomycetes.

Acidophilic actinomycetes can be divided into two main groups, which are neutrotolerant acidophiles and strict acidophiles. Typical neutrotolerant acidophiles grow in media at pH 4.5–7.5

with optimum growth between pH 5.0 and 5.5. Members of strictly acidophilic group typically grow in media between pH 3.5 and 6.5, with an optimum growth at pH 4.5.8, 9 In acidic habitats,

the actinomycetes commonly found belong to the genus _Streptomyces._10 Rice is a staple crop that supports about half of the world’s population. However, fungal rice diseases are major

problems in rice cultivation.11 The use of chemical synthetic compounds is regarded as an effective method for prevention and therapy, but has deleterious effects on health and the

environment.12, 13 It is necessary to find an environmental-friendly alternative for sustainable protection management. Actinomycetes have been considered as potential biocontrol agents

against various phytopathogenic fungi because of their production of bioactive metabolites14, 15, 16 or of enzymes that hydrolyzed fungal cell walls.17, 18, 19, 20 Most of the previous works

were focused on the ability of actinomycetes isolated from neutral pH isolation media. However, acidophilic actinomycetes were reported to inhibit fungi better than neutrophilic

actinomycetes under acidic condition.21 Crawford _et al._22 also reported that acidophilic actinomycetes exhibited strong antagonism toward multiple fungal root pathogens. In addition,

Basilio _et al._14 demonstrated that actinomycetes isolated under alternative selective pH conditions possess a significant capacity to produce compounds with antimicrobial activity. In rice

field soil, the pH is usually slightly acidic, which is favorable for the growth of pathogenic fungi. The use of acidophilic actinomycetes in the rice field would be more effective than

neutrophilic strains. However, little attention has been given to determining the diversity and antifungal activity of acidophilic actinomycetes. Therefore, in this study, actinomycetes were

isolated from rhizospheric soils under acidic selective conditions and their ability to inhibit the rice pathogenic fungi _Fusarium moniliforme_, _H. oryzae_ and _R. solani_ was assessed.

The diversity of the actinomycetes obtained was also investigated. MATERIALS AND METHODS SAMPLE COLLECTION AND SELECTIVE ISOLATION OF ACTINOMYCETES Rhizospheric soil samples (17 samples from

rice plants and 4 samples from rubber trees) were collected from several provinces of Thailand (Chiang Mai, Nakhon Sawan, Pathum Thani, Phatthalung, Phetchaburi, Phra Nakhon Si Ayuttaya,

Ratchaburi and Suphan Buri). The samples were air dried, and sieved to remove unwanted particles. The pH of soil was determined according to Davet.23 Air dried soil sample was mixed with

distilled water at a ratio of 1:2.5, thoroughly mixed and left to stand for 30 min before pH measurement with pH meter. The isolation of actinomycetes was conducted using a dilution plate

technique. One gram soil sample was diluted in 4 ml of 0.85% (w/v) NaCl solution, mixed well and then heat treated at 55 °C for 10 min in a water bath to eliminate fast growing bacteria.

Serial 10–fold dilutions were prepared for each pretreated sample and aliquots (0.1 ml) of each dilution were spread onto acidified starch casein agar (SCA)24 and acidified Gause no. 1,25 pH

5.5, supplemented with nalidixic acid, nystatin and ketoconazole at final concentration of 25 μg ml−1, 50 μg ml−1 and 100 μg ml−1, respectively. Media of pH 5.5 was prepared by mixing

sterile double strength media with an equal volume of sterile citrate/phosphate buffer of pH 5.5 (0.1 M citrate/0.2 M Na2HPO4). The method for pH adjustment using buffer was applied to all

media used in this experiment, otherwise indicated. The plates were incubated at 28 °C for 28 days, and then the number of actinomycete colonies, recognized by their morphological

characteristics, was counted. The colonies were then selected and restreaked on acidified International _Streptomyces_ Project (ISP) medium 226 (pH 5.5) for purity checking and maintained on

the same medium at room temperature. Spores and mycelial suspensions were stored in 20% (v/v) glycerol solution at −20 °C as stock culture. ACTINOMYCETE CHARACTERIZATION Putative

actinomycete isolates were characterized by their morphological, chemical and physiological characteristics. Their morphological characteristics were observed on acidified ISP medium 326 (pH

5.5), after incubation at 28 °C for 14 days. The color of spore mass and diffusible pigment production were determined and recorded by comparison with a color chart.27 The isomeric form of

2,6-diaminopimelic acid (DAP) of whole organism hydrolysates was determined using paper chromatography according to the method as described by Hasegawa _et al._28 and Becker _et al._29 The

assignment of actinomycetes as acidophilic or neutrophilic strains was based on their ability to grow on ISP medium 2 at pH 4.5 and 7.5. The growth of each isolate was observed after being

incubated at 28 °C for 3, 7 and 14 days. The isolates that grew better or grew only on acidified media were regarded as acidophiles and the isolates that grew better or grew only on media of

pH 7.5 were regarded as neutrophiles.30 _IN VITRO_ ANTIFUNGAL ACTIVITY Antagonistic activity of the isolates against three rice pathogenic fungi _F. moniliforme_ (bakanae), _H. oryzae_

(brown spot) and _R. solani_ (sheath blight), were evaluated on potato dextrose agar (PDA, pH 5.5) using a dual culture technique.31 These fungi were maintained on PDA at room temperature.

Two 7-day-old actinomycete discs (5 mm) grown on acidified ISP medium 2 (pH 5.5) were placed on opposite sides, 3 cm away from the center of the PDA plate. After incubation at 28 °C for 7

days, a fungal mycelia disc (5 mm in diameter) was placed in the center of the plate. PDA plates with fungal mycelia discs in the center of the plate (without actinomycete) served as

control. The plates were incubated at 28 °C until the fungal mycelium of control reached the edge of the plate. The percentage of inhibition was calculated as [(_r_1−_r_2)/_r_1] × 100.

Where, _r_1 is the radial mycelia growth in control and _r_2 is the radial mycelia growth that occurs toward the actinomycetes.32 The experiment was conducted in duplicate. SCREENING FOR

SIDEROPHORE PRODUCTION Discs (5 mm) containing good lawns of actinomycete isolates grown on ISP medium 2 (pH 5.5) at 28 °C for 7 days were placed on chrome azurol S agar.33 Sterile ISP

medium 2 (pH 5.5) discs were also placed on chrome azurol S agar as controls. The inoculation plates were incubated at 28 °C for 5 days. An orange halo around the agar discs was considered

as siderophore-producing activity and the diameter of the halo was recorded. The experiment was conducted in duplicate. SCREENING FOR PHOSPHATE-SOLUBILIZING ACTINOMYCETES All actinomycete

isolates were cultured on ISP medium 2, at pH 5.5 and incubated at 28 °C for 7 days. Discs (5 mm) containing good lawn of the isolates were placed on Pikovskaya agar.34 The inoculation

plates were incubated in the dark at 28 °C for 14 days. The presence of a clear zone around the agar discs was considered as phosphate-solubilizing activity and the diameter of clear zone

was measured. The experiment was conducted in duplicate. DNA EXTRACTION, PCR AMPLIFICATION AND SEQUENCING OF 16S RIBOSOMAL RNA (RRNA) GENE Genomic DNA was extracted as described by Kieser

_et al._35 PCR amplification of 16 S rRNA gene was carried out using the primers: STR1F (5′-TCACGGAGAGTTTGATCCTG-3′) and STR1530R (5′-AAGGAGATCCAGCCGCA-3′).36 The PCR consisted of an initial

denaturation step at 94 °C for 4 min, followed by 30 cycles of 94 °C for 1 min, 57 °C for 1 min, 72 °C for 1 min and a final extension step for 10 min at 72 °C. Sequencing of 16S rDNA was

performed using the service of Macrogen (Seoul, Korea) and 1st BASE Laboratory (Selangor, Malaysia), with the same primers as those used for PCR amplification. The resultant sequences were

compared with other sequences of related type strains available in the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/). The 16S rDNA sequences were aligned with related species using

CLUSTALW program.37 A neighbor-joining38 phylogenetic tree was generated using MEGA version 5.0 software,39 evaluated by bootstrap analysis of 1000 replications; a distance matrix was

generated using Kimura’s 2-parameter model.40 RESULTS AND DISCUSSION SELECTIVE ISOLATION OF ACTINOMYCETES Both rice plants and rubber trees can grow in acid soil and the characteristic

acidic pH of paddy soils and of rubber tree plantation soils have been reported.41, 42 In this study, the pH of rice rhizosphere soils (R1 to R17) ranged from strongly acidic to slightly

alkaline (pH 3.1–7.6) and the rubber tree rhizosphere soils (R18 to R21) were highly acidic to slightly acidic (pH 4.7–6.5) (Table 1). In a preliminary test, five soil samples were used to

isolate acidophilic actinomycetes at pH 4.5. One gram of each soil sample was suspended in 4 ml of 0.85% NaCl, heated at 55 °C for 10 min before being spread on acidified SCA medium with pH

adjusted to 4.5, supplemented with nalidixic acid (25 μg ml−1) and nystatin (50 μg ml−1). However, after a few days of incubation, most isolation plates were covered with fungal mycelium,

which overgrew the small colonies of actinomycetes. As Muramatsu _et al._43 observed, the isolation of acidophilic actinomycetes is difficult due to fungal contamination, which grow well

under acidic conditions. In addition, most antifungal agents such as nystatin are unstable at low pH. Therefore, the isolation media used were evaluated with those soil samples to

selectively isolate acidophilic actinomycetes by adjusting the pH of the media within the range of 4.0–6.0 and supplemented with fungal inhibitors such as salt (sodium chloride and sodium

propionate), dyes (rose Bengal and Congo red) and antibiotics (nalidixic acid, nystatin and ketoconazole). The results showed that acidified SCA and Gause no. 1 agar plates of pH 5.5,

supplemented with 25 μg ml−1 antibiotic nalidixic acid , 50 μg ml−1 nystatin and 100 μg ml−1 ketoconazole had fewer fungal contamination and gave higher number of actinomycete colonies (data

not shown). Therefore, these two media were then applied for all soil samples. After incubating the plates at 28 °C for 28 days, colonies of actinomycetes were counted. Most soil samples

showed actinomycete colonies <30 CFU per plate on Gause no. 1 medium, which had been spread with soil suspension at a dilution of 1:4. The highest number of actinomycete colonies (2.48 ×

104 CFU g−1 soil) was found in rice plant rhizospheric soil sample (R2) on SCA medium (Table 1). In total, 351 actinomycete isolates were obtained from 21 soil samples (276 isolates from

rice rhizospheres and 75 isolates from rubber tree rhizospheres). The highest number of actinomycete isolates (_n_=28) were obtained from sample R11, and the lowest number of only one

isolate from sample R12. CHARACTERIZATION OF THE ISOLATES The detection result of 2,6-DAP from whole-cell hydrolysates showed that 325 isolates (93.2%) contained _LL_-DAP. From their

morphological characteristics and the DAP type, this majority of isolates were then assigned to the streptomycete group. This number included members of the genus _Streptacidiphilus_, which

has similar colony morphology and DAP type as the genus _Streptomyces_. These 325 isolates were classified into five color groups based on their spore color on ISP medium 3. The isolates

which produced gray spore color were dominant (53.5%), followed by brown (18.5%), white (4.9%), yellow (4.6%) and green (1.5%). Fifty-five isolates (16.9%) in the streptomycete group did not

produce spores on acidified ISP medium 3 (pH 5.5). The remaining 26 isolates (6.8%), containing _meso_-DAP, were assigned to the non-streptomycete or rare actinomycete group.

Representatives of these non-streptomycetes (23 isolates) and streptomycetes (35 isolates) were identified to the genus level by 16S rRNA gene sequences analysis. All 351 isolates were

evaluated for their pH requirement for growth. The results showed that 212 isolates could grow better on pH 4.5 medium when compared with pH 7.5 and were assigned to acidophilic

actinomycetes. However, they were not strictly acidophilic actinomycetes because these isolates could grow on medium pH 7.5. Ninety isolates grew better on pH 7.5 medium when compared with

pH 4.5 and the remaining 49 isolates which grew only on pH 7.5 medium were assigned to neutrophilic actinomycetes. A previous study reported that acidophilic actinomycetes were present in

soils in which the pH did not exceed 6.8 and were not found in slightly alkaline soil.44 However, in this study acidophilic actinomycetes were found in all soil samples including those with

slightly alkaline pH. One possible explanation is that soils are heterogeneous systems with multiple microzones, where pH may considerably differ from the average value. In acidic soil,

neutrophilic actinomycetes grew and generated spores in microsites of ammonia adsorption on organic fragments and the spores survived.8 On the other hand, acidophilic actinomycetes could

grow in alkaline soils around plant roots that release H+ for nutrient exchange, causing acid microsite, and they were recovered on an acidified isolation media. Among 325 streptomycete

isolates, 205 isolates were classified to acidophilic strains and 120 isolates to neutrophilic strains. Nineteen isolates of non-streptomycetes were classified to neutrophilic strains,

whereas the other seven isolates were acidophilic strains. According to previous studies, streptomycetes were the most notable acidophilic actinomycetes, which is probably related to the

fact that they are predominant in most soils.10, 43, 45 ANTIFUNGAL ACTIVITY The _in vitro_ antifungal activity assay showed that, 203 isolates (57.8%), 114 isolates (32.5%) and 177 isolates

(50.4%) suppressed _F. moniliforme, H. oryzae_ and _R. solani_, respectively. Two hundred and thirty-nine isolates (68.1%) inhibited at least one tested pathogenic fungus and 91 isolates

(25.9%) exhibited antifungal activity against all tested fungi. All antagonistic isolates belong to the streptomycete group except R8-39 and R13-3, which were non-streptomycete strains.

Isolate R8-39 inhibited the growth of all tested fungi and R13-3 inhibited only _F. moniliforme_. The percentage of inhibition activity of isolate R8-39 to inhibit _F. moniliforme_, _H.

oryzae_ and _R. solani_ were 74%, 84% and 71%, respectively. Nine streptomycete isolates exhibited >80 percent of inhibition against all pathogenic fungi. Among these, eight strains

(R9-4, R14-1, R14-5, R14-6, R14-10, R18-16, R20-5 and R21-2) were acidophiles and only one strain (R19-3) was neutrophile. Strains R9-4, R14-1, R14-5 and R20-5, which gave the highest

antagonistic activity, were selected for 16S rRNA gene analysis. Previous studies using actinomycetes isolated from neutral pH isolation media, reported lower percentages of antagonistic

behavior toward _F. moniliforme_ than that found in this study. Baniya and Vaidya46 isolated 28 actinomycetes from vemicompost, of which only 4 isolates (10.5%) were active against _F.

moniliforme._ Hatamy _et al._47 studied the inhibition activity of 100 actinomycetes isolated from greenhouse soil in Iran, reported that 18 strains showed high activity against _F.

moniliforme_, and other neutrophilic actinomycetes showed antagonistic activity against _H. oryzae_ and _R. solani_. Kathiresan _et al._48 isolated 160 actinomycetes from sediment samples in

India and found that about 51% and 31% of isolates were effective against _H. oryzae_ and _R. solani_, respectively. Acidophilic actinomycetes are usually scattered in acid terrestrial

systems,45, 49 in which they were important saprophytes and antagonistic microorganisms.21, 30 It is important to explore actinomycetes having antifungal activity in acidic condition for

direct use as biocontrol in acidic environment. Therefore, both acidophilic and neutrophilic actinomycetes were evaluated for their antifungal activity on acidic medium. Comparison of

antifungal activity between acidophilic and neutrophilic actinomycetes (Table 2) showed that acidophilic isolates gave higher percentage of inhibition with all tested pathogenic fungi. In

this experiment, all actinomycetes were evaluated for their antifungal activity on acidified agar, pH 5.5, and a greater proportion of acidophilic actinomycete isolates showed antifungal

activity compared with neutrophilic strains under acidic condition. This may be due to the acidic condition that supported the growth of acidophilic more than neutrophilic isolates.

SIDEROPHORE PRODUCTION Streptomycete isolated from roots of a Thai jasmine rice plant with siderophore production was reported to promote plant growth.50 In this experiment, the ability of

all 351 isolates to produce siderophore was tested. Three hundred and thirty-eight isolates exhibited yellow to orange halos on chrome azurol S agar plate, and were thus considered to be

siderophore producers. Two hundred and forty-five isolates (69.8%) exhibited positive zone diameters in the range of 1.1–2.0 cm, and 13 isolates exhibited zone diameters >3.0 cm. There

were reports suggesting that the siderophore-producing bacteria could inhibit fungi by Fe ion uptake competition. The produced siderophore could inhibit the growth of fungi or induce the

resistance of the plant to phytopathogenic fungi.51, 52, 53 Rhizospheric soil actinomycetes were also reported to inhibit the growth of phytopathogens and produce siderophore.54 In this

study, 70.4% of siderophore-producing strains exhibited antifungal activities. On the other hand, 12 of 13 strains shown no siderophore production and could not inhibit any pathogenic fungi

(_F. moniliforme, H. oryzae_ and _R. solani_). The comparison of acidophilic and neutrophilic actinomycetes for siderophore production showed that the percentage of siderophore-producing

acidophilic strains was slightly higher than neutrophilies. The proportion of siderophore production by acidophilic strains was 98.6% compared with 92.8% in neutrophiles. PHOSPHATE

SOLUBILIZATION Among 351 actinomycetes, 266 isolates (75.8%) showed an ability to solubilize phosphate on Pikovskaya agar with the diameters of clear zones ranging from 0.7–1.6 cm. Only six

isolates produced clear zones with diameters >1.5 cm. Five isolates were acidophiles and one isolate was neutrophile. Phosphorus is an important element for plant growth. However, most of

the phosphorus in soil are in insoluble forms, which are unavailable for plants. Deficiency of phosphate has negative effect on agricultural yield. Actinomycetes were reported to possess

the ability to solubilize phosphate and thus significantly promote the growth of plants.55, 56 In acidic soils, phosphorus limitation is a serious problem because phosphorus forms complexes

with metal ions.57 In this experiment, it was found that 93.9% of the acidophilic group and 48.2% of the neutrophilic group were able to solubilize phosphate. This indicated that

phosphate-solubilizing acidophilic actinomycetes might have an important role in the phosphorus cycle in acidic soils. 16S RRNA GENE ANALYSIS The 16S rRNA gene sequencing analysis was

carried out on 23 non-streptomycete isolates, which were preliminary identified on the basis of morphological characteristics and chemotaxonomic analysis. The resultant sequences were

determined and compared with the sequences available in the EzTaxon-e database. The percentages of 16S rRNA gene sequence identity of these isolates to the closest type strain are presented

in Table 3. The results revealed that they were members of the following genera: _Allokutzneria_ (one isolate, family _Pseudonocardiaceae_), _Amycolatopsis_ (five isolates, family

_Pseudonocardiaceae_), _Nocardia_ (two isolates, family _Nocardiaceae_), _Nonomuraea_ (nine isolates, family _Streptosporangiaceae_), _Mycobacterium_ (four isolates, family

_Mycobacteriaceae_), _Saccharopolyspora_ (one isolate, family _Pseudonocardiaceae_) and _Verrucosispora_ (one isolate, family _Micromonosporaceae_). The genus _Nonomuraea_ was the most

frequently found. Most of non-streptomycete isolates were neutrophiles; the five exceptions were members of the genus _Amycolatopsis_ (isolates R8-21, R12-7, R13-25, R13-26 and R15-35),

which were acidophiles. Phylogenetic tree constructed based on the 16S rRNA gene sequences, which was supported by high bootstrap values, confirmed their affiliation to these families is

shown in Figure 1. Based on colonial characteristics and morphological characteristics under light microscope, another non-streptomycete isolate R13-3, which showed ability to inhibit _F.

moniliforme_ was presumed a member of isolates R8-21, R13-25, R13-26 and R15-35, which identified to the genus _Amycolatopsis_ by 16S rRNA gene sequence analysis. In this study, one strain

of the genus _Allokutzneria_ (strain R8-39) was isolated from a rice rhizosphere soil. The genus _Allokutzneria_ encompasses only two species, _Allokutzneria albata_ and _Allokutzneria

multivorans_, which were isolated from soils in the Philippines and China, respectively.58, 59, 60 Only _A. albata_ has been reported to produce a highly active antiviral antibiotic

(cycloviracin).58 The strain R8-39 shared the highest 16S rRNA gene similarity of 98.8% to _A. albata_ and clustered with _A. albata_ and _A. multivorans_ in the 16S rRNA gene tree, which

formed a well separated clade supported by a 99% bootstrap value in the family _Pseudonocardiaceae_ (Figure 1). This observation suggests that the strain R8-39 may represent a new species in

the genus _Allokutzneria_. However, detailed polyphasic taxonomic characterization of the strain is required to clarify its status. Analysis of 16S rRNA gene sequences of 35 representative

_LL_-DAP-containing isolates and 4 isolates ,which showed the highest antifungal activity, could be confirmed and assigned to the genus _Streptomyces_. The isolates with high similarity to

_S. misionensis_ were frequently found in different soil samples (Table 4). Phylogenetic tree of the resulting 16S rRNA gene sequences assigned them into clusters in the _Streptomyces_ genus

(Figure 2). The biggest cluster consisted of 13 acidophilic strains and the closest neighbor was _S. misionensis_, which had 16S rRNA sequence similarities of 99.5–100%. The 16S rRNA gene

sequences of four effective antifungal isolates (R9-4, R14-1, R14-5 and R20-5) also fell into this cluster although these strains had been isolated from different soil samples. These four

isolates produced whitish aerial mycelium, grayish spore and yellowish to grayish brown reversed color on acidified ISP medium 3 (pH 5.5). The isolates R9-4, R14-5 and R20-5 produced yellow

diffusible pigment which was not detected in R14-1. Melanoid pigment was not produced on ISP medium 7. Microscopic observation revealed the formation of compact spiral chain of spores.

Acidic environments are major selective pressures of members of the genus _Streptacidiphilus._49 A previous work using soil samples collected from rice field soil from Thailand reported the

isolation of _Streptacidiphilus oryzae_ JCM 13271T.61 However, no member of the genus _Streptacidiphilus_ was recovered in this study. This may be due to the fact that the pH of the

isolation media not being acidified to the optimum pH for members of the _Streptacidiphilus_. _S. misionensis_ strains have been previously reported to exhibit antimicrobial activity such as

_S. misionensis_ strain NRRL 3609, which produced substance that could inhibit growth of various fungi including _Candida albicans_ and _Cryptococcus neoformans._62 _S. misionensis_ strain

PMS101 isolated from the rhizosphere of healthy lily bulbs was reported to have antagonistic activity, reducing the incidence of seedling blight caused by _Fusarium proliferatum_ and

effectively control basal rot caused by _Fusarium oxysporum_ f. sp. _lilii_.63 There is no report on pathogenicity of _S. misionensis_ to human, animals or plants. This suggests that _S.

misionensis_ strains could be used as safe biocontrol agents. In rice field soil, the pH is usually slightly acidic. _S. misionensis_ strains R9-4, R14-1, R14-5 and R20-5 were neurotolerant

acidophiles that exhibited efficient antagonistic activity against rice pathogenic fungi (_F. moniliforme, H. oryzae_ and _R. solani_). In addition, all except R9-4 produced siderophore, and

all of them solubilized phosphate. As a result, _S. misionensis_ strains R9-4, R14-1, R14-5 and R20-5 are potential candidates for use in a sustainable development of agriculture especially

in acidic paddy soil. The evaluation of active metabolites, produced by actinomycetes in acidic conditions, indicates the importance to isolate certain groups of species that have useful

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was supported by the scholarship to promote international publication from the Graduate School Kasetsart University to NP. This work was also supported by the Higher Education Research

Promotion and the National Research University Project of Thailand, Office of the Higher Education Commission. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Microbiology,

Faculty of Science, Kasetsart University, Bangkok, Thailand Nalin Poomthongdee & Kannika Duangmal * Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart

University, Bangkok, Thailand Kannika Duangmal * Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand Wasu Pathom-aree Authors * Nalin Poomthongdee View

author publications You can also search for this author inPubMed Google Scholar * Kannika Duangmal View author publications You can also search for this author inPubMed Google Scholar * Wasu

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Poomthongdee, N., Duangmal, K. & Pathom-aree, W. Acidophilic actinomycetes from rhizosphere soil: diversity and properties beneficial to

plants. _J Antibiot_ 68, 106–114 (2015). https://doi.org/10.1038/ja.2014.117 Download citation * Received: 29 January 2014 * Revised: 16 July 2014 * Accepted: 30 July 2014 * Published: 27

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