Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight

Accepted Manuscript Title: Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight Author: Winanda Himaman Arinthip Thamchaipenet Wasu Pathom-aree Kannika Duangmal PII: DOI: Reference:

S0944-5013(16)30166-5 http://dx.doi.org/doi:10.1016/j.micres.2016.04.011 MICRES 25885

To appear in: Received date: Revised date: Accepted date:

25-2-2016 22-4-2016 24-4-2016

Please cite this article as: Himaman Winanda, Thamchaipenet Arinthip, Pathomaree Wasu, Duangmal Kannika.Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Actinomycetes from Eucalyptus and their biological activities for controlling Eucalyptus leaf and shoot blight

Winanda Himamana,b, Arinthip Thamchaipenetc, Wasu Pathom-areed, Kannika Duangmala,b* a

Department of Microbiology, Faculty of Science, Kasetsart University, Chatuchak, Bangkok

10900, Thailand b

Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University,

Chatuchak, Bangkok 10900, Thailand c

Department of Genetics, Faculty of Science, Kasetsart University, Chatuchak, Bangkok

10900, Thailand d

Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200,

Thailand

*Corresponding author: Kannika Duangmal, E-mail: [email protected]

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Abstract In Thailand, Eucalyptus plantations rapidly expand across the country. Leaf and shoot blight caused by Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria destructans is a serious disease in Eucalyptus plantations. In this study, a total of 477 actinomycete strains were successfully isolated from roots and rhizosphere soil of Eucalyptus. Four hundred and thirty nine isolates were classified as streptomycetes and 38 isolates were non-streptomycetes. Among these isolates, 272 (57.0%), 118 (24.7%) and 241 (50.5%) isolates were antagonistic to Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria destructans, respectively. All isolates were tested for their abilities to produce siderophores, indole acetic acid (IAA) and solubilise phosphate. Most isolates (464, 97.3%) produced siderophores. The majority of isolates (345, 72.3%) solubilised phosphate. In addition, almost half of these isolates (237, 49.7%) produced indole acetic acid. Strain EUSKR2S82 which showed the strongest inhibitory effect against all tested fungi with plant growth promoting ability was selected to test with Eucalyptus. This strain could colonize plant roots and increase Eucalyptus roots length. In a detached leaves bioassay, the disease severity of EUSKR2S82-inoculated Eucalyptus leaves was only 30% compared to 95% in the control treatment. The 16S rRNA gene sequence analysis revealed that the strain EUSKR2S82 was related to Streptomyces ramulosus NRRL-B 2714T (99.44% similarity). Identification of non-streptomycete isolates using 16S rRNA gene sequences classified them into 9 genera: Actinoallomurus,

Actinomadura,

Amycolatopsis,

Cryptosporangium,

Microbispora,

Micromonospora, Nocardia, Nonomuraea and Pseudonocardia. It is evident that Eucalyptus tree harbored several genera of actinomycetes. The selected isolate, EUSKR2S82 showed potential as a candidate for biocontrol agent of leaf and shoot blight of Eucalyptus and to promote growth.

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Keywords Actinomycetes; Biocontrol; Eucalyptus; Leaf and Shoot Blight; Plant Growth Promoter; 16S rRNA gene 1. Introduction Actinomycetes are Gram-stain positive saprophytic bacteria that are widely distributed in soil and other terrestrial environments. They contribute significantly to the turnover of complex biopolymers of organic matter, such as chitin and lignocellulose in ecosystems (Cao et al., 2005). They are also well known as producers of many secondary metabolites, with application in the agricultural, pharmaceutical and medical industries, such as antibiotics, antitumor, anti-infection agents and novel natural products (Hasegawa et al., 2006; Igarashi, 2004; Qin et al., 2011). There are several reports of using actinomycetes for the biological control of plant pathogenic bacteria and fungi (Cao et al., 2005; El-Tarabily et al., 2000; Kunoh, 2002; Prapagdee et al., 2008; Mingma et al., 2014). Moreover, actinomycetes can produce plant growth promoters to enhance plant growth and help the establishment of plants under stressed conditions (Srivastava et al., 2015). Hence, it is important to explore unique environments for potential actinomycete strains. The rhizosphere is an interesting area in which interactions occur between plants and microbes, including actinomycetes. The specific structure and diversity of the rhizosphere bacterial community varies between plant species and root zones (Yang and Crowley, 2000). An endophytic environment is one habitat that harbors several bioactive actinomycetes. Both rhizospheric and endophytic actinomycetes have been proved to play vital roles as plant growth promoters (Compant et al., 2005), disease resistance inducers (Trejo-Estrada et al., 1998), drought tolerance stimulators (Hasegawa et al., 2004) and act as biocontrol agents (Kanini et al., 2013; Kunoh, 2002; Poomthongdee et al., 2015). Eucalyptus has recently become an important timber species in Thailand, particularly in relation to timber supply within the pulp industry (Manavakun, 2014). There are currently

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19.6 million hectares of Eucalyptus plantations worldwide, of which 0.5 million hectares are located in Thailand (Petermann, 2011). The demand for wood products is increasing dramatically every year. However, the problems of pests and pathogens in all areas of Eucalyptus plantation are also increasing. Pongpanich et al. (2010) reported that the most serious disease in Eucalyptus plantations was leaf and shoot blight caused by Cryptosporiopsis eucalypti, Cylindrocladium reteaudii and Teratosphaeria destructans. These fungal infections can eventually lead to a drastic reduction in the yield and quality of Eucalyptus plantations. Disease management using chemicals is not suitable for controlling diseases in the wide plantation areas, due to concerns about the impacts upon people's health and environments. Systematic breeding strategies for Eucalyptus species' resistance to common diseases have been implemented with limited success. However, this approach is time-consuming. Therefore, the use of actinomycetes as biological control agents to control leaf and shoot blight in Eucalyptus is of interest for study. This present study was aimed at selecting actinomycetes from roots and rhizosphere soils of Eucalyptus, with the potential for use as biocontrol for fungal pathogen that cause leaf and shoot blight of Eucalyptus and investigate their ability to promote plant growth. The selected isolates were evaluated to reduce the infection of Cylindrocladium sp., the important causal agents of leaf and shoot blight of Eucalyptus in nurseries and plantations. The taxonomic diversity of non-streptomycetes from Eucalyptus was also investigated. 2. Materials and methods 2.1. Samples collection Roots (23 samples) and rhizospheric soil (27 samples) of healthy Eucalyptus (Eucalyptus camaldulensis) were collected from Eucalyptus plantation areas from different provinces in Thailand (Bangkok, Kamphaengphet, Nakhonratchasima, Prachin Buri, Sing Buri, Sukhothai and Suphan Buri) during the period from May 2011-April 2012. Roots were

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dug out carefully to ensure that maximal amounts of root materials were collected and transferred to the laboratory. Rhizospheric soil was separated from the roots and air dried at room temperature before processing. Soil pH was determined. 2.2. Isolation of actinomycetes from roots and rhizospheric soils Eucalyptus root samples were surface sterilized as described by Mingma et al. (2014) with some modifications. Roots were washed in running water to remove soil particles and surface sterilized by shaking in 0.1% Tween 20 for 5 min, followed by a solution of 1.0% NaOCl for 10 min, 2.5% of Na2S2O3 for 10 min and then 75% ethanol solution for 5 min. Samples were then washed three times in sterile water to remove surface sterilization agents. The effectiveness of the surface sterilization method was determined by spreading 100 µl of sample from the last wash on ISP2 agar plates (Shirling and Gottlieb, 1966) and incubated at 28°C for 1–2 days to examine microbial growth. The surface sterile samples were then soaked in 10% NaHCO3 for 10 min before being crushed with a sterile glass rod and diluted with 0.85% normal saline. The prepared root suspensions were spread on two selective media; starch casein (SC) agar (Küster and Williams, 1964) and humic acid-vitamin (HV) agar (Hayakawa and Nonomura, 1987), supplemented with nalidixic acid (25 µg/ml) and ketoconazole (100 µg/ml). The plates were incubated at 28°C for 14–21 days. Rhizospheric actinomycetes were isolated from air-dried rhizospheric soil samples using the dilution plate method. One gram of each soil sample was suspended in 9 ml of sterile distilled water. The resultant 10-1 dilution was heated at 55°C for 6 min and then serially diluted up to 10-5. Soil suspensions (0.1 ml) from 10-3 to 10-5 were applied onto SC and HV agar plates which were supplemented with antibiotics as previously described. After incubation at 28°C for 14 days, the total numbers of actinomycete colonies were counted. Colonies with different morphological characteristics were purified on ISP2 agar and kept for

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long term preservation in 20% (v/v) glycerol at –20°C. The isolates from Eucalyptus roots and from rhizospheric soil were named with "EUR" and "EUS" abbreviations, respectively. 2.3. Fungal pathogens Eucalyptus leaves with symptoms of leaf and shoot blight disease were collected from Eucalyptus plantation area. The diseased leaves were kept in plastic bags and brought to the laboratory for fungal pathogen isolation. The three mentioned fungal pathogens, (Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria destructans), were isolated from the Eucalyptus leaves using Potato Dextrose Agar (PDA). Briefly, leaf samples were incubated in Petri dishes over the wet filter paper for 24 h. Spore masses of C. eucalypti and T. destructans which exuded from the conidiomata of infected leaves, were streaked onto PDA. A single spore of Cylindrocladium sp. was isolated onto PDA under a stereo microscope and cultured on PDA plates. The taxonomic identification of these fungal pathogens was achieved by standard keys, based on their cultural, morphological and sporulation characteristics (Old et al., 2003). All three pathogenic isolates on PDA slants were maintained by overlaying with sterile liquid paraffin for long term preservation. 2.4. In vitro antagonistic bioassay All actinomycete isolates obtained from both roots and rhizospheric soils were screened for their antagonistic activity against C. eucalypti and Cylindrocladium sp., using a dual culture technique. The well-grown actinomycete colony on ISP2 agar was cut into a mycelium disc (5 mm in diameter) and transferred to the PDA plates, placed 3 cm away from the center of the plate. After incubation at 25°C for 7 days, a fungal disc (5 mm in diameter) was placed in the center of the plate. PDA plates with a fungal mycelia disc in the center of the plate were served as a control. The plates were incubated at 28°C until the radial growth of the fungal mycelium of the control plate reached 3 cm. The percentage of inhibition was calculated as [(r1– r2)/ r1] x 100, where r1 is the radial of fungal mycelial growth in the

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control and r2 is the radial of fungal mycelial growth that occurs towards the actinomycetes. The experiment was conducted in duplicate. Due to the slow-growth of Teratosphaeria destructans, the antifungal activity test against this pathogen was evaluated by an agar overlay technique, as described by Anand et al. (2006) with a slight modification. T. destructans was grown on a Carrot Agar (CA) plate (fresh carrot 200 g/l and agar 15 g/l) at 28°C until sporulation. Then, fungal spore suspension of approximately 5x105 spores/ml was prepared in distilled water. For this procedure, 5 ml of soft PDA (Potato Dextrose Broth with 0.6% agar) was mixed with 1 ml of prepared fungal spore suspension and poured over the plate of 7 days old actinomycetes on PDA. The diameter of the inhibition zone and the actinomycete colonies was measured after being incubated at 28°C for 7 days. The ratio of the inhibition zone to the colony size was recorded. The effects of the antagonistic isolates with the highest antifungal activity on mycelial morphology of C. eucalypti and Cylindrocladium sp., including spore germination of T. destructans on antagonistic plates were observed under the compound microscope (Olympus, Tokyo, Japan). The edge of the fungal colony and the inhibition zone on test plate were cut and inoculated on fresh PDA and incubated for 1 week. The survival of the pathogens was observed. 2.5. Screening for plant growth promoting traits Plant growth promoting traits, which were siderophores, indole acetic acid and phosphate solubilization activity, were examined for all 477 isolates. Chrome Azurol Sulphonate (CAS) assay (Schwyn and Neilands, 1987) was used to detect siderophore production. Actinomycete discs (5 mm) cut from 7 days culture on ISP2 were placed onto a CAS agar plate and incubated at 28°C for 3 days. The siderophore production was indicated by a change in color of the agar from blue to orange around the disc. The diameter of the orange halo was recorded.

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The production of IAA was determined according to the method of Khamna et al. (2009) with some modifications. Two actinomycete discs (5 mm) of 7 days culture on ISP2 were inoculated into 5 ml of GYE broth (glucose 10 g/l and yeast extract 10 g/l) supplemented with 0.2% (w/v) L-tryptophan, on a rotary shaker at 170 rpm for 7 days in the dark at 28°C. The cultures were centrifuged at 5,800 g for 10 min. The supernatant (1 ml) was mixed with 1 ml of Salkowski's reagent (Gordon and Weber, 1951) and incubated in the dark for 30 min at room temperature. The development of a pink color indicated IAA production. The reaction mixture was read in a spectrophotometer at 530 nm absorbance. The amount of IAA produced per milliliter of culture (μg/ml) was estimated using a standard curve of indole acetic acid (0.5–100 μg/ml). Phosphate solubility was examined on Pikovskaya’s medium (PVK) as described by Gaur (1990). Actinomycete discs (5 mm) of 7 days old culture on ISP2 were placed on PVK agar plate supplemented with 0.5% tricalcium phosphate as the sole phosphorus source. The plates were incubated at 28°C for 2 weeks. The colony which produced halo zone was considered a phosphate solubilizing isolate and the diameter of the zone was recorded. All the experiments were carried out in duplicate. 2.6. Root colonization assay Based on their in vitro antifungal activity, siderophore and IAA production and phosphate solubilization activity, actinomycete isolates with high activities were selected for root colonization assay. The competence of a selected antagonist to colonize the root of Eucalyptus was determined. A root colonization assay was carried out in vitro by the method modified from Mafia et al. (2009a). Eucalyptus seeds used from Eucalyptus camaldulensis Dehnh. were supplied by the Royal Forest Department, Thailand. The seeds were washed thoroughly with tap water and surface-disinfected with 50% ethanol for 30 sec, followed by 5% NaOCl for 1 min, 2.5% Na2S2O3 for 10 min and then rinsed three times with sterile

9

distilled water. The efficiency of the disinfection procedure was checked by placing the disinfected seeds onto PDA. Aliquots of the final rinsing solution were also spread on the same medium to check the efficiency of the surface sterilization procedure. The seeds were then soaked in 108 spores/ml of actinomycetes for 24 h. Individual seed was aseptically transferred to a test tube (2.5x20 cm) containing 0.4% water-agar (20 ml) and maintained at room temperature for 60 days. Root colonization was observed visually as a turbid zone around the roots. The root colonization was confirmed by transferring the roots onto new Petri dishes containing ISP2 supplemented with nalidixic acid (100 µg/ml) and incubated at 28°C. The result was compared with plants originating from sterile seeds (the control). To determine endophytic colonization, roots were surface sterilized with wash solutions and then washed three times in distilled water. The samples were then crushed with 1 ml of sterile distilled water and vortexed for 30 sec. The homogenates were spread on ISP2 in order to determine the actinomycete isolates inside the surfaced-sterilized roots. 2.7. Detached leaves bioassay Eucalyptus leaves were detached from healthy plants, washed in running tap water and surface-sterilized according to the procedure of Palaniyandi et al. (2011). The detached leaf assay was carried out using 4 treatments as follows: (1) Eucalyptus leaves were soaked with actinomycete spores at 108 spores/ml for 60 min; (2) Eucalyptus leaves were soaked with actinomycete spores, as described before, and then sprayed with spore suspension of Cylindrocladium sp. (105 spores/ml) to fully cover the leaf; (3) Eucalyptus leaves were sprayed with spore suspension of Cylindrocladium (105 spores/ml) to fully cover the leaf and (4) Eucalyptus leaves were sprayed only with sterile distilled water, as a control. All treatments were kept in sterile Petri dishes with wet filter paper and the plates were sealed with paraffin film to keep the moisture in the leaves, at 28°C up to 5 days. Fifteen leaves were used per treatment. The effect of actinomycetes on disease suppression on the detached

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leaves was assessed by recording the percentage of infected leaf area according to Xue et al. (2013), with modifications. The degree of disease severity as a percentage of the lesion area on the leaf surface was determined from a score of 0–4. Scores were assigned as follows: Score 0, no symptoms; Score 1, 1–25% of leaf area was infected; Score 2, 26–50% of leaf area was infected; Score 3, 51–75% of leaf area was infected and Score 4, more than 75% of leaf area was infected. Disease severity and biocontrol efficacy were calculated according to the following formula as described by Xue et al. (2013): Disease severity = [∑ (the disease score x the number of leaves with that score)/(total number of leaves investigated x 4)] x 100; where the number 4 represents the highest disease score. 2.8. Morphological characteristics, DAP analysis and 16S rRNA gene sequencing The actinomycete isolates were streaked onto ISP3 (Shirling and Gottlieb, 1966) and incubated at 28°C for 14 days. The colors of the aerial spore mass, substrate mycelium and soluble pigments were observed. The cell wall type was also determined on the basis of the occurrence of isomers of diaminopimelic acid (DAP) in whole-organism hydrolysates using a modified method from Hasegawa et al. (1983). All meso-DAP containing actinomycetes and the strains which show the highest antagonistic activity against all pathogenic fungi, and/or having plant growth promoting activity with observed root colonization, were characterized by 16S rRNA gene sequencing. Genomic DNA of the isolate was extracted by the modified method of Kieser et al. (2000). 16S rRNA gene fragments were amplified by PCR using a TopTaq Master Mix kit (Qiagen) with

the

primers,

1F

(5'-TCACGGAGAGTTTGATCCTG-3')

and

1530R

(5'-

AAGGAGGTGWTCCARC C-3') (Kataoka et al., 1997), or occasionally Mg4F (5'ATTCCTGGTGTAGC GGT-3') and 782R (5'-ACCAGGGTATCTAATCCTGT-3') (Chun, 1995). The PCR conditions consisted of an initial hot start incubation (10 min at 94°C) followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and an

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extension at 72°C for 1 min followed by a final extension at 72°C for 10 min. PCR products were purified with a Qiagen QIAquick PCR clean-up kit (Qiagen, Chatsworth, CA), following the manufacturer’s protocol and sequenced by a commercial sequencing company, Macrogen, Korea (http://dna.macrogen.com). The 16S rRNA gene sequencing results were aligned against corresponding sequences of the type strains retrieved from the EzTaxon-e database (http://www.ezbiocloud.net/eztaxon). The EzTaxon-e server (Kim et al., 2012) was employed in order to assess the degree of DNA similarity. 3. Results 3.1. Isolation of actinomycetes The objective of this study was to isolate and screen actinomycete strains from rhizosphere soils and the roots of Eucalyptus with antagonistic and plant growth promoting activities. Rhizosphere soils of Eucalyptus collected from many provinces in Thailand were sandy loams or loamy sands with acidic to neutral pH (3.84 to 6.94). The total actinomycete counts of 27 soil samples showed the numbers on SC agar and HV agar ranged from 9.4x104– 3.45x106 and 1.0x105–2.16x106 cfu/g of dry soil, respectively (Table 1). Sample number 8 gave the highest number of rhizospheric actinomycete isolates (32 isolates). For the isolation of endophytic actinomycetes, the fine roots samples of Eucalyptus with a diameter of 3–5 mm, taken from a depth of 10–25 cm below ground, were used in this study. The numbers of endophytic actinomycetes from each sample were varied. The highest number of 19 isolates was recovered from sample number 21. However, not all samples yielded endophytic isolates. In total, 477 actinomycete isolates (95 endophytes and 382 rhizosphere) were recovered from 24 root samples and 27 rhizospheric soils (Table 1). Most isolates were obtained from HV agar.

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3.2. In vitro antagonistic bioassay Eucalyptus leaf and shoot blight fungal pathogens (Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria destructans), isolated from symptom leaves and tested for pathogenicity, were used for antagonistic assay. All actinomycete strains were screened against C. eucalypti and Cylindrocladium sp. by the dual culture technique and against T. destructans by the agar overlay technique. The results revealed that the degree of antifungal activity varied greatly among the isolates. Comparison for antagonistic activity between endophytic actinomycetes and the isolates from the rhizosphere soil (Supplementary material Table A1) showed that the number of root endophytic actinomycetes with antagonistic activity for controlling C. eucalypti (69.5%) was higher than those from rhizospheric soils (53.9%). Activity against Cylindrocladium sp. was the least frequent with 15.8% from root endophytic strains, compared to 27.0% of those from rhizospheric soils. From a total of 477 isolates, only 48 isolates (10.1%) displayed activity against all three fungal pathogens. Among these, strain EUSKR2R82 showed the highest activity by completely inhibiting the growth of all tested fungal pathogens. Two hundred and seventy two isolates (57.0%) inhibited C. eucalypti in which 17 isolates showed a maximum inhibition of 100%. One hundred and eighteen isolates (24.7%) inhibited Cylindrocladium sp. with 3 isolates exhibiting a maximum 100% of growth inhibition. A study of actinomycetes on spore germination of T. destructans showed that 241 isolates (50.5%) could inhibit spore germination. Among these 477 isolates, 380 isolates (79.7%) inhibited at least one of these three serious pathogens while 96 isolates (20.1%) did not show any antifungal activity. Antagonistic activities of all 477 actinomycete isolates are shown in Supplementary material Table A2. Isolate EUSKR2S82, which showed the highest antifungal activity to inhibit C. eucalypti and Cylindrocladium sp. with high growth inhibitory against T. destructans (Fig. 1),

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was selected for further study. The edge of fungal mycelia which faced to the actinomycete colony on the antagonistic plate was observed. The result showed that the mycelia of C. eucalypti and Cylindrocladium sp. were stunted, while spore germination of T. destructans was not occurred after being placed onto the fresh PDA. Compound light microscope observations revealed that EUSKR2S82 had a direct effect on the mycelial morphology of C. eucalypti and Cylindrocladium sp., reflecting its potential for permanently stopped the growth and germination of T. destructans spores (Fig. 2). The results exhibited that metabolites of EUSKR2S82 were effective in restricting the growth of tested phytopathogenic fungi by mean of fungicidal activity. It could be summarized from the results that the antibiosis mechanism involved in the biocontrol of plant pathogens appears mainly to be antifungal metabolites production. This study provided the first evidence of the fungal pathogens, C. eucalypti, Cylindrocladium sp. and T. destructans, of Eucalyptus leaf and shoot blight being controlled by antagonistic actinomycetes. 3.3. Screening for plant growth promoting traits The screening of plant growth promoting traits for 477 actinomycete isolates showed that 464 isolates (97.3%), 237 isolate (49.7%) and 345 isolate (72.3%) produced siderophores, IAA and solubilised phosphate, respectively. In addition, 183 isolates (38.4%) produced all three plant growth promoting agents with only 7 isolates (1.5%) did not produce any tested plant growth promoters. Among 464 siderophores producing actinomycetes, 153 isolates (33.0%) produced wide orange zone on CAS plate of > 20 mm. Two hundred and thirty eight actinomycetes (49.9%) produced IAA in the range of 0.16 –215.64 µg/ml. Determination of phosphate solubilization on Pikovskaya’s medium in the present study revealed that only 26 isolates (5.5%) were able to produce a wide clear zone (>15 mm) on tricalcium-phosphate plates. The remaining majority of isolates (53.0%) were either unable to solubilise phosphate or produced a narrow clear zone (<10 mm). The production of plant

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growth promoting substances of 477 actinomycete isolates is shown in a Supplementary material Table A3. From the results of in vitro antagonistic bioassay and screening for plant growth promoting traits, 3 isolates namely EUSKR2S82, EUSNT1H43 and EURKR1S17, were selected as potential biocontrol agents for further study. These isolates could inhibit all three pathogenic fungi causing the leaf and shoot blight disease of Eucalyptus and also produced IAA, siderophores with phosphate solubilizing ability (Table 2). 3.4. Root colonization assay Root colonization ability was studied with 3 selected isolates, EUSKR2S82, EUSNT1H43 and EURKR1S17. After planting surface sterilized Eucalyptus seeds which were inoculated with these actinomycete strains, the turbid zone was observed around the plant roots. The root colonization in Eucalyptus by these isolates was visually observed and confirmed after transferring root segments on to the fresh culture medium. The inoculated actinomycetes were found to be predominant strains from each root segment. Interestingly, isolate EUSKR2S82 was recovered from surface sterilized inoculated Eucalyptus roots. This observation confirmed that the strain EUSKR2S82 had the ability to colonize plant roots as an endophyte, as it was found in both the inside and the outside of the plant roots. Moreover, the seedlings inoculated with EUSKR2S82 gave longer root length when compared to the control seedlings, and the lateral roots were also well developed with more root hairs (Fig. 3) 3.5. Detached leaf bioassay Among three isolates from the root colonization assay, isolate EUSKR2S82 showed the highest antifungal activity with rhizospheric and endophytic properties. Therefore, isolate EUSKR2S82 was used in the subsequent experiments to assess its efficiency in controlling Cylindrocladium sp. causing leaf spot, leaf blight and seedling blight which regarded as serious diseases in nurseries.

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The observation of all treatments revealed that control leaves were healthy (Fig. 4a), whereas all the other Eucalyptus leaves treated with Cylindrocladium sp. showed serious symptoms of leaf spot followed by leaf blight (Fig. 4c). However, these symptom developments did not occur when leaves were inoculated with the isolate EUSKR2S82 (Fig. 4b). When detached leaves treated with EUSKR2S82 spores were inoculated with Cylindrocladium sp., the fungal growth was inhibited (Fig. 4d). This indicated that isolate EUSKR2S82 could limit the spread of fungal pathogens. The disease severity was reduced from 95% in the control treatment to 30% at a concentration of 108 spores/ml of EUSKR2S82. 3.6. Morphological characteristics, DAP analysis and 16S rRNA gene sequencing An analysis of DAP types and morphological characteristics of 477 actinomycete isolates confirmed that most isolates (92.0%) contained LL-DAP and belonged to the streptomycete group within the genus Streptomyces. The remaining 8.0% contained mesoDAP which was regarded as a non-streptomycete group. Among 382 isolates from rhizospheric soils of Eucalyptus, 355 isolates (92.9%) contained LL-DAP whereas 27 isolates (7.1%) contained meso-DAP. Of the 95 endophytic actinomycetes, 11 isolates (7.3%) showed the presence of meso-DAP. Based on the spore mass color of the isolates on ISP3, Streptomyces 439 isolates were assigned into 6 distinct groups, which consisted of the colors of gray (55.8%), white (15.3%), green (13.9%), yellow (9.6%), pink (2.7%) and brown (2.7%) spores. A comparison of nearly complete 16S rDNA sequences of 38 non-streptomycete strains (accession number LC085562-LC085599) that showed whole-cell hydrolysates rich in meso-DAP revealed the phylogenetic affiliations to 9 different genera, representing 6 families. They were affiliated with the genus Actinoallomurus (n = 1, family Thermomonosporaceae),

Actinomadura

(n

=

3,

family

Thermomonosporaceae),

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Amycolatopsis (n = 4, family Pseudonocardiaceae), Cryptosporangium (n = 1, family Cryptosporangiaceae), Microbispora (n = 2, family Streptosporangiaceae), Micromonospora (n = 17, family Micromonosporaceae), Nocardia (n = 3, family Nocardiaceae), Nonomuraea (n

=

5,

family

Streptosporangiaceae)

and

Pseudonocardia

(n

=

2,

family

Pseudonocardiaceae) (Table 3). The genus Micromonospora was the most frequently found in both root and rhizosphere samples. Three strains were isolated from roots and 14 strains were isolated from soils. These Micromonospora species were identified to 11 different species. Of the endophytic isolates, 11 isolates (28.9%) belonged to the genus Micromonospora (n = 3) and Nocardia (n = 3), Actinomadura (n = 2) and Cryptosporangium (n = 1), Actinoallomurus (n = 1) and Amycolatopsis (n = 1). Phylogenetic trees constructed using neighbour-joining tree based on 16S rRNA gene sequences of the non-streptomycete isolates and the related type strains was shown in the Supplementary material Fig. A1. Strain EUSKR2S82, the most active isolate, was analyzed for its relatedness to known species, based on 16S rDNA sequence. It was evident that strain EUSKR2S82 was closely related to Streptomyces ramulosus NRRL B-2714T (DQ026662) (99.44%). The 16S rRNA genes from the other two selected isolates for root colonization assay (EUSNT1H43 and EURKR1S17) showed that the strain EUSNT1H43 shared 99.08% similarity to Streptomyces himastatinicus ATCC 53653T and strain EURKR1S17 shared 100% similarity to Streptomyces malaysiensis NRBC 16446T. The percentage of 16S rRNA gene sequence similarity of meso-DAP containing isolates and strain EUSKR2S82, EUSNT1H43 and EURKR1S17 to the closest type strains is presented in Table 3. The similarity values of these isolates to the closest type species ranged between 98.6 and 99.9%. 4. Discussion Nowadays, there are widespread studies of using actinomycetes as a biological control agent and biofertilizer in various agricultural crops (El-Tarabily et al., 2008; Xue et al., 2013;

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Goudjal et al., 2014) to reduce the use of chemicals and fertilizers. In forestry, the promotion of plant growth and the reduction of disease infection by the use of microorganisms that play a role in plant disease biocontrol are also of interest. A few studies have been reported on the use of actinomycetes to control forestry diseases such as Neofusicoccum parvum in Brazil pines (Dalmas et al., 2013), brown rot disease in teak plantations (Yanti et al., 2012). However, there are no report on the isolation of actinomycetes from Eucalyptus and the potential of these microorganisms to control leaf and shoot blight in Eucalyptus. In this study, 477 isolates of actinomycetes were obtained from 24 Eucalyptus roots and 27 rhizospheric soil samples. Based on the preliminary characterization of these isolates, it was revealed that members of the genus Streptomyces were dominant (92.0%), compared to non-streptomycetes or rare actinomycetes (8.0%). Generally, the genus Streptomyces was usually obtained from plant roots and rhizospheric soils (Khamna et al., 2009; Mingma et al., 2014) and they are best known for their ability to produce secondary metabolites, especially antibiotics. However, non-streptomycetes are also accepted as valued targets for the discovery of novel bioactive compounds and new antibiotics, due to their excellent metabolic diversity. In this study, non-streptomycete isolates made up to 11.6% of all the endophytic isolates. Kaewkla and Franco (2013) recommended the possible ways to obtain the rare group by using isolation media with low concentrations of nutrients as actinomycetes' requirements inside the plants, plating with a large amount of plant samples and a long incubating time with regularly removed visible emerging colonies. The isolation of endophytic actinomycetes from Eucalyptus is difficult, due to bacterial contamination which overgrows on the media, although the media are supplemented with nalidixic acid (25 µg/ml). Endophytic microorganisms from Eucalyptus, which include fungi, bacteria and actinomycetes, had been reported (Ferreira et al., 2008; Kharwar et al., 2010; Kaewkla and Franco, 2013) and led to the finding of interesting or novel species with valuable secondary metabolites. In this study,

18

most actinomycetes isolated from both roots and rhizosphere soils were obtained from HV agar. This may be due to the medium component which contained soil humic acid as a sole carbon and nitrogen source, which was suitable for the recovery of actinomycetes from soil samples (Hayagawa and Nonomura, 1987). The knowledge of using actinomycetes to inhibit plant pathogens as biocontrol agents is largely based on studies of endophytic and rhizospheric actinomycetes. In order to inhibit pathogens, the biocontrol microbes use several mechanisms such as antibiosis, the production of cell wall-degrading enzymes, parasitism and the induction of host resistance (Palaniyandi et al., 2013). In this study, all 477 isolates were tested for their antagonistic activity to Cryptosporiopsis eucalypti, Cylindrocladium sp. and Teratosphaeria destructans. Forty eight isolates (10.1%) showed antagonistic activity against all three fungal pathogens. Three potent streptomycete strains, EUSKR2S82 and EUSNT1H43 (isolated from rhizosphere soil), and EURKR1S17 (isolated from the root), were found to have the highest in vitro antifungal activity against all test fungal pathogens. Moreover, these strains have the ability to produce IAA, siderophores and solubilise phosphate. Identification of the isolates EUSKR2S82, EUSNT1H43 and EURKR1S17 showed that they were members of the genus Streptomyces. Several investigations indicated that Streptomyces are the promising microbe that inhibited the growth of several plant pathogens including fungi and bacteria (Golińska and Dahm, 2013; Kobayashi et al., 2012; Mingma et al., 2014). Interestingly, isolate EUSKR2S82 showed the strongest inhibitory effect against all the fungi tested. The study on the effect of EUSKR2S82 on the mycelium of C. eucalypti and Cylindrocladium sp. showed that this strain was able to completely inhibit the fungal growth and change the mycelium color of both the pathogenic fungi, as compared to the control treatment. A mode of action observed was likely to be antibiosis which appeared in the co-inoculated dishes as an inhibition zone. Our observation of fungal mycelium in the antagonistic plates suggested that antifungal

19

agents from EUSKR2S82 were fungicidal, while those of EUSNT1H43 were fungistatic, because the fungal growth was temporarily suppressed. Regarding plant growth enhancement, the efficiency of various microorganisms, including actinomycetes, to promote the growth of Eucalyptus, were reported (Mafia et al., 2009a, 2009b; Paz et al., 2012; Peralta et al., 2012; Salla et al., 2014). IAA is the major phytohormone which stimulates root elongation and cellular division (Kloepper et al., 2004). It is widely used in agriculture to promote plant growth and development. In the present study, the preliminary assay of IAA production based on Salkowski’s reaction, pink-red color formation was recorded for IAA producing strains. The range of IAA production was 0.2– 215.6 µg/ml. Many problems of nutrient accumulation in soil, especially calcareous soil, were concerned, as reported by Osotsapar (1999), that iron deficiency limited legume production in that type of soil. In recent years, siderophores production by actinomycetes is focused on its ability to indirectly promote plant growth by capturing ferric iron in soil and provide nutrients by the specific uptake system to stimulate plant growth (Rungin et al., 2012). In the screening for siderophores production by CAS agar assay, 153 active isolates could produce an orange halo zone of siderophores wider than 20 mm in diameter. Many researchers have reported siderophore production by actinomycetes (Muangham et al., 2014; Rungin et al., 2012; Poomthongdee et al., 2015). Phosphate solubilization is also one of the microbial mechanisms to improve phosphorus availability to plants, thus enhancing the growth and yield. Recently, the capability of phosphate-solubilizing actinomycetes has attracted interest as reported by several researchers (El-Tarabily et al., 2008; Salcedo et al., 2014). The ability of strain EUSKR2S82 to colonize the Eucalyptus roots and its endophytic property was our strategy for selecting potential antagonistic strains. Both root colonization and endophytic properties are beneficial for plant root systems, especially when the mode of

20

action used by these antagonists is antibiosis or competition for habitats and nutrients in soil (Xue et al., 2013). Among the three fungal pathogens in this study, Cylindrocladium sp. had been recorded with a variety of symptoms on seedlings (root and collar rot, shoot blight, leaf blight and foliar spot) in nurseries and plantations (Old et al., 2003). To the best of our knowledge, there is no biocontrol agent from actinomycetes available against seedling diseases of Eucalyptus. Amongst the antagonists, Streptomyces isolate EUSKR2S82 displayed the highest activity against Cylindrocladium sp. in in vitro assay. Its ability to control the fungi was also confirmed in detached leaf assay. This strain is a potential candidate for future application as a promising biocontrol and plant growth promoting agent in Eucalyptus nursery conditions. Normally, plants have been treated with fungicides at all stages of the Eucalyptus growth to control fungal diseases. An attempt has been made to control Cylindrocladium leaf spot of Eucalyptus seedlings in Thailand by using fungicides such as Carbendazim (Saksirirat et al., 2013). Conversely, a report of Maciel et al. (2012) showed that strong antagonists of Trichoderma spp. could give protection against the leaf spot caused by Cylindrocladium candelabrum. Mafia et al. (2009b) reported Pseudomonas sp. FL2 and Bacillus subtilis 3918, S1 and S2 were effective against mycelial growth of Cylindrocladium candelabrum and Rhizoctonia solani. Additionally, they also suggested that integrated disease management is important for the use of actinomycetes to control fungal pathogens. The 16S rDNA analysis revealed that strain EUSKR2S82 was phylogenetically closely related to Streptomyces ramulosus NRRL B-2714 T with 99.44% similarity. Several studies have reported the role of S. ramulosus as an effective actinomycete to control pathogenic microorganisms by producing antibiotics (Atta et al., 2011; Uhr et al., 1985). Based on results in morphological characteristics as well as on the DAP analysis from whole-cell extracts, all actinomycete antagonists with plant growth promote activities were identified as shown in Supplementary material Table A1. More than 90% of the active

21

isolates were identified as member to the genus Streptomyces. Some non-streptomycete isolates were significant antagonism of fungal pathogens but limited to produce plant growth promoting substances. An isolate EURKPP3H10 member of the genus Cryptosporangium showed a high ability to inhibit C. eucalypti (87.3% inhibition). The species of Cryptosporangium have never been reported for biocontrol activities. In addition, three isolates in genus Micromonospora (EUSSB4S10, EUST2H6 and EUSKY1H23) were antagonistic to Cylindrocladium sp. with 45.5−54.5% inhibition. Regarding to the antagonistic activity against T. destructans, the ratio of inhibition zone to the colony size of all non-streptomycete isolates was low. Interestingly, 16 out of 38 non-streptomycete strains showed antagonistic activity against at least one of the tested fungal pathogens. None of the non-streptomycete isolates was active against all tested pathogens. Considering with 153 active siderophore producers, 8 isolates (5.2%) were classified as non-streptomycetes. It was clear that most active non-streptomycete isolates in this research demonstrated narrow spectrum inhibitory activity against tested fungi with low plant growth promoting activities (Supplementary material Table A4). Most isolates in the genus Streptomyces showed remarkable activities in in vitro bioassay against fungal pathogens and abilities to produce plant growth promoting agents in high quantity. However, search for novel nonstreptomycete strains having antagonistic activities and plant growth promoting activity is also of interest. Kaewkla and Franco (2013) reported on the isolation of endophytic actinobacteria from Eucalyptus camaldulensis using several media. They obtained 39 actinobacterial strains belonging to Gordonia, Micromonospora and Streptomyces from leaves, stems and roots. However, in our study, E. camaldulensis roots were found to harbor higher diversity of non-streptomycetes as we could be able to isolate members of genera Actinoallomurus, Actinomadura, Amycolatopsis, Cryptosporangium, Micromonospora and Nocardia. As mentioned above, non-streptomycete isolates showed limited biopotential

22

activity both in antimicrobial activity and plant growth promoting. However, several studies revealed that non-streptomycete group could act as promising biocontrol agents (El-Tarabily et al., 2010; Tian et al., 2013) and plant growth promotors (Shutsrirung et al., 2013). The present study has exhibited for the first time that the selected actinomycete strain characterized molecularly as Streptomyces ramulosus strain EUSKR2S82 had strong antagonistic ability against C. eucalypti, Cylindrocladium sp. and T. destructans in in vitro bioassay and detached leaf assay. This strain could colonize the Eucalyptus roots and increased Eucalyptus root length. S. ramulosus strain EUSKR2S82 is likely to be the potential candidate for biocontrol application for Eucalyptus plantations. Conflict of interest The authors declare no conflict of interest related to this work. Acknowledgements This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission; Kasetsart University Research and Development Institute.

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Shutsrirung, A., Chromkaew, Y., Pathom-Aree, W., Choonluchanon, S., Boonkerd, N., 2013. Diversity of endophytic actinomycetes in mandarin grown in northern Thailand, their phytohormone production potential and plant growth promoting activity. Soil Sci. Plant Nutr. 59, 322–330. Srivastava, S., Patel, J.S., Singh, H.B., Sinha, A., Sarma, B.K., 2015. Streptomyces rochei SM3 induces stress tolerance in chickpea against Sclerotinia sclerotiorum and NaCl. J. Phytopathol. 163, 583–592. Tian, S.-Z., Pu, X., Luo, G., Zhao, L.-X., Xu, L.-H., Li, W.-J., Luo, Y., 2013. Isolation and characterization of new p-terphenyls with antifungal, antibacterial, and antioxidant activities from halophilic actinomycete Nocardiopsis gilva YIM 90087. J. Agric. Food Chem. 61, 3006–3012. Trejo-Estrada, S.R., Paszczynski, A., Crawford, D.L., 1998. Antibiotics and enzymes produced by the biocontrol agent Streptomyces violaceusniger YCED-9. J. Ind. Microbiol. Biotechnol. 21, 81–90. Uhr, H., Zeeck, A., Clegg, W., Egert, E., Fuhrer, H., Peter, H.H., 1985. The structure of acetomycin. Spectroscopic characterization and X-ray analysis of a bromo derivative. J. Antibiot. 38, 1684–1690. Xue, L., Xue ,Q., Chen, Q., Lin, C., Shen, G., Zhao, J., 2013. Isolation and evaluation of rhizosphere actinomycetes with potential application for biocontrol of Verticillium wilt of cotton. Crop Prot. 43, 231–240. Yang, C.H., Crowley, D.E., 2000. Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Appl. Environ. Microbiol. 66, 345–351. Yanti, Y., Zainon, M.N., Marshida, A.H., 2012. Antagonistic activity of three actinomycetes, MG01, MG02 and KT2F towards Phellinus noxius. In: Business, Engineering and

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Industrial Applications (ISBEIA), 2012 IEEE Symposium, Bandung, Indonesia, 729– 732.

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FIGURES LEGEND Fig. 1. In vitro antifungal activity of the isolate EUSKR2S82 against: (a) Cryptosporiopsis eucalypti; (b) Cylindrocladium sp.; and (c) Teratosphaeria destructans, (right) in comparison to controls (left).

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Fig. 2. Micrographs of fungal pathogens in the presence of EUSKR2S82: (a) Cryptosporiopsis eucalypti hyphae in control plate; (b) Lysis of hyphal growth of C. eucalypti by EUSKR2S82; (c) Cylindrocladium sp. hypha in control plate; (d) Cylindrocladium sp. hypha showing bulbous structures; (e) Germinating T. destructans spore in control plate; and (f) Dead spores of T. destructans from the inhibitory zone.

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Fig. 3. In vitro Eucalyptus root colonization assay: (a) Differences in shoot and root length of each inoculated seedling after maintained at room temperature for 60 days; (b) Confirmation of roots colonization by EUSKR2S82 on ISP2 agar; and (c) Growth of endophytic EUSKR2S82 from surface-sterilized Eucalyptus roots.

(d)

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Fig. 4. Detached leaves assay of Eucalyptus sp. with Cylindrocladium sp.: (a) leaf from control treatment; (b) leaf inoculated with isolate EUSKR2S82; (c) leaf inoculated with Cylindrocladium sp. (showing the serious symptom of leaf blight disease): and (d) leaves pretreated with isolate EUSKR2S82 before inoculated with Cylindrocladium sp. The leaves were observed after incubation at 28oC for 5 days.

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Table 1 Occurrence and number of actinomycetes from roots and rhizospheric soils of Eucalyptus. No. of actinomycetes (cfu/g dry soil)

Sample No.

on SCA

on HV

1

2.1x105

2.8x105

2

2.95x105

3 4

3.0x10

5

Soil pH

No. of isolates from

No. of streptomycete

No. of non-streptomycete

Roots

Rhizosphere soils

6.63

ND

7

6

1

3.1x105

6.94

ND

7

7

0

2.4x105

2.8x105

5.51

ND

9

9

0

5

5

6.79

5

5

6

4

1.5x105

1.0x105

6.94

2

15

14

3

6

3.45x105

3.1x105

5.7

0

14

11

3

7

1.68x105

3.7x105

6.09

2

20

21

1

8

3.6x105

6.6x105

5.71

0

32

31

1

9

1.6x10

5

6.44

2

21

21

2

10

7.15x105

4.5x106

4.86

11

26

35

2

11

1.81x106

6.0x106

5.4

6

26

32

0

12

6.5x105

1.4x106

4.47

10

20

28

2

13

3.3x105

2.95x105

6

1.0x10

4.55x10

1.80x10

5

6.1

4

13

11

6

6

5.19

0

13

13

0

14

1.18x10

15

9.55x105

1.88x106

4.95

4

12

15

1

16

1.08x106

1.25x106

4.3

5

11

15

1

17

3.05x105

2.1x105

4.27

3

5

8

0

18

7.15x105

1.05x106

4.88

0

10

9

1

19

5.35x10

5

5

5.56

0

7

7

0

20

7.5x105

1.48x106

3.84

1

10

11

0

21

3.56x105

8.76x105

4.40

19

12

30

1

22

1.55x106

1.46x106

4.68

4

15

18

1

23

3.4x105

2.16x106

24

9.4x10

4

25

5.85x10

6.73

3

26

26

3

6

6.69

3

11

13

1

3.45x106

5.3x106

6.89

8

17

23

2

26

1.63x105

4.10x105

5.69

1

6

6

1

27

5.0x105

1.33x106

6.08

2

12

13

1

95

382

439

38

Total

ND: not determine

1.1x10

36

Table 2 Antagonistic activities against Eucalyptus leaf and shoot blight disease of potential actinomycete isolates and their plant growth promotion traits. Isolate

Inhibition (%)

Inhibition ratio

Siderophores (mm)

Phosphate solubilization (mm)

IAA (μg/ml)

Cryptosporiopsis eucalypti

Cylindrocladium sp.

T. destructans

EUSKR2S82

100

100

4.8

17

11

28.03

EUSNT1H43

100

65.5

5

14.5

7

2.33

EURKR1S17

100

87.3

6.3

14

10

10.59

37

Table 3 The closest type strain of meso-DAP containing actinomycete isolates and isolate EUSKR2S82, EUSNT1H43 and EURKR1S17 based on nearly complete 16S rRNA gene sequences. Isolates

Accession number

Geographic origin (Province)

EURKR1H50H EURKPP1H17 EURSB3S3 EUSN1H28 EURKY2H15 EUSKPP4S84 EUSKY4S4 EUSKPP2H19 EURKPP3H10

LC085562 LC085563 LC085564 LC085565 LC085566 LC085567 LC085568 LC085569 LC085570

Nakhonratchasima Kamphaengphet Sing Buri Bangkok Prachin Buri Kamphaengphet Prachin Buri Kamphaengphet Kamphaengphet

EUSN4H13 EUSSB4S58 EUSSB4S10 EURSB1S3 EUSN2S54 EUST1S18 EUST2H18 EUSKY1H23 EUST1S2 EUST1H6 EUSN1S8 EUST2H12 EURSB3S48 EUSN3S99 EURKPP2H80 EUST2H9 EUSSB4S7 EUSN1H4 EUS1S102 EURN4S5 EURSB1H9 EURSB4H5 EUSNTS40 EUSKR2S45 EUSNTS44 EUST1H8 EUSSB4S2 EUSNTH2 EUSSB4H15 EUSKR2S82 EUSNT1H43 EURKR1S17

LC085571 LC085572 LC085573 LC085574 LC085575 LC085576 LC085577 LC085578 LC085579 LC085580 LC085581 LC085582 LC085583 LC085584 LC085585 LC085586 LC085587 LC085588 LC085589 LC085590 LC085591 LC085592 LC085593 LC085594 LC085595 LC085596 LC085597 LC085598 LC085599 LC085600 LC097196 LC097195

Bangkok Sing Buri Sing Buri Sing Buri Bangkok Bangkok Bangkok Prachin Buri Bangkok Bangkok Bangkok Bangkok Sing Buri Bangkok Kamphaengphet Bangkok Sing Buri Bangkok Sukhothai Bangkok Sing Buri Sing Buri Nakhonratchasima Nakhonratchasima Nakhonratchasima Bangkok Sing Buri Nakhonratchasima Sing Buri Nakhonratchasima Nakhonratchasima Nakhonratchasima

a

Closest type strains

% Similarity (nt diff./nt total)a

Actinoallomurus vinaceus K10-0528T (AB668307) Actinomadura geliboluensis A8036T (HQ157187) Actinomadura geliboluensis A8036T (HQ157187) Actinomadura geliboluensis A8036T (HQ157187) Amycolatopsis echigonensis LC2T (AB248535) Amycolatopsis echigonensis LC2T (AB248535) Amycolatopsis echigonensis LC2T (AB248535) Amycolatopsis eurytherma NT202T (AJ000285) Cryptosporangium minutisporangium IFO 15962T (AB037007) Microbispora hainanensis 211020T (FJ261972) Microbispora hainanensis 211020T (FJ261972) Micromonospora aurantiaca ATCC 27029T (CP2162) Micromonospora avicenniae 268506T (JQ867183) Micromonospora avicenniae 268506T (JQ867183) Micromonospora avicenniae 268506T (JQ867183) Micromonospora chaiyaphumensis MS5-1T (AB196710) Micromonospora eburnea LK2-10T (AB107231) Micromonospora eburnea LK2-10T (AB107231) Micromonospora echinaurantiaca DSM 43904T (X92618) Micromonospora echinospora ATCC 15837T (US8532) Micromonospora echinospora ATCC 15837T (US8532) Micromonospora endolithica DSM 44398T (AJ560635) Micromonospora endolithica DSM 44398T (AJ560635) Micromonospora costi CS1-12T (AB9810481) Micromonospora inositola DSM43819T (X92610) Micromonospora krabiensis MA2T (AB196716) Micromonospora maritima D10-9-5T (HQ704071) Micromonospora maritima D10-9-5T (HQ704071) Nocardia vaccinii DSM 43285T (Z36927) Nocardia vaccinii DSM 43285T (Z36927) Nocardia vaccinii DSM 43285T (Z36927) Nonomuraea coxensis JCM 13931T (AB505224) Nonomuraea kuesteri GW14-1925T (AJ746362) Nonomuraea monospora PT708T (FJ347524) Nonomuraea salmonea DSM 43678T (X97892) Nonomureae jabiensis A4036T (HQ157186) Pseudonocardia halophobica DSM 43089T (Y08534) Pseudonocardia kujensis A4038T (J989287) Streptomyces ramulosus NRRL B-2714T (DQ026662) Streptomyces himastatinicus ATCC 53653T (EF408736) Streptomyces malaysiensis NRBC 16446T (AB249918)

99.08 (12/1303) 99.86 (2/1444) 99.51 (7/1430) 99.93 (1/1397) 99.36 (9/1397) 98.81 (17/1425) 99.48 (7/1357) 99.85 (2/1364) 99.11 (13/1453)

nt diff./nt total = Nucleotide difference/total nucleotide used in analysis

99.51 (7/1425) 99.43 (8/1405 ) 99.93 (1/1396) 99.29 (10/1408) 99.09 (13/1426) 99.31 (10/1440) 99.71 (4/1360) 99.72 (4/1420) 98.80 (17/1411) 98.97 (15/1461) 99.64 (5/1408) 99.72 (4/1408) 98.67 (19/1425) 98.73 (18/1418) 100 (0/1404) 99.36 (9/1416) 99.86 (2/1388) 99.71 (4/1369) 99.51 (7/1425) 98.81 (17/1424) 98.81 (17/1424) 98.60 (18/1290) 99.79 (3/1431) 99.02 (14/1431) 99.78 (3/1374) 99.79 (3/1407) 99.26 (10/1357) 99.64 (5/1408) 99.52 (7/1461) 99.44 (8/1429) 99.08 (14/1409) 100 (0/1417)