Trichoderma metabolites as biological control agents against Phytophthora pathogens

Accepted Manuscript Trichoderma metabolites as Biological Control Agents against Phytophthora Pathogens Soo-Jung Bae, Tapan Kumar Mohanta, Jun Young Chung, Minji Ryu, Gweekyo Park, Sanghee Shim, Seung-Beom Hong, Hyunchang Seo, Dong-Won Bae, Inhwan Bae, Jong-Joo Kim, Hanhong Bae PII: DOI: Reference:

S1049-9644(15)30035-9 http://dx.doi.org/10.1016/j.biocontrol.2015.10.005 YBCON 3333

To appear in:

Biological Control

Received Date: Accepted Date:

19 August 2015 9 October 2015

Please cite this article as: Bae, S-J., Mohanta, T.K., Chung, J.Y., Ryu, M., Park, G., Shim, S., Hong, S-B., Seo, H., Bae, D-W., Bae, I., Kim, J-J., Bae, H., Trichoderma metabolites as Biological Control Agents against Phytophthora Pathogens, Biological Control (2015), doi: http://dx.doi.org/10.1016/j.biocontrol.2015.10.005

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Trichoderma metabolites as Biological Control Agents against Phytophthora Pathogens

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Soo-Jung Baea,1, Tapan Kumar Mohantaa,1, Jun Young Chungb, Minji Ryua, Gweekyo Parka,

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Sanghee Shimc, Seung-Beom Hongd, Hyunchang Seoe, Dong-Won Baef, Inhwan Baeg,*,

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Jong-Joo Kima,*, Hanhong Baea,*

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a

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b

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Republic of Korea

School of Biotechnology, Yeungnam University, Gyeongsan 712-749, Republic of Korea Department of Orthopedic Surgery, School of Medicine, Ajou University, Suwon 442-749,

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c

College of Pharmacy, Duksung Women's University, Seoul 132-714, Republic of Korea

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d

Korean Agricultural Culture Collection, National Academy of Agricultural Science, Rural

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Development Administration, Suwon 441-853, Republic of Korea

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e

Department of Food and Nutrition, Shingu College, Seongnam 462-743, Republic of Korea

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f

Central Instrument Facility, Gyeongsang National University, Jinju 660-701, Republic of

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Korea

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g

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* To whom correspondence should be addressed:

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Inhwan Bae

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Tel: +8231-371-5049; Fax: +8231-371-5049; E-mail: [email protected]

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Jong-Joo Kim

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Tel: +8253-810-3027; Fax: +82-53-810-4769; E-mail: [email protected]

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Hanhong Bae

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Tel: +8253-810-3031; Fax: +82-53-810-4769; E-mail: [email protected]

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Soo-Jung Bae and Tapan Kumar Mohanta contributed equally to work

College of Pharmacy, Chungang University, Seoul 156-756, Republic of Korea

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ABSTRACT

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Trichoderma species are well-known biological control agents. In this study, metabolites

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from 128 Trichoderma isolates were extracted from liquid cultures using ethyl acetate and

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tested for their activities against seven Phytophthora isolates. Following preliminary analysis,

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eight Trichoderma isolates were selected for further tests. Among them, the metabolites from

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T. atroviride/petersenii (KACC, Korea Agricultural Culture Collection, 40557) and T. virens

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(KACC 40929) showed the strongest inhibitory activities against Phytophthora isolates.

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Treatment with KACC 40557 extract inhibited Phytophthora growth, induced defense-related

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genes, and caused plant hormonal changes during Phytophthora infection in the detached

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leaves of pepper and tomato plants. Our results showed the potential for use of Trichoderma

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metabolites as biological control agents against Phytophthora pathogens.

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Key words: Trichoderma, Phytophthora, metabolites, ethyl acetate extract, biological control

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1. Introduction The genus Trichoderma belongs to ascomycetic fungi found in the soil (Samuels, 2006).

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Trichoderma spp. are well-known biocontrol agents against phytopathogens (Romão-

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Dumaresq et al. 2012). For example, T. harzianum, T. virens and T. viride are currently

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marketed as biocontrol agents. The mechanisms for anti-phytopathogen activities include

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antibiosis, mycoparasitism, induced resistance and niche exclusion (Bae, 2011). Antibiosis

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involves the production of various antimicrobial compounds that function as inhibitors of

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phytopathogen growth (Vinale et al. 2008). More than 100 antimicrobial compounds have

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been identified from Trichoderma spp. (Vinale et al, 2008). During the mycoparasitism

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process, phytopathogen cell walls are degraded by cell wall-degrading enzymes produced

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from Trichoderma (Reithner et al. 2011). There is competition between Trichoderma spp.

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and phytopathogens for infection sites and nutrients, which is known as niche exclusion.

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The genus Phytophthora is a devastating plant pathogen that infects almost all plant

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species (Hansen et al. 2012). In 1861, deBary identified Phytophthora infestans as the causal

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agent of late potato blight, which was responsible for the Irish potato famine (Raffaele et al.

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2010). Phytophthora species are oomycetes, which are fungi-like eukaryotic microorganisms

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also known as water molds. More than 100 species of Phytophthora are potential plant

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pathogens. Phytophthora mycelia contain non-partitioned hyphae with several diploid nuclei.

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While chitin is the primary component of fungal cell walls, β-glucan and cellulose are the

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major components of Phytophthora cell walls. Phytophthora species produce different types

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of spores (oospores, chlamydospores and zoospores), but cannot synthesize sterols, which are

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the target of many fungicides (Gaulin et al. 2010). As a result, Phytophthora pathogens are

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difficult to control using the majority of currently available fungicides. In addition,

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Phytophthora species can overcome chemical control agents and resistance to plant hosts via

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genetic flexibility.

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In this study, the possibility for use of Trichoderma metabolites as biocontrol agents

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against Phytophthora was investigated. A total of 128 Trichoderma isolates were screened

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for their anti-Phytophthora activities using ethyl acetate extracts isolated from liquid cultures.

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Further confirmation of eight selected Trichoderma isolates was conducted by minimum

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inhibitory concentration, disk diffusion and antibiosis tests. In addition, we investigated the

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molecular and biochemical responses of Phytophthora and plants (pepper and tomato) after

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application of the ethyl acetate extract of KACC 40557.

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2. Materials and Methods

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2.1. Trichoderma and Phytophthora isolates

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A total of 128 Trichoderma isolates were obtained from the Rural Development

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Administration (RDA) Genebank Information Center (GIC) (Suwon, Republic of Korea)

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(data not shown). Trichoderma isolates were grown on potato dextrose agar (PDA) (Difco,

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Sparks, MD, USA) at 25°C for 14 days under dark conditions. Seven Phytophthora isolates

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were maintained on 20% clarified V8 (cV8) juice agar (20% V8 juice, 8% CaCO3, 1.5%

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Bacto agar). The following seven species were used in this study: P. cactorum (KACC,

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Korea Agricultural Culture Collection, 40166), P. capsici (KACC 40157), P. drechsleri

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(KACC 40463), P. infestans (KACC 43071), P. melonis (KACC 40197), P. nicotianae

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(KACC 44717), and P. sojae (KACC 40412). These isolates were grown at the following

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temperatures to optimize their growth: P. infestans, 18°C; P. cactorum and P. sojae, 25°C; P.

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capsici, P. drechsleri, P. melonis and P. nicotianae, 30°C. The isolates were cultivated for 5–

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13 days under dark conditions. 4

962.2. Extraction of Trichoderma metabolites using ethyl acetate 97

After 14-days of growth, sterile water (8 mL) was poured into the plates, and mycelia

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were harvested by scratching using a glass stick. The amount of harvested mycelia was

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dependent on isolates; however, the same concentration of extracts was used for the treatment.

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The harvested mycelia were poured into 500 mL of minimal salts broth (MIN) media in 1,000

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mL Erlenmeyer flasks and grown for 14 days at 25°C at 150 rpm (Bae et al., 2011). Half of

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the volume of ethyl acetate (EtOAc, 250 mL) was added into the liquid culture and shaken at

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150 rpm for 10 min. After 1 h of incubation without shaking, the top layer was transferred

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into a flask, concentrated, and dried using a rotary vacuum evaporator at 36°C (Rouini et al.,

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2006). The dried extracts were weighed and dissolved in acetone-water (1:9, v/v).

106 1072.3. Preliminary screening for anti-Phytophthora assay 108

The EtOAc extracts from 128 Trichoderma isolates were screened for anti-Phytophthora

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activity. Phytophthora plugs (0.6 cm diameter) were cut from actively growing edges of cV8

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plates using a sterile cork borer and then placed mycelial side up in the middle of a plate with

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a 6 cm diameter containing 10 mL PDA. cV8 plates were used for P. infestans and P. sojae

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instead of PDA because of slow growth in the PDA media. Two layers of sterile Whatman

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filter paper discs with a 0.6 cm diameter that contained either Trichoderma EtOAc extracts

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(100 µg/10 µL) as treatment, acetone-water (1:9, v/v) as a negative control, or propamocarb

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Pestanal® (1 µg/10 µL; Sigma, St. Louis, MO, USA) as a positive control were placed on top

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of the Phytophthora plug. The plates were then incubated at 18–30°C for 5–13 days until

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Phytophthora growth in the negative control plates reached the edge of the plates, during

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which time the colony diameter was measured daily. The optimal temperature of

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Phytophthora growth is dependent on the species mentioned above. Each experiment was 5

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repeated twice, with three biological replications per experiment. The percent inhibition of

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mycelial growth was calculated as follows: growth inhibition (%) = [(control mycelia

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diameter – treated mycelia diameter) / control mycelia diameter]  100 (Satish et al., 2007).

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Trichoderma isolates selected through preliminary screening that showed 100% inhibitory

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activity against at least three Phytophthora species via EtOAc extract with a concentration of

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100 µg/10 µL were further tested for anti-Phytophthora activity using: i) minimum inhibitory

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concentration (MIC) test using the EtOAc extract, ii) disk diffusion test using the EtOAc

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extract, and iii) antibiosis test using the liquid culture filtrate of potato dextrose broth (PDB).

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2.4. Additional tests

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The MIC of the EtOAc extract against Phytophthora was determined by the modified

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agar diffusion method (Espinel-Ingroff et al., 2007). The MIC was determined using the same

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method used for preliminary screening with three different concentrations of EtOAc extracts,

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100, 10 and 1 µg, in 10 µL of acetone-water (1:9, v/v). Seven Phytophthora species were

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treated with the extracts from eight Trichoderma isolates, which were selected through

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preliminary screening. Each experiment was repeated two times, with three biological

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replications per experiment.

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For the disk diffusion test, a small plate (6 cm diameter) containing 10 mL PDA or cV8

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was used for the disk diffusion test. cV8 plates were used for P. infestans and P. sojae instead

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of PDA plates owing to slow growth in the PDA plate. Two layers of sterile filter paper discs

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(0.6 cm diameter) that contained EtOAc extracts [100 µg/10 µL of water (1:9, v/v)] as

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treatment and acetone-water (1:9, v/v) as a control were placed on the surface of PDA or cV8

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plates at 3 cm from a Phytophthora plug (0.6 cm diameter) that had been cut from the

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actively growing edge of and placed on the plate mycelial side down . The plate was then 6

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incubated for 5–13 days at 18–30°C until Phytophthora growth in the control reached the

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edge of the plate. The colony diameters of each Phytophthora were measured daily, after

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which the growth inhibition (%) in response to the EtOAc extract relative to the control was

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calculated using the formula provided above (Satish et al., 2007). Each experiment was

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repeated twice, with three biological replications per experiment.

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For the antibiosis test, liquid cultures of the selected Trichoderma isolates were grown

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for 14 days in MIN media at 25°C with shaking at 150 rpm. Mycelia were then removed

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using four layers of sterile cheese cloth. The liquid cultures were filtered using a 0.22 μm

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syringe filter system (Millipore, Billerica, MA, USA). Agar plates were prepared by adding

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4.5 mL of liquid culture filtrate and 4.5 mL of MIN 3% Bacto agar to small plates (6 cm

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diameter). Control plates contained uninoculated MIN broth instead of Trichoderma liquid

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culture filtrate (Bae et al., 2011). Phytophthora plugs (0.6 cm diameter) were then placed

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mycelial side down in the middle of each plate and incubated at 18–30°C for 5–13 days. The

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colony diameters of each Phytophthora isolate were measured daily. Growth inhibition (%)

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by the extract relative to the control was calculated as a percentage using the formula given

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above. Experiment was repeated twice, with three biological replications per experiment.

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2.5. Field emission-scanning electron microscopy (FE-SEM) Morphological changes in Phytophthora were observed after treatment with Trichoderma

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extract using FE-SEM. Specimens were prepared using the same culture method as in the

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disk diffusion test, with the following modification. A small piece of membrane filter (2  2

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cm, Sigma) was placed between the Phytophthora inoculum and filter paper disc that

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contained the EtOAc extract (Ahameethunisa and Hopper, 2010). The plate was then

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incubated at 18–30°C until Phytophthora grew on the membrane filter. Osmium tetroxide 7

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(1%) was used to fix the mycelia on the membrane filter for 24 h at 4°C. The fixed specimens

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were subsequently dehydrated in serially diluted ethanol (50%, 70%, 80%, 90%, 95% and

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twice at 100%) for 20 min each, after which they were placed in iso-amyl acetate for 30 min

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twice and dried in a critical point dryer with CO2. Next, the dried samples were coated with

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gold/palladium using an ion-sputterer in a high-vacuum chamber. Finally, samples were

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observed by FE-SEM/energy-dispersive spectroscopy (EDS) (S-4100, Hitachi Ltd., Tokyo,

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Japan).

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2.6. Analysis of gene expression P. sojae was cultured on PDA media for 10–14 days. Three agar plugs (0.6 cm diameter)

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were cut from the actively growing edges of the plates and inoculated into 5 mL PDB. The

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inoculated PDB were then incubated by shaking at 150 rpm and 25°C for 3 days. One-fifth

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volume (1 mL) of PDB culture was subsequently removed from the liquid culture and

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replaced with 1 mL (10 µg/ µL) of the EtOAc extract of KACC 40557. The same volume of

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acetone-water (1:9, v/v) was added to the liquid culture instead of the extract as a control.

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The cultures were incubated with shaking at 150 rpm and 25°C for 0.5, 1, 6 and 12 h, after

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which the mycelia were harvested and RNA was extracted using an RNeasy plant mini kit

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(Qiagen, Valencia, CA, USA).

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Pepper (Capsicum annum cv. Bugang) and miniature dwarf tomato (Solanum

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lycopersicum, Micro-Tom) were grown in a growth room under continuous light (150 µmol

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m-2 s-1) at 23°C. Leaves with similar developmental stage and size were harvested and placed

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abaxial side up on wet Whatman filter paper in a plate. The EtOAc extract of KACC 40557

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(100 µg/10 µL) was applied onto the center of leaves in two positions. Acetone-water (9:1)

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was used as a control. P. capsici plugs (0.6 cm diameter) were harvested from actively 8

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growing edges of the PDA plate using a sterile cork borer. Two plugs were placed on top of

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the extracts (EP, extract + P. capsici plug) or acetone-water (AP, acetone-water + P. capsici

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plug) directly in the middle of the leaf tissues so that the mycelial side faced the extract or

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acetone-water. PDA plugs without P. capsici were placed on top of the extract as an extract

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control (EA, extract + PDA plug without P. capsici ). The plates were then covered with lids,

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sealed with parafilm, and incubated at 23°C in the dark. The percent total infection (lesion)

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was measured after 48, 72 and 96 h of inoculation, and the leaves were harvested in liquid

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nitrogen to extract RNA and plant hormones.

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The molecular responses to the EtOAc extract of KACC 40557 were analyzed in the

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detached leaves of pepper or tomato with or without P. capsici using real-time quantitative

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reverse transcription polymerase chain reaction (real-time qRT-PCR). Complementary DNA

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(cDNA) was synthesized using 1 μg of RNA and the GoScript Reverse Transcription System

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(Promega, Madison, WI, USA). Real-time qRT-PCR was conducted as previously described

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(Bae et al., 2006) using the Mx3000P qPCR system (Agilent, Santa Clara, CA, USA) with

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SYBR Green real-time PCR Master Mix (LPS solution, Daejeon, Korea). PCR primers were

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designed using the Primer 3 software from Biology Workbench (Table 1; Subramaniam,

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1998). ACTIN (ACT), a constitutively expressed gene, was used as a reference control for

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normalization during real-time qRT-PCR analysis. Analysis of relative transcript expression

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level was conducted as previously described by Livak (2001). Each experiment was

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performed using three or four independent biological replicates.

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2.7. Analysis of plant hormone Plant hormones were analyzed using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system to monitor changes in response to the EtOAc 9

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extract of KACC 40557 in the detached pepper leaves with or without P. capsici (Pan et al.,

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2010). Five hormones were analyzed: auxin (IAA), gibberellin (GA3), abscisic acid (ABA),

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jasmonic acid (JA) and salicylic acid (SA). Briefly, the leaf tissues were ground into fine

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power, after which 50 mg of the ground samples were used for hormone analysis. Internal

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standard (IS) solution (50 µL) was also added to the samples to give a final concentration of 1

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mg/mL. Next, extraction solvent (500 µL of 2-propanol/H2O/concentrated HCL, 2:1:0.002,

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v/v/v) was added to the sample, after which the samples were placed in a shaker at 100 rpm

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and 4°C for 30 min. After adding 1 mL of dichloromethane, samples were returned to the

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shaker for 30 min at 4°C. This step was followed by centrifugation of samples at 14,000 rpm

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and 4°C for 5 min. The supernatant was then removed and concentrated in a nitrogen

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evaporator. Finally, samples were dissolved in 0.1 mL methanol and stored at -20°C until

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further analysis. HPLC for hormone analysis was conducted using an Agilent 1100 series

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HPLC system equipped with a degasser, pump, auto-sampler and column oven.

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Chromatographic separations were performed using a SunFireC18 column (2.1 × 10 mm)

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(Waters, Milford, MA, USA). The isocratic mobile phase of 15:85 v/v, 0.1% formic acid in

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water and 0.1% formic acid in methanol was applied at a flow rate of 300 µL/min, column

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temperature of 30°C and an injection volume of 10 L in all experiments. Measurements for

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MS analysis were made using a 500°C source temperature, 5.5 KV (positive) and -4.5 KV

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(negative) ion spray voltages, 3 collision gas (CAD), 15 curtain gas (CUR), 45 ion source gas

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and dwell times of 1, 2 and 150 ms. Calibration curves of the standards were prepared using

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0.25, 0.5, 1.0, 2.5, 5.0, 50 and 500 ng/mL ABA, JA and SA, 0.5, 1.0, 2.0, 5.0, 10, 100 and

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1000 ng/mL GA3, and 12.5, 25, 50, 125, 250 and 2500 ng/mL IAA. Calibration curves were

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estimated using the ratio of ABA, GA3, IAA, JA, and SA area/IS area versus the ratio of

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ABA, GA3, IAA, JA and SA/IS concentration. Each calibration curve was assayed

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individually using a 1/x weighted linear regression.

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2.8. Statistical analysis

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Comparison among groups was conducted by one-way analysis of variance (ANOVA) with

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Turkey’s test using the SPSS software, version 22.0 (IBM Corp, Armonk, NY, USA), at a

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significance level of 5%.

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3. Results and Discussion

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3.1. Anti-Phytophthora activities of Trichoderma isolates

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Eight Trichoderma isolates were selected based on the results of preliminary screening,

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which showed 100% inhibitory activity against at least three Phytophthora species using the

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concentration (100 µg/10 µL) of EtOAc extracts of Trichoderma isolates (Table 2). Further

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tests were conducted using MIC, disk diffusion and antibiosis methods with the eight selected

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Trichoderma isolates.

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In the MIC test, high concentrations (100 µg/10 µL) of the EtOAc extracts of three

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Trichoderma isolates (KACC 40552, 40557 and 40929) showed broad inhibitory effects

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against seven Phytophthora species, while KACC 40553 and 40931 showed inhibitory effects

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against five and six Phytophthora species, respectively (Table 2). The EtOAc extracts of

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KACC 41717, 40871 and 41707 showed inhibitory activity against four, three and three

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Phytophthora species, respectively. Using medium concentration of 10 µg/10 µL, KACC

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40929 extract exerted 100% inhibition against five Phytophthora species, but less than 100%

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inhibition against P. catorum and P. nicotianae (83% ± 1 and 60% ± 17, respectively).

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KACC 40931 also exerted strong activity against all Phytophthora species except P.

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nicotianae.

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The efficacy as an anti-Phytophthora agent was evaluated by the disk diffusion method.

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KACC 40929 extract exerted broad inhibitory effects against seven Phytophthora species

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(18–100%), with the strongest inhibition being observed against P. sojae (100%), while

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KACC 40931 extract exerted inhibitory effects against six Phytophthora species (9–93%),

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with the strongest inhibition against P. infestans (93% ± 0) and P. drechsleri (79% ± 3).

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KACC 41717 extract showed anti-Phytophthora activity against three species with 16% (P.

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meloni) to 87% (P. infestans) inhibition. The remaining extracts showed mild inhibitory

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effects against Phytophthora species.

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According to the antibiosis test, the culture filtrates of five isolates (KACC 40552, 40553,

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405575, 40929 and 40931) showed inhibitory activity against at least five Phytophthora

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species, while the remaining three (KACC 40871, 41707 and 41717) exerted inhibitory

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effects against three or less Phytophthora species. None of the culture filtrates showed greater

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than 50% inhibitory activity against P. drechsleri and P. sojae. However, the growth of P.

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capsici and P. nicotianae was inhibited by 80% and 64% in response to the culture filtrates of

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KACC 40929 and KACC 40552, respectively. Additionally, the growth of P. melonis was

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inhibited by more than 50% by KACC 40552, 40553, 40557, 40929 and 41717, while P.

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cacturum growth was inhibited by more than 50% by KACC 40552, 40553, 40557 and 40929.

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P. infestans was inhibited by more than 50% by KACC 40553, 40557, 40929, 40931 and

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41717. Overall, KACC 40929 was found to be the best isolate for antibiosis against

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Phytophthora pathogens. Antibiosis occurs during interactions with pathogens and low-

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molecular-weight diffusible compounds or antibiotics, which are produced by Trichoderma

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(Benítez et al., 2004). 12

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Overall, while KACC 40929 showed the strongest activity against Phytophthora

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pathogens, KACC 40557 and 40931 showed good potential as biocontrol agents, which is in

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accordance with the results of previous studies (Etebarian et al. 2000; Srivastav et al. 2011).

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There have been many previous studies of T. virens (KACC 40929) as a biocontrol agent;

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therefore, we used KACC 40557 (T. atroviride/petersenii) for further analysis because it has

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not yet been investigated in detail, despite showing good potential as a biocontrol agent.

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3.2. Morphological changes in Phytophthora by Trichoderma extracts Treatment with Trichoderma extracts caused significant morphological changes in

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Phytophthora hyphae, including swelling, knotting, crumpling, flattening, shriveling, bursting

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and necrosis (Fig. 1). Morphological changes of seven Phytophthora mycelia treated with

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Trichoderma extracts were observed using FE-SEM as follows: KACC 40929 extract against

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P. melonis (A), P. sojae (B), P. capsici (C), P. cactorum (D), P. infestans (E); KACC 40553

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extract against P. cactorum (F); KACC 40552 and KACC 40557 extracts against P.

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nicotianae (G and H, respectively); KACC 41717 extract against P. drechsleri (I). These

301

changes might be due to the loss of cell wall integrity owing to interference with cell wall

302

biosynthesis and/or cell wall degradation. Trichoderma spp. produce diverse metabolites that

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interfere with cell wall synthesis directly or indirectly, leading to drastic morphological

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changes (Robert-Seilaniantz et al., 2011; Mukherjee et al., 2013). These results also

305

confirmed the strong activity of the extracts against Phythophthora.

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3.3. Molecular response of P. sojae upon Trichoderma extract treatment

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The ability of some Trichoderma species to suppress plant disease and stimulate the

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growth and development of plants explains their widespread and long term use as biocontrol 13

310

agents during cultivation of many crops (Hermosa et al., 2000; Schubert et al., 2008). The

311

beneficial effects of Trichoderma on plants have been attributed to their capacity to

312

antagonize pathogens such as Phytophthora via a combination of antibiosis, mycoparasitism

313

or competition for space and substrate. The anti-Phytophthora activity of Trichoderma may

314

be caused by altering the gene expression of Phytophthora pathogens. Therefore, analysis of

315

gene expression was conducted to better understand the molecular responses of P. sojae in

316

liquid culture by treatment with KACC 406557 extract. Specifically, we investigated

317

expressional changes in the genes encoding cellulose synthase 1 (CES1), putative P-type

318

adenosine triphosphatase (ATPase), mitogen-activated protein kinase (MAPK), and necrosis-

319

inducing-like protein (NLP) (Table 1; Fig. 2). The overall expression pattern was similar in

320

four P. sojae genes in response to the extract treatment.

321

Cellulose, which is a homopolymer consisting of β-1,4-glucan chains synthesized at the

322

plasma membrane, is one of the major components of cell walls. Cellulose synthesis is

323

carried out by cellulose synthase 1 (CES1) localized in the plasma membrane. Real-time

324

qRT-PCR results showed that CES1 was significantly up-regulated by KACC 40557 extract

325

relative to a control acetone-water treated sample. Up-regulation of P. sojae CES1 started

326

from 6 h after treatment (736% and 46% relative to P. sojae ACT in treatment and control,

327

respectively). Previous reports suggested that cellulose synthases played significant roles in

328

osmotic stress tolerance in a reactive oxygen species (ROS)-mediated manner (Zhu et al.,

329

2011). Potential fungicides bind to cellulose synthase and directly regulate fungal

330

pathogenesis (Blum et al., 2012). Accordingly, CES1 might be up-regulated to avoid the

331

stress generated by the extract.

332 333

Transmembrane ATPases are a class of plasma membrane-bound enzyme that catalyze the decomposition of ATP to ADP, leading to the liberation of free phosphate ions (Heguera 14

334

and Beauge, 1997). ATPases are also involved in the import and export of various

335

metabolites for different cellular functions and play important roles in overcoming various

336

types of stresses to maintain cellular metabolism (Malofeeva et al., 2012). P. sojae ATPase

337

was significantly up-regulated from 6 h after treatment (48% and 9% in treatment and control,

338

respectively), indicating that the extract might act as stress in P. sojae.

339

In eukaryotic cells, a family of serine/threonine protein kinases known as mitogen-

340

activated protein kinases (MAPKs) are involved in diverse signal transduction pathways,

341

including biotic and abiotic stress responses (Sinha et al., 2011). The MAPK of P. sojae was

342

significantly up-regulated from 6 h after treatment (10% and 0% in treatment and control,

343

respectively). In fungi, MAPKs are important to maintenance of cell wall integrity, hyper-

344

osmoregulation and spore formation. Up-regulation of P. sojae MAPK was found to be

345

required for formation of zoospores and cysts in a stress-mediated manner (Li et al., 2010).

346

To overcome the stress caused by the extract, P. sojae might strengthen its cell wall or

347

shorten its life cycle through the formation of spores via up-regulation of MAPK. However,

348

we did not detect increased sporulation during treatment.

349

NLPs are necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins

350

widely distributed in eukaryotic and prokaryotic plant pathogens that play critical roles in

351

various stress responses (Qutob et al., 2006). The NLP of P. sojae treated with extract was

352

induced at 12 h (37% and 0% in treatment and control, respectively). The expression of NLP

353

transcript was modulated by cellular toxicity and immune responses, and NLP is known to

354

elicit programmed cell death in a ROS-mediated manner (Qutob et al., 2006). Accordingly,

355

the extract might elicit ROS-mediated immune response, leading to cell death of P. sojae.

356 357

3.4. Molecular and hormonal responses of plants upon Phytophthora infection and 15

358 359

Trichoderma extract treatment We analyzed the molecular responses of pepper and tomato leaf tissues after infection

360

with P. sojae and treatment with KACC 40557 extract. The percent total infection was

361

measured up to 96 h after treatment (Fig. 3). Severe damage was observed in pepper leaves

362

from 72 h after treatment in AP (24%), while no significant damage was detected in PDE

363

(1%) and EP (0%). The damage was increased by up to 62% in AP 96 h after treatment, while

364

there was minor damage in EA (2%) and no damage in EP (0%). Severe damage was

365

observed in tomato leaves from 48 h after treatment in AP (35%), while small spots were

366

detected in EA (2%) and EP (2%). The damage was accumulated up to 74% in AP 96 h after

367

treatment, while minor damage was observed in EA (4%) and EP (10%). These results

368

indicate that the extract functioned as an anti-Phytophthora compound against P. sojae in

369

pepper and tomato leaves without severe harmful effects on the plants. Anti-Phytophthora

370

activity was more efficient in pepper leaves than tomato leaves, possibly due to the different

371

interactions between plants, pathogens and extracts. The same leaf tissues were used for the

372

analysis of plant hormones.

373 374 375

3.4.1. Molecular responses of pepper leaves Eight pepper genes were analyzed to understand their roles in P. capsici-mediated

376

pathogenesis and their modulation upon treatment with KACC 40557 extract: catalase (CAT),

377

ascorbate peroxidase (APX), cys2/his2-type zinc finger transcription factor (ZFP1), capsaicin

378

synthase (CSY1), methionine-R-sulfoxidereductase B2 (MSRB2), baxinhibitor 1 (BI-1),

379

receptor-like protein kinase (RLK), and mitogen-activated protein kinase kinase (MAPKK)

380

(Fig. 4).

16

381

CAT and APX are well-known for their ability to scavenge ROS (Bailey-Serres and

382

Mittler, 2006). The CAT transcript level was significantly up-regulated by treatment with the

383

extract (1130% and 546% relative to pepper ACT in EA and EP, respectively) relative to the

384

control (157% in AP) at 48 h. The expression of CAT was decreased in EA and EP (815%

385

and 169%, respectively) at 72 h, while it was up-regulated in AP (429%). These findings

386

indicate that the induction of CAT might no longer be required due to the death of P. capsici

387

in response to the extract in EP, while its induction may still be necessary owing to pathogen

388

growth in AP. Conversely, the expression of APX was up-regulated by the extract (181% and

389

186% in EA and EP, respectively) relative to AP (152%) at 48 h, and remained static during

390

treatment. Overall, these results indicate that the extract was able to induce CAT and APX

391

transcripts, which ultimately led to protection of leaf tissues from P. capsici. As previously

392

shown, the extract directly inhibited Phytophthora growth.

393

ZFP1 responds to different environmental and stress related stimuli (Courchesne et al.,

394

2009), and transgenic rice over-expressing ZFP19 showed increased tolerance to oxidative

395

stress through ROS scavenging (Sun et al., 2010). In this study, ZFP1 was significantly up-

396

regulated in P. capsici infected pepper leaves (28% in AP) relative to the other samples at 48

397

h (8% and 3% in EA and EP, respectively). ZFP1 transcripts in EA and EP were up-regulated

398

during treatment in a similar manner. P. capsici could grow in the pepper leaves (AP) due to

399

the absence of extract, which created stress conditions in plant cells and eventually induced

400

ZFP1.

401

Capsaicin is a unique alkaloid of the plant kingdom that is restricted to the genus

402

Capsicum and generally characterized as a counter irritant, analgesic or antioxidant agent

403

(Barceloux, 2009). Significant up-regulation of CSY1 was observed 48 h after inoculation in

404

EA and EP (0.14% and 0.28%, respectively) relative to AP (0.03%). The highest transcript 17

405

level was detected in EP at 48 h, indicating that CSY1 was highly up-regulated by the

406

combination of P. capsici and extract (EP), while P. capsici infection (AP) had minor effects

407

on the expression of CSY1. The transcript level in EA and EP returned to baseline from 72 h,

408

while the CSY1 level of AP remained almost unchanged during treatment. Taken together,

409

these findings indicate that CSY1 might be an important factor in protection of leaf tissues

410

from P. capsici.

411

MSRB2 enzyme catalyzes the reduction of free and protein-bound methionine sulfoxide

412

to methionine, which confers biotic and abiotic stress responses (Siddiqui et al., 2014).

413

MSRB2 transcript was significantly up-regulated in AP (277%) and EA (203%) at 48 h, while

414

it was moderately induced in EP (73%). At later time points, the MSRB2 levels of AP (20%

415

and 19% at 72 h and 96 h, respectively) and EP (57% and 50% at 72 h and 96 h, respectively)

416

were reduced to a similar level of EP (63% and 43% at 72 h and 96 h, respectively). These

417

findings indicate that P. capsici in AP and the extract in EA might act as a stimulus to induce

418

MSRB2. Simultaneous treatment with P. capsici and extract (EP) did not induce MSRB2,

419

possibly because the extract was primarily used to inhibit P. capsici growth.

420

The BI-1 protein is an evolutionarily conserved endoplasmic reticulum (ER) localized

421

protein that protects plants against stress-induced apoptosis (Kiviluoto et al., 2012). The B1-1

422

regulates important cellular stress responses via interactions with a broad range of partners to

423

inhibit many facets of apoptosis, including production of ROS, cytosolic acidification and

424

calcium levels (Robinson et al., 2011). The expression levels of BI-1 in EA and EP (56% and

425

55%, respectively) were significantly higher than in AP (36%) at 48 h, indicating the

426

induction of BI-1 transcript by the extract. At 96 h, the transcript levels of the three samples

427

were similar owing to a gradual decrease in EA and EP and an increase in AP during

428

inoculation. The BI-1 level was the lowest in the EP sample (38%) at 96 h. 18

429

The plant immune system is activated by microbial infection, which is recognized by

430

immune receptors localized in the cytoplasm or plasma membrane (De Smet et al., 2009).

431

RLK is a receptor localized in the plasma membrane. Upon initiation of stress signaling, RLK

432

receives the signal and transmits it to downstream effector proteins, which conduct necessary

433

action against stress conditions (Alam et al., 2010). In this study, the transcript levels of RLK

434

were similar in all samples at 48 h. RLK was up-regulated in EA (45% and 41% at 72 h and

435

96 h, respectively) and EP (37% and 37% at 72 h and 96 h, respectively) at later time points,

436

while RLK level did not change significantly in AP (12% and 16% at 72 h and 96 h,

437

respectively). This difference might have been due to the severe damage to pepper leaf

438

tissues in AP.

439

MAPKs are important signaling molecules that modulate various biotic/abiotic stress

440

responses in plants (Sinha et al., 2011). MAPKs act in a relay signaling module that starts

441

from mitogen-activated protein kinase kinase kinase (MAPKKK). MAPKKK passes the

442

signals to mitogen-activated protein kinase kinase (MAPKK), which passes the signal to

443

MAPKs. MAPKs subsequently act on their downstream effector proteins to regulate

444

downstream responses (Sinha et al., 2011). In the present study, the transcript level of

445

MAPKK was significantly higher in EA (82%) at 48 h than in AP (58%) and EP (54%). At 96

446

h, MAPKK transcript levels were higher in EA (44%) and EP (64%) than in AP (15%),

447

possibly because the pepper leaf tissues were severely damaged in AP.

448 449 450

3.4.2. Molecular responses of tomato leaves The expression patterns of two tomato genes were also analyzed to investigate

451

Phytophthora-mediated pathogenesis and their modulation upon treatment with KACC 40557

452

extract. Two genes encoding the following proteins were analyzed: pathogenesis-related 19

453

protein 1 (PR-1) and receptor-like protein kinase (RLK) (Fig. 5). During pathogen infection,

454

plants usually undergo induction of genes involved in defense (Dao et al., 2011; Liu and

455

Ekramoddoullah, 2006). It is well-known that PRs are induced by various pathogens and

456

biotic stresses (Liu and Ekramoddoullah, 2006). In this study, PR-1 was significantly up-

457

regulated by P. capsici (AP) during inoculation (1636%, 4994% and 11,415% at 48 h, 72 h

458

and 96 h, respectively), but not in EP sample (0.07, 2.87 and 9.30% at 48 h, 72 h and 96 h,

459

respectively). The absence of changes in the EP sample during treatment indicates biocontrol

460

activity of the extract against P. capsici. Treatment with extract inhibited P. capsici growth;

461

therefore, there was no induction of PR-1 in response to P. capsici. Treatment with extract

462

alone (EA) also induced PR-1. The plant immune system is activated by a microbial pattern

463

recognized by immune receptors that are cytoplasmic or localized at the plasma membrane

464

(De Smet et al., 2009), including receptor-like protein kinase (RLK). In this study, RLK was

465

significantly up-regulated in EP (0.25% and 0.80% at 72 and 96 h, respectively) from 72 h

466

after inoculation; however, its expression was not changed in EA or AP. Overall, these results

467

confirmed that the extract was able to control P. capsici growth in tomato.

468 469 470

3.4.3. Hormonal responses of pepper leaves Plant hormones comprise a collection of structurally unrelated signaling molecules derived

471

from different metabolic pathways involved in diverse arrays of biotic/abiotic stress

472

responses (Hitomi et al., 2013; Robert-Seilaniantz et al., 2011). In this study, various plant

473

hormones were analyzed in detached pepper leaves in response to P. capsici and the extract

474

(Fig. 6).

475 476

The IAA levels were 0.29, 0.12 and 0.88 µg/g in the leaves of AP, EA and EP samples at 72 h, respectively. The IAA level of EP was significantly higher than that of the other two 20

477

samples. At 96 h, the IAA levels were 1.21, 0.96 and 1.03 µg/g in AP, EA and EP,

478

respectively. In addition, the IAA level of EP was not significantly different from 72 h to 96

479

h, while the levels in the other two samples were significantly up-regulated during treatment.

480

A synergistic effect on IAA was observed in response to combined treatment with P. capsici

481

and the extract (EP) at 72 h, while the IAA level was low at 72 h in EA. The combination of

482

P. capsici and the extract (EP) could induce IAA at early time points, which might be

483

important to protection of plants from pathogens.

484

At 72 h, the GA3 levels were 0.66, 0.31 and 0.65 µg/g in AP, EA and EP, respectively,

485

with similar values being observed in AP and EP samples, but significantly lower values in

486

EA. At 96 h, the GA3 levels were 3.62, 1.17 and 1.90 µg/g in AP, EA and EP, respectively.

487

Significant induction of the AP sample was observed at 96 h. These findings indicate that P.

488

capsici infection induced GA3 production, while the extract inhibited P. capsici growth and

489

led to minor GA3 induction in EP.

490

ABA was present at 0.19, 0.13 and 0.09 µg/g in AP, EA and EP at 72 h, respectively,

491

with a significantly higher level occurring in AP than that in EP. The absence of the extract in

492

AP sample allowed plants to undergo P. capsici growth, leading to the induction of ABA.

493

This induction is a well-known defense mechanism against pathogens that occurs through the

494

ROS scavenging system (Fath et al., 2001). ABA maintains a high amount of APX and

495

superoxide dismutase (SOD), leading to programmed cell death (Fath et al. 2001). Enhanced

496

ABA has also been reported to lead to pathogen susceptibility (Ton et al., 2009). At 96 h,

497

ABA was detected at 0.12, 0.62 and 0.31 µg/g in AP, EA and EP, respectively, while the

498

extract in EA and EP led to significant up-regulation of ABA, which might be important for

499

plant protection.

21

500

Plant responses to different biotic/abiotic stresses are locally or systemically orchestrated

501

by signaling molecules such as JA, which inhibits plant growth, induces plant defense, and

502

regulates fungal pathogen attack (Kunkel and Brooks, 2002). In this study, the JA levels were

503

0.37, 0.23 and 0.38 µg/g in AP, EA and EP at 72 h, respectively. Similar JA levels were

504

detected in AP and EP. At 96 h, the JA levels were 1.46, 1.09 and 0.50 µg/g in AP, EA and

505

EP, respectively. The JA levels were significantly up-regulated in AP and EP samples during

506

treatment, while no significant changes were observed in EP. In plants, enhanced JA levels

507

led to pathogen susceptibility (Thaler et al., 2012). In this study, the presence of the extract in

508

plants suppressed P. capsici growth (EP); however, pathogen infection induced necrosis in

509

leaf tissues (AP), which led to development of systemic acquired resistance (SAR) via up-

510

regulation of JA. Arabidopsis triple mutant fad3 (fatty acid desaturase3), fad7 and fad8 with

511

impaired JA production or jar1 (jasmonic acid resistant1) exhibited enhanced susceptibility

512

to various pathogens, including Alternaria brassiciola, Botrytis cinera and Pythium species

513

(Stintzi et al., 2001). One mutant, cev1 (constitutive expression of VSP1), exhibited increased

514

resistance to Erisyphe sp. (Ellis and Turner, 2001). Mutant cev1 stimulated both the JA and

515

ethylene signal pathways, regulating a defense pathway.

516

SA is also an important signaling molecule that plays a central role in plant defense

517

against pathogens (Kunkel and Brooks, 2002). We detected SA at 0.06, 0.28 and 0.10 µg/g in

518

AP, EA and EP at 72 h, respectively. Similar levels of SA were detected in AP and EP, while

519

significantly higher levels were found in EA. At 96 h, the SA levels were 1.07, 1.37 and 1.11

520

µg/g in AP, EA and EP, respectively. SA was significantly up-regulated at later time points

521

for each treatment without significant differences among treatments. SA levels were also

522

significantly up-regulated after pathogen infection, and SA treatment of plants resulted in

523

enhanced resistance to a broad range of pathogens (Vlot et al., 2009). SA is essential for the 22

524

rapid activation of defense response, which is mediated by several resistance genes for SAR.

525

Several PR genes with SA dependent expression have been investigated to better understand

526

the SA dependent defense mechanism. For example, Arabidopsis mutants (eds1, eds4, eds5,

527

pad4 and sid2) that are unable to produce SA showed high disease susceptibility relative to

528

wild type plants (Kunkel and Brooks, 2002). In the present study, no significant differences

529

were observed between AP and EP at each time point; however, the extract alone (EA)

530

significantly up-regulated SA at 72 h. Overall, these findings confirm that the extract highly

531

up-regulated SA in plants, which might prevent pathogen infection of the host plant.

532

In conclusion, metabolites extracted from Trichoderma spp. showed strong anti-

533

Phytophthora activities that were confirmed using minimum inhibitory concentration, disk

534

diffusion, and antibiosis tests. Treatment with the extract of KACC 40557 inhibited P. capsici

535

growth via changes in plant hormone levels and induction of defense-related genes in leaf

536

tissues. These results demonstrate that Trichoderma metabolites contain compounds with

537

strong biocontrol and antimicrobial activities against Phytophthora.

538 539

Acknowledgements

540

This research was supported by a Yeungnam University Research Grant in 214A367009.

541 542

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Stintzi, A., Weber, H., Reymond, P., Browse, J., Farmer, E.E. 2001. Plant defense in the absence of jasmonic acid: the role of cyclopentenones. Proc. Natl. Acad. Sci. USA. 98, 12837–12842.

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Subramaniam. 1998. The biology workbench - A seamless database. Proteins. 32, 1–2.

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Sun, S., Guo, S., Yang, X. 2010. Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice. J. Exp. Bot. 61, 2807–2818.

665 666

Thaler, J.S., Humphrey, P.T., Whiteman, N.K. 2012. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci. 17, 260–270.

667 668

Ton, J., Flors, V., Mauch-Mani, B. 2009. The multifaceted role of ABA in disease resistance. Trends Plant Sci. 14, 310–317.

669 670

Vinale, F., Sivasithamparam, K., Ghisalberti, E. L., Marra, R., Woo, S. L., and Lorito, M. 2008. Trichoderma–plant–pathogen interactions. Soil Biol. Biochem. 40:1–10

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Vlot, A.C., Dempsey, D.A., Klessig, D.F. 2009. Salicylic acid, a multifaceted hormone to combat disease. Annu. Rev. Phytopathol. 47, 177–206.

673 674 675

Zhu, J., Lee, B., Dellinger, M., Cui, X., Zhang, C., Wu, S., Nothnagel, E.A., Zhu, J. 2011. A cellulose synthase like protein is required for osmotic stress tolerance in Arabidopsis. Plant J. 63, 128–140.

676 677 678 679 680 681 682 27

683

Figure legends

684

Fig. 1. Scanning electron micrographs of seven Phytophthora mycelia treated with ethyl

685

acetate extracts of the selected Trichoderma isolates. Hyphal morphology of Phytophthora

686

spp. treated with the extracts of Trichoderma KACC 40929 against P. melonis (A), P. sojae

687

(B), P. capsici (C), P. cactorum (D), P. infestans (E). Hyphal morphology of P. cactorum

688

treated with the extract of Trichoderma KACC 40553 (F). Hyphal morphology of P.

689

nicotianae treated with the extracts of Trichoderma KACC 40552 and KACC 40557 (G, H).

690

Hyphal morphology of P. drechsleri treated with the extract of Trichoderma KACC 41717 (I).

691

The parameters for electron micrographs were as follows: 15.0 kV2.00 K15.0 μm for A–E,

692

G–I; 15.0 kV1.50 K20.0 μm for F.

693 694

Fig. 2. Gene expression patterns of Phytophthora sojae in response to the ethyl acetate

695

extract of Trichoderma KACC 40557. Three-day-old P. sojae liquid cultures were treated

696

with extract, after which mycelia were harvested for RNA extraction. Acetone in water was

697

used as a control. The transcript level was normalized against that of the P. sojae ACTIN

698

(ACT) gene. Significant differential expression (P < 0.05) was observed between control and

699

treated samples at 12 h after treatment. Bars indicate the means ± standard errors of three

700

biological replications.

701 702

Fig. 3. Comparison of Phytophthora capsici infection in the detached leaves of pepper and

703

tomato plants. Percent total infection was measured in leaves treated with acetone-water +

704

PDA plug with P. capsici (AP), Trichoderma KACC 40557 extract + PDA plug without P.

705

capsici (EA), and Trichoderma KACC 40557 extract + PDA plug with P. capsici (EP). Bars

28

706

indicate the means ± standard errors of three biological replications. Significant differences

707

were evaluated at P ≤ 0.01 (**) and P < 0.05 (*), respectively.

708 709

Fig. 4. Gene expression patterns of pepper leaves following treatment with ethyl acetate

710

extract of Trichoderma KACC 40557 and P. capsici. Transcript levels were measured in

711

leaves treated with acetone-water + PDA plug with P. capsici (AP), Trichoderma KACC

712

40557 extract + PDA plug without P. capsici (EA), and Trichoderma KACC 40557 extract +

713

PDA plug with P. capsici (EP). The transcript level was normalized against that of the pepper

714

ACTIN (ACT) gene as an internal control. Bars indicate the means ± standard errors of three

715

biological replications. The letters on the right side of each group indicate significant

716

differences between the mean values (P < 0.05). NS, not significant.

717 718

Fig. 5. Gene expression patterns of tomato leaves following treatment with the ethyl acetate

719

extract of Trichoderma KACC 40557 and P. capsici. Transcript levels were measured in

720

leaves treated with PDA plug with acetone-water + PDA plug with P. capsici (AP),

721

Trichoderma KACC 40557 extract + PDA plug without P. capsici (EA), and Trichoderma

722

KACC 40557 extract + PDA plug with P. capsici (EP). The transcript level was normalized

723

against that of the tomato ACTIN (ACT) gene. Bars indicate the means ± standard errors of

724

three biological replications. The letters on the right side of each group indicate significant

725

differences between the mean values (P < 0.05).

726 727

Fig. 6. Analysis of plant hormones in response to the ethyl acetate extract of Trichoderma

728

KACC 40557 and P. capsici. Hormone levels were measured in pepper leaves with acetone-

729

water + PDA plug with P. capsici (AP), Trichoderma KACC 40557 extract + PDA plug 29

730

without P. capsici (EA), and Trichoderma KACC 40557 extract + PDA plug with P. capsici

731

(EP). Bars indicate the means ± standard errors of three biological replications. Significant

732

differences were evaluated at P ≤ 0.01 (**) and P < 0.05 (*), respectively.

733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 30

754

Table 1. List of genes and primer sequences used for real-time quantitative reverse

755

transcription polymerase chain reaction (real-time qRT-PCR).

756 757

Table 2. Percent inhibition of the mycelial growth and anti-Phytophthora activity of the

758

selected Trichoderma metabolites against seven Phytophthora pathogens.

759 760

31

Table 2. Percent inhibition of the mycelial growth and anti-Phytophthora activity of the selected Trichoderma metabolites against seven Phytophthora pathogens. P. melonis MIC T. atroviride T. petersenii a (KACC 40552) T. gamsii T. koningiopsis (40553) T. atroviride T. petersenii (40557)

b

DD

%

µg

100

100

-

100

100

100

c

P. cactorum AB

d

MIC µg

69±3

100

100

-

-

87±1

100

100

100

-

82±0

100

-

-

-

-

T. harzianum (40871)

-

T. virens (40929)

100

10

75±9 83±1 82±15

T. brevicompactum 51±3 (40931)

10

30±1

T. brevicompactum 46±1 (41707)

10

23±4

10

16±4 83±1

T. virens (40717)

e

79±6

DD

%

P. drechsleri AB

MIC

DD

%

µg

79±2

100

100

-

-

83±0

100

100

100

-

83±0

100

-

-

-

P. sojae AB

MIC

DD

%

µg

21±1

100

100

-

-

39±1

-

-

100

-

33±2

100

-

-

-

-

P. capsici AB

MIC

DD

%

µg

5±2

100

100

7±5

-

-

100

100

100

17±5

5±2

100

100

100

-

3±2

P. nicotianae AB

MIC

DD

P. infestans DD

AB

100

8±1

27±1

100

100

7±2

62±3

24±2 10±1

100

100

13±2 57±1

100

18±2

8±2

100

100

16±1

7±5

18±2 13±1

100

10

69±4

100

%

µg

26±0

100

100

7±0

33±2

-

-

100

5±2

33±2

100

100

-

-

-

-

100

AB

28±1 64±2

-

-

MIC %

µg

100

10

69±3 80±0

100

10

59±2 41±1

100

10

100

35±3

100

10

27±1 80±1 60±17

10

3±0 82±15 100

24±2 13±3

100

100

79±3 26±1 58±5

10

27±5 41±3

100

10

9±2

-

-

-

-

-

37±3

1

93±0 66±2

-

-

-

-

-

-

-

-

-

-

-

100

100

-

-

74±3 39±4

-

-

-

-

40±2

10

5±2

-

-

-

-

-

29±6

1

81±1 34±4

-

-

-

-

33±3

1

-

14±1

-

-

-

-

37±2

1

87±2 67±4

a

Korean Agricultural Culture Collection; bMinimum inhibitory concentration (X µg/10µL); cDisc diffusion test (100 µg/10 µL); dAntibiosis test (100 µg/10 µL); eNo antifungal activity

1

Table 1. List of genes and primer sequences used for real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR). Gene

GenBank ID

Forward primer sequence (5’ 3’)

Reverse primer sequence (5’ 3’)

Phytophthora sojae CES1

EF563997

CGTAAGTCCAAGTGGTTGAACAAG

CAGGTTGTACGCGATGTAGACAC

ATPase

EU938005

ACGTTCGTCATGTTCGATATGTTC

AATGCAGCAGACGTACATCAAGT

MAPK

FJ349603

AGCTTTGACTTCGACTTTGAGAAC

CGAAGAAGAAGAGGAGGAAGATGTA

NLP

AF511649

CAGCACGTTGTAGTAGTCGATCTT

CATCATGTACTCCTGGTACTTCCC

ACT

X15900

TCATGGTCGGCATGGACCA

GGCCGTGGTCGTGAACGAG

Capsicum annum CAT

DV643234

GGGTCCTGTCTACAACAATGTATCT

GTAGAGAAGCGACAAATAACAGGTG

APX

AAL35365

CCTACTGAAACTACCCACAGACATT

ATACCAGTAACTCAGTGCCACAACT

ZFP1

AY196704

GGAGAAGGAGAAGATGGTGTATAAG

ACACTTGTGACAAATCGAACACTC

CSY1

DQ349223

GTTTACGGATATTGTCAAGCAAGAG

CTCAGTAACTCCAATCTCCACAAGT

MSRB2

EF144172

ATTCTAAGACAGAAAGGAACCGAGT

ACAAGCTGCACAAGTAATCTCAATC

BI-1

FJ19768

AGGGAGTACTTGTACCTTGGAGG

CATGCTTGACGTAATCCATATCAC

RLK

EF397556

AGATCTACTTTCCACACTGGCTCTA

GAATATAGGTAAGGTTTGGGAGGAA

MAPKK

GQ249256

AGTGGATTAGACTACTTGCACAACC

AGCCATTGTAGTTGACTCCATAAGT

ACT

GQ339766

GACGTGACCTAACTGATAACCTGAT

CTCTCAGCACCAATGGTAATAACTT

Solanum lycopersicum PR-1

NM001247429

GTAGACAAGTTGGAGTCGGTCCTAT

CCAGCTTGACAAGTATTCGAGTTAT

RLK

SLU58473

GTTAATCCTTGAGATCTTGACTGGA

CCTCACAACAACTTAGTCCAATCTT

ACT

FJ532351

GGTCGTGATTTAACTGATAACCTGA

CTCTCAGCACCAATGGTAATAACTT

2

A

B

C

D

E

F

G

H

I

Fig. 1. Scanning electron micrographs of seven Phytophthora mycelia treated with ethyl

acetate extracts of the selected Trichoderma isolates. Hyphal morphology of Phytophthora spp. treated with the extracts of Trichoderma KACC 40929 against P. melonis (A), P. sojae (B), P. capsici (C), P. cactorum (D), P. infestans (E). Hyphal morphology of P. cactorum treated with the extract of Trichoderma KACC 40553 (F). Hyphal morphology of P. nicotianae treated with the extracts of Trichoderma KACC 40552 and KACC 40557 (G, H). Hyphal morphology of P. drechsleri treated with the extract of Trichoderma KACC 41717 (I). The parameters for electron micrographs were as follows: 15.0 kV2.00 K15.0 μm for A–E, G–I; 15.0 kV1.50 K20.0 μm for F.

6000

CES1

4000 2000

Expression relative to ACTIN (%)

0 300

ATPase

200 100 0 90

MAPK

60 30 0 60

NLP

40

Control Treatment

20 0 0

0.5

1

6

12

Time after treatment (h)

Fig. 2. Gene expression patterns of Phytophthora sojae in response to the ethyl acetate extract of Trichoderma KACC 40557. Three-day-old P. sojae liquid cultures were treated with extract, after which mycelia were harvested for RNA extraction. Acetone in water was used as a control. The transcript level was normalized against that of the P. sojae ACTIN (ACT) gene. Significant differential expression (P < 0.05) was observed between control and treated samples at 12 h after treatment. Bars indicate the means ± standard errors of three biological replications.

Tomato

AP EA EP

75 50 25

* *

0

**

100

% total infection

Pepper

100

*

*

75 50

*

** **

**

72

96

25 0

AP

EA

EP

48 72 96 h 48

72

96

48

Time after treatment (h)

Fig. 3. Comparison of Phytophthora capsici infection in the detached leaves of pepper and tomato plants. Percent total infection was measured in leaves treated with acetonewater + PDA plug with P. capsici (AP), Trichoderma KACC 40557 extract + PDA plug without P. capsici (EA), and Trichoderma KACC 40557 extract + PDA plug with P. capsici (EP). Bars indicate the means ± standard errors of three biological replications. Significant differences were evaluated at P ≤ 0.01 (**) and P < 0.05 (*), respectively.

1500

240

CAT

1000

APX

c b a

160

b AP

80 Capsicumabannum genes EA

Expression relative to ACTIN (%)

500 0 45

EP

0 0.36

ZFP1

30

CSY1

0.24

15

a b b 0.12

0 360

0.00 75

NS MSRB2

240

50

120

25

BI-1

b a b

NS

0 75

0 120

RLK

50

b ab

25

MAPKK

BI-1

80

b ab

40

a 0

a 0

48

72

96

48

72

96

Time after treatment (h) Fig. 4. Gene expression patterns of pepper leaves following treatment with ethyl acetate extract of Trichoderma KACC 40557 and P. capsici. Transcript levels were measured in leaves treated with acetone-water + PDA plug with P. capsici (AP), Trichoderma KACC 40557 extract + PDA plug without P. capsici (EA), and Trichoderma KACC 40557 extract + PDA plug with P. capsici (EP). The transcript level was normalized against that of the pepper ACTIN (ACT) gene as an internal control. Bars indicate the means ± standard

errors of three biological replications. The letters on the right side of each group indicate significant differences between the mean values (P < 0.05). NS, not significant.

Expression relative to ACTIN (%)

15000 10000

150 100 50 0

PR-1

a

5000

b b

0 1.2

RLK AP EA EP

0.8 0.4

a

b b

0.0 48

72

96

Time after treatment (h)

Fig. 5. Gene expression patterns of tomato leaves following treatment with the ethyl

acetate extract of Trichoderma KACC 40557 and P. capsici. Transcript levels were measured in leaves treated with PDA plug with acetone-water + PDA plug with P. capsici (AP), Trichoderma KACC 40557 extract + PDA plug without P. capsici (EA), and Trichoderma KACC 40557 extract + PDA plug with P. capsici (EP). The transcript level was normalized against that of the tomato ACTIN (ACT) gene. Bars indicate the means ± standard errors of three biological replications. The letters on the right side of each group indicate significant differences between the mean values (P < 0.05).

1.6

IAA

1.2

*

**

0.8 0.4 0.0 6.0

GA3

4.5 3.0

**

Concentration (ng/mg)

1.5

** **

0.0 1.6 1.2

ABA

0.8

**

0.4

** *

**

0.0 2.0

JA

1.5

**

1.0

**

0.5

* 0.0 2.0

72 h SA 96 h *

1.5 1.0 0.5

**

0.0 PHA AP

PDM EA

PHM EP

Treatment Fig. 6. Analysis of plant hormones in response to the ethyl acetate extract of Trichoderma KACC 40557 and P. capsici. Hormone levels were measured in pepper leaves with acetone-water + PDA plug with P. capsici (AP), Trichoderma KACC 40557 extract + PDA plug without P. capsici (EA), and Trichoderma KACC 40557 extract + PDA plug with P. capsici (EP). Bars indicate the means ± standard errors of three biological replications. Significant differences were evaluated at P ≤ 0.01 (**) and P < 0.05 (*), respectively.

HIGHLIGHTS 

Trichoderma spp. were screened for anti-Phytophthora activity.



Trichoderma atroviride/petersenii (KACC 40557) showed the best anti-Phytophthora activity.



Trichoderma metabolite treatment caused plant hormonal and transcriptional changes.

Graphical Abstract

Pepper

Tomato

P. capsici

P. capsici + Trichoderma extract