Antifungal activity of plant essential oils and selected pseudomonas strains against Phomopsis theicola

Mira Starović1, Danijela Ristić1, Goran Aleksić1*, Snežana Pavlović2, Mehmet Musa Özcan3, Magdalena Knezević4 and Dragana Jošić4 1Institute for Plant Protection and Environment, Teodora Drajzera 9, 11040 Belgrade, Serbia 2Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia 3Department of Food Engineering, Faculty of Agriculture, Selçuk University, 42079 Konya, Turkey 4Institute of Soil Science, Teodora Drajzera 7, 11040 Belgrade, Serbia *Corresponding author: algoran@sezampro.rs Received: 1 April, 2017 Accepted: 23 May, 2017


INTRODUCTION
The genus Phomopsis (Sacc.) Bubák has a wide geographical distribution in the world, with over 800 species that occur as endophytes, saprotrophs and parasites in a very diverse range of host plants, including woody and herbaceous hosts. Pathogenic Phomopsis spp. can cause considerable economic losses in different crops and they are often associated with shoot blights, leaf spots, fruit rots, stem cankers, and dieback (Udayanga et al., 2011). Species of Phomopsis and their Diaporthe sexual states are very dangerous pathogens of young apple trees. Phomopsis spp. produce cankers and dieback shoot blight on apple trees, and it may take years before damage to internal wood is severe enough to kill a fruit tree. Stem canker and dieback are important factors that limit the longevity of apple trees and reduce their yield in Serbia. Phomopsis spp. have been described as a cause of dieback and canker in apple fruit growing regions of South Africa (Van Niekerk et al., 2004;Cloete et al., 2011).
Diaporthe canker is controlled by sanitation along with chemical treatment. Sprays of thiophanate-methyl, applied three times after petal fall (10, 20 and 30 days), are recommended to control the diseases on European pear in Japan. Apple cultivars vary in their susceptibility to Diaporthe canker. 'Jonagold' and 'Jonatan' are more susceptible than 'Tsugaru', 'Starking Delicious' and 'Indo' (Sutton et al., 2014).
Over the years, chemical pesticides have made a great contribution to efforts to control plant diseases. However, intensive applications of pesticides have resulted in a development of fungal resistance and extensive damage to the environment. Therefore, an eco-friendly alternative is required to preserve the quality and generate quantity of agricultural products.
Essential oils (EOs) of plant origin are one of the significant products of agriculture-based industry. They have a wide application in folk remedies but in recent years their potential antimicrobial activity has been increasingly recognized. Numerous studies have documented the antifungal properties of plant products (Carmo et al., 2008;Tavassoli et al., 2011). A few of them have confirmed their antifungal properties against fungal pathogens of fruit.
Plant growth-promoting rhizobacteria (PGPR) colonize plant roots and promote growth of diverse plant species. The PGPR include diverse genera but Bacillus and Pseudomonas are predominant (Podile & Kishore, 2006). Pathogen control capacity has been attributed to several substances produced by antagonistic rhizobacteria. The bacteria perform antagonistic activity toward pathogens using several mechanisms: synthesis of hydrolytic enzymes that can lyse pathogenic fungal cells; competition for nutrients; colonization of niches at the root surface or production of siderophores and antibiotics (Kamilova et al., 2005;Neeraja et al., 2010;Maksimov et al., 2011). Antibiotics that have diverse mechanisms of action, including the inhibition of synthesis of pathogen cell walls and interference with membrane structures of cells have been produced by antagonistic bacterial strains (Maksimov et al., 2011). Several indigenous Pseudomonas spp. from the rhizosphere of different plants have been confirmed as PGPRs in Serbia (Jošić et al., 2012;Pivić et al., 2015;Jošić et al., 2015).
The aim of this study was to investigate the possibility of biological control of the phytopathogenic fungus Ph. theicola using several essential oils and indigenous Pseudomonas spp. strains.

Antifungal activity of EOs
The phytopathogenic fungus Ph. theicola was isolated from sunken canker tissue of apple trees cv. 'Golden Delicious' (Figure 1) in the locality Trstenik, Serbia (43º 37' N, 21º 00' E, and 164 m above the sea level). Essential oils (EOs) extracted by hydro-distillation from several medicinal plants: Turkish pickling herb (Echinophora tenuifolia), oregano (Origanum vulgare), basil (Ocimum basilicum) and myrtle (Myrtus communis) (Table 1), were used in antagonistic assays with a Ph. theicola isolate from the collection of the Institute for Plant Protection and Environment, Belgrade, Serbia ( Figure 2). Fungal spores were washed from the surface of potato dextrose agar (PDA) plates with sterile 0.85% saline containing 0.1% Tween 80 (v/v). The spore suspension was adjusted to a concentration of approximately 5.0x10 4 in a final volume of 100 μl per well.
Minimum inhibitory concentrations (MIC) were determined by microdilution method in 96 well microtiter plates (Daouk et al., 1995). Microtiter plates were incubated for 5 days at 28°C. The experiment was repeated four times. Fluconazole was used as a positive control. The lowest concentrations without visible growth were defined as the minimal concentrations which inhibited fungal growth ( Figure 3).
Separation of minimal inhibitory concentration (MIC) means was carried out by Duncan's multiple range tests. An analysis of variance was performed on MIC data for four EOs applied to Ph. theicola. Significance was evaluated at p<0.05. STATISTICA v.7 (StatSoft, Inc.) was used for statistical analyses.

Antifungal activity of Pseudomonas spp.
The assay for antagonism in vitro was performed on Waksman agar medium by dual culture method (Wolf et al., 2002). Overnight cultures (ONC) of bacteria were optimized to 10 7 CFUmL -1 and used for preparation of 3 fractions: cell-free supernatant (CFS), CFS treated with EDTA (ethylenediaminetetraacetic heat-treated cell-free supernatant (HS-CFS). ONCs were centrifuged twice at 13000 rpm for 5 min., without and with filtration (filter tubes with microporous membrane 0.22 μm) (Merck Millipore Ltd.); one aliquot was treated with 1mM EDTA, while another aliquot was heated at 70 o C for 30 min. Fungal mycelium was placed as a 6 mm plug in the center of each Petri dish, while bacteria (10 μL) were placed on its edges. Control variants contained only mycelia of Ph. theicola and the fungus with 1mM EDTA added instead of bacterial culture/fraction. The cultures were incubated at 25°C for 9 days. Morphological changes of Ph. theicola were observed in dual culture. The percentage inhibition (PI) of Ph. theicola growth was calculated using the following formula: PI=100 × (1-R2/R1), where R1 was the radial distance growth of the fungus in the control plate, and R2 was the radial distance growth of the fungus in bacterial treatment. All fungal inhibition assays were performed in four replicates and repeated three times.
Among all oils tested, oregano proved to be the best inhibitor of the apple pathogen Ph. theicola, followed by basil EO.

Antifungal activity of Pseudomonas spp.
All tested Pseudomonas spp. strains showed inhibition of Ph. theicola growth (Table 3). Morphological abnormalities of Ph. theicola, such as mycelial deviations, were observed in a dual culture using different fractions of Pseudomonas spp. strains. All treatments of E65 strain, as well as the CFS -EDTA and HS-CFS of M1 and K113 caused mycelial deformation and color change from dark brown to dark green. The same effects were observed in the HS-CFS of L1 and CFS-EDTA of B25 strains.
Differences in the effectiveness of Pseudomonas spp. strains on Ph. theicola growth inhibition are shown in Figure 4. The percentage of growth inhibition ranged from 13.5 (HS-CFS of E65) to 62.5% (ONC of K113). The highest inhibition was observed in all three fractions of K113 strain, while the CFS-EDTA fraction of L1 strain was the more effective inhibitor than the same fraction of K113 strain.

DISCUSSION
The results obtained in vitro could be useful from the practical point of view. Turkish pickling herb, oregano, basil and myrtle essential oils used in this study can be used in future field trial evaluation of the efficacy of control of Ph. theicola.
The results of this study may serve as a guide for selecting essential oils and their concentrations in further in vivo trials aimed at fungicide development. The data obtained in the present work suggest that the selected essential oils originating from Turkey can be applied as inhibitors to prevent growth of phytopathogenic fungi. In accordance with numerous previous reports, oregano and basil oils originating from Serbia had been found to inhibit the growth of Phomopsis species at concentrations of 70 μg/mL and 5950.00 μg/mL, respectively (Stević et al., 2014). Myrtle oil demonstrated bioactive properties, especially antifungal activity to Fusarium sp., Drechslera sp. and Macrophomina phaseolina (Starović et al., 2016). Oregano oil was found to control Botrytis cinerea and Monilinia laxa growth on stone fruit and Phomopsis sp. (Lopez-Reyes et al., 2013), while basil oil controlled Phomopsis sp., Fusarium spp., and Phoma sp. (Stević et al., 2014) using concentrations of 750μg/mL and 5.1-7.65 mg/mL, respectively.
Morphological deformation of the phytopathogenic fungus Ph. theicola was a significant feature of the activity of Pseudomonas spp. strains tested in this study.
Bacillus subtilis that produces diffusible and volatile compounds had been reported earlier to induce structural deformations in phytopathogenic fungi (Chaurasia et al., 2005). Ethyl acetate extracts of P. aeruginosa and B. subtillis culture filtrates caused similar effects on the germination and morphology of Ph. azadirachtae conidia (Girish et al., 2009).
In this study, the width of inhibition zones ranged from about 7 to 30 mm, which is similar to the results of Zalewska et al. (2004), who reported 8.6-23.5 mm inhibition zones of Ph. viticola caused by different Pseudomonas spp. A P. putida strain caused inhibition of Ph. viticola mycelial growth of 6-12 mm (Haggag et al., 2013), which is lower than the results for Ph. theicola inhibition by Pseudomonas spp. in this present study. The highest inhibition effect on Ph. theicola growth was shown by K113, followed by L1 strain, exceeding 60% and 57% for ONC and CFS, while the lowest inhibition values were observed for the ONC of B25 strain (45%) and CFS of M1 (39.5%). PI value decreased with EDTA treatment of CFS (30-39.5%), as well as with heat treatment of CFS (13.5-29.5%), suggesting that all tested strains produced thermosensitive extracellular metabolites. These results are consistent with the report by Girish et al. (2009), where ethyl acetate extract of P. oleovorans caused 42% of growth inhibition of Ph. azadirachtae. The same authors reported that Ph. azadirachtae inhibition by the same extract of P. aeruginosa reached the maximal PI value. Biological control activity of Pseudomonas spp. and Bacillus sp. against several Phomopsis species have been reported. Srinivas et al. (2005) reported higher effectiveness of P. fluorescens than T. harzianum and fungicide treatments in reducing Ph. vexans infection and increasing brinjal seed germination, vigor index and field emergence. Combinations of two systemic fungicides and P. aeruginosa culture filtrate were effective in in vitro growth inhibition of Ph. azadirachtae and had no significant negative effect on neem seed germination (Girish et al., 2012). Patkowska and Błażewicz-Woźniak (2013) applied post-culture liquids of antagonistic bacteria Pseudomonas sp. Ps 255 and Bacillus sp. B 73 to the surface of soybean seeds and limited plant infection with Ph. sojae and other fungi previously isolated from seeds. P. putida, the producer of fluorescent siderophore pseudobactin, was very effective as a biocontrol agent in reducing the dieback and phomopsis diseases of grapevine (Haggag et al., 2013). To the best of our knowledge, this is the first report on growth inhibition of Ph. theicola and biological control of this pathogen by indigenous Pseudomonas spp. in Serbia.
Pseudomonas spp. strains K113 and L1 were moderately effective in inhibiting Ph. theicola mycelial growth, showing more than 60% and 57% inhibition when ONC and CFS were used, respectively. Further assessments of in vivo effectiveness of K113 and L1 cultures in protecting apple from Ph. theicola will be useful for estimation of their applicability as an alternative to chemical protection.