In vitro sensitivity of Alternaria solani to conventional fungicides and a biofungicide based on tea tree essential oil

A study of in vitro sensitivity of five Alternaria solani isolates to cooper-oxychloride, chlorothalonil, difenoconazole, pyraclostrobin and a biofungicide based on tea tree essential oil was carried out. The isolates were obtained from infected tomato leaves collected from five different locations in Serbia. The tested isolates showed the highest sensitivity to pyraclostrobin with EC50 values ranging from 0.0014 to 0.0041 μg ml-1. The EC50 values of difenoconazole were 0.018-0.037 μg ml-1, chlorothalonil 2.99-4.54 μg ml-1, and cooper-oxychloride 13.27-15.63 μg ml-1. All tested A. solani isolates were the least sensitive to tea tree oil (1323.97-3307.08 μg l-1).


INTrODuCTION
Early blight of tomato, caused by the necrotrophic fungus Alternaria solani Sorauer, is one of the most common foliar diseases of tomato (Solanum lycopersicum L.). The disease can occur over a wide range of climatic conditions but is most intense in areas with heavy dew, rainfall and high relative humidity. It causes dampingoff of tomato seedlings, as well as collar rot, leaf spots, stem lesions and fruit rot in later stages. Plant infection may result in a complete loss of yield through foliage destruction and direct fruit damage by the pathogen, as well as sun blotch on defoliated plants (Rotem, 1998).
Typical symptoms of tomato early blight are dark spots with concentric rings of spores surrounded by a halo of chlorotic area on leaves. Early blight lesions first occur on older senescing leaves, which become chlorotic and abscise prematurely, eventually spreading to other foliage in plant canopy under conditions conducive for disease development (Franc & Christ, 2001;Ganie et al., 2013). Periods of profuse moisture from rain, dew or overhead irrigation are required for spore germination and infection, while temperatures ranging from 5 to 30°C are favourable for pathogen sporulation and disease development (Gudmestad et al., 2013).
Most currently grown tomato cultivars are susceptible to early blight to varying degrees, so that foliar fungicides are frequently used to manage the disease. The most effective early blight control measure consists of frequent applications of fungicides starting early in the growing season before the first symptoms appear (Pscheidt & Stevenson, 1988).
A number of foliar fungicides can be used to manage early blight in tomato. Copper-oxychloride, mancozeb and chlorothalonil are the most frequently used protectant fungicides for early blight management but they provide insufficient control under high disease pressure (Holm et al., 2003;Pasche & Gudmestad, 2008). Quinol-oxidizing inhibitor (QoI) fungicides have been used successfully since their introduction in 1999, and provided a very high level of disease control (Stevenson & James, 1999;Pasche & Gudmestad, 2008). Unfortunately, QoI resistance due to the F129L mutation in the cytochrome b gene was first detected in North Dakota and Nebraska in 2001 (Pasche et al., 2004;Pasche et al., 2005). The F129L mutation conveys a moderate level of resistance to QoI fungicides such as azoxystrobin and pyraclostrobin. Development of resistance of pathogenic fungi towards synthetic fungicides is a great problem that may affect significantly the efficacy of chemical fungicides.
As an alternative to synthetic fungicides, and in congruence with improvements in integrated pest management, a search for natural products has become a very important task. Many researchers have examined the influence of essential oils on fungi that are plant pathogens, and fungi important in food industry, and proved that such plant compounds could provide a solution (Sitara et al., 2008;Soković et al., 2009;Tanović et al., 2009;Parveen et al., 2010;Mihajlović et al., 2013). Essential oils are plant volatiles containing monoterpenes, sesquiterpenes and phenyl propionoids. The essential oil of Melaleuca alternifolia, commonly known as tea tree oil, has a long history of use as a topical antiseptic (Markham, 1999). Tea tree oil is produced by steam distillation of leaves and terminal branches of M. alternifolia and consists largely of cyclic monoterpenes (Brophy et al., 1989), of which about 50% are oxygenated and about 50% are hydrocarbons. Tea tree oil exhibits a broad-spectrum antimicrobial activity which may be principally attributed to terpinen-4-ol (Markham, 1999, Carson et al., 2006.
The aim of this study was to evaluate in vitro sensitivity of Alternaria solani isolates originating from Serbia to several fungicides and a tea tree oil-based product.

Isolation and identification of Alternaria solani
The isolates of A. solani were derived from symptomatic tomato leaves collected from five locations in Serbia. Fragments of infected leaf tissue were placed on potato dextrose agar (PDA) medium and incubated at 25ºC for seven days to allow mycelial growth. Fragments excised from the mycelia developed were transferred onto PDA medium and purified by monospore isolation. A. solani cultures were identified on the basis of colony morphology and microscopic observation of conidia, and the identity of isolates was confirmed by polymerase chain reaction (PCR). The obtained isolates were stored on PDA medium at 4ºC in the culture collection of the Institute of Pesticides and Environmental Protection, Belgrade.
Pyraclostrobin was first dissolved in dimethylsulfoxide (DMSO), and then a set of stock solutions for each tested fungicide and tea tree essential oil were made using sterile distilled water. Freshly-made stock solutions were prepared to give specific concentrations of each active ingredient in µg ml -1 . Volumes of stock solution were added to molten (50 o C) PDA medium prior to pouring, thereby producing active ingredient concentrations ranging from 0.0001 to 6000 µg ml -1 (Löcher & Lorenz, 1991).

In vitro fungicide sensitivity tests
A. solani isolates were grown on PDA medium amended with the fungicides: copper-oxychloride, chlorothalonil, difenoconazole, pyraclostrobin and tea tree oil, and used in sensitivity tests. Based on preliminary data, the following concentrations were selected for further study: 3.125, 6.25, 12.5, and 25 µg ml -1 of copper oxychloride; 0.01, 0.1, 1, 10 and 100 µg ml -1 of chlorothalonil; 0.001, 0.01, 0.1, 1 and 10 µg ml -1 of difenoconazole; 0.0001, 0.001, 0.01, 0.1, 1, and 10 µg ml -1 of pyraclostrobin; and 750, 1500, 2500, 3000, 4000, 5000 and 6000 µg ml -1 of tea tree oil. Control plates were not amended with fungicides. Tests for each isolate were replicated three times per concentration of each fungicide. Mycelial plugs of 7-days old culture (10 mm in diameter) were removed from the edges of colonies grown on PDA medium, placed upside down on fungicide-amended and fungicide-free PDA media in Petri dishes, and incubated at 25 o C in the dark. After 7 days, colony diameter of each isolate was measured in three directions (minus the diameter of inoculation plug) and the percent inhibition (PI) values per each fungicide rate were calculated using the formula below: where a = colony diameter of control plates and b = colony diameter of fungicide-amended plates. PI values were subjected to regression analysis against the logarithmic values of fungicide rates. The EC 50 (fungicide concentration which inhibits mycelial growth by 50%) was determined for each isolate and data on fungicide concentration and relative inhibition were analysed using probit analysis, according to Finney (1971).

rESuLTS
The sensitivity of A. solani isolates to fungicides and tea tree oil is shown in Tables 2, 3, and 4. Of all tested fungicides, pyraclostrobin exibited the highest toxicity. The growth of all tested isolates was significantly reduced (approximately 43%) even by the lowest concentration of pyraclostrobin (0.0001 µg ml -1 ). Growth inhibition of over 82% was achieved by pyraclostrobin concentration of 10 µg ml -1 . Concentrations which inhibited mycelial growth by 50% (EC 50 ) ranged from 0.0014 to 0.0041 µg ml -1 .
The tested A. solani isolates were also highly sensitive to difenoconazole. All isolates were able to grow well at 0.001 µg ml -1 difenoconazole concentration (<10% growth inhibition), but they were partially inhibited by the next two higher concentrations (0.01 and 0.1 µg ml -1 ) and significantly inhibited by the highest concentration of 10 µg ml -1 (>95% growth inhibition). The EC 50 values for difenoconazole ranged between 0.018 and 0.037 µg ml -1 .
Among all tested conventional fungicides, copperoxychloride exhibited the lowest toxicity to A. solani isolates. All isolates were partially inhibited by the two lowest concentrations (3.125 and 6.25 µg ml -1 ), while inhibition by the highest concentration of copper-oxychloride (25 µg ml -1 ) was more than 55%. The obtained EC 50 values for copper-oxychloride were higher than those of the other investigated fungicides, ranging between 13.27 and 15.63 µg ml -1 .
All A. solani isolates showed the lowest sensitivity to tea tree oil, in contrast to the conventional fungicides. Mycelial growth of the isolate AS-3 was significantly inhibited by the concentration of 3000 µg ml -1 , while AS-1 showed a corresponding value of over 5000 µg ml -1 . The EC 50 values for tea tree essential oil ranged from 1323.97 to 3307.08 µg ml -1 .

DISCuSSION
The results of this study showed that the tested fungicides exhibited different levels of toxicity to A. solani isolates. Pyraclostrobin and difenoconazole demonstrated the highest toxicity to all tested A. solani isolates. Töfoli et al. (2003) had reported that tebuconazole and difenoconazole provided elevated inhibition of mycelial growth and partial inhibition of conidium germination. In that study, azoxystrobin and pyraclostrobin+methiram showed intermediary inhibitory effect on mycelial growth and complete inhibition of conidium germination starting from 1 μg ml -1 , while chlorothalonil and mancozeb demonstrated minor inhibitory levels but superior nevertheless to the control. Pasche et al. (2004) evaluated azoxystrobin sensitivity in an in vitro spore germination assay. They detected no significant differences in mean EC 50 values between baseline isolates and all other isolates collected in 1999. Mean azoxystrobin EC 50 values of A. solani isolates collected in 2000 and 2001 were significantly higher than means from previous years, and mean azoxystrobin EC 50  Nearly all isolates examined in that azoxystrobin assessment were classified into two groups: one that included 76 isolates with EC 50 s below 0.10 μg ml -1 , which was designated as clearly "azoxystrobin sensitive", and another one with EC 50 values above 1.450 μg ml -1 , designated as "azoxystrobin reduced-sensitive". Wang et al. (2008) had tested sixty field A. solani isolates for their sensitivity to difenoconazole employing the mycelial lineal growth method. The results showed that the isolates were all sensitive to difenoconazole, no strain was found with sharply decreasing sensitivity, and the averaged EC 50 value (0.3050±0.1361 μg ml -1 ) could therefore be used as the baseline-sensitivity of A. solani to difenoconazole. Isolates from Serbia were even more sensitive to difenoconazole than those from China, having EC 50 s below 0.037 μg ml -1 .
Antimicrobial activity of the essential oils of various aromatic and medicinal plants has been recognized for a long time. However, strong antimicrobial effects of some other essential oils have also been reported (Tanović et al., 2005;Soylu et al., 2006;Lee et al., 2007;Tanović et al., 2009;Đorđević et al., 2011). In our present study, tea tree oil, which is a biofungicide, had higher EC 50 values than the synthetic fungicides. Regarding EC 50 values, the most sensitive was isolate AS-3 (EC 50 =1323.97 µg ml -1 ), while AS-1 had the highest EC 50 value (3307.08 µg ml -1 ). In another study, Abbo et al. (2009) had reported that tea tree essential oil had a highly suppressive effect against Alternaria sp. and could be used as a successful inhibitory agent against tomato early blight pathogen. These results are similar to the findings that Gustafson et al. (1998) andCarson et al. (2006) had reported on inhibitory effects of that oil against Gram-positive and Gram-negative bacteria, yeast and fungi.
A study conducted by El-Mougy (2009) showed that carnation, caraway and thyme oils had inhibitory effects on A. solani in vitro. Mycelial growth of A. solani was completely inhibited by 1% concentration of carnation oil, while inhibition by the same concentration of caraway and thyme oils was 85.4% and 79.3%, respectively. These results showed that carnation, caraway and thyme oils had greater toxicity to A. solani than tea tree oil.
Babagoli and Behdad (2012) also examined the effect of essential oils from Carum copticum, Zataria multiflora and Satureja hortensis on the mycelial growth of A. solani. They concluded that, since Carum copticum was able to inhibit 99.5% of fungus growth by a concentration of 200 µg ml -1 , and 100% by 400 µg ml -1 , it could potentially replace chemical fungicides currently used against early blight of tomato caused by this fungus.
Among the five fungicides analyzed in our present study, pyraclostrobin and difenoconazole demonstrated the highest toxicity to A. solani. The tested isolates were medium sensitive to chlorothanlonil. Copper-oxychloride showed low toxicity to the pathogen. Tea tree oil did not exhibit a significant antifungal activity in vitro, but its efficacy in the field will nevertheless be the subject of a detailed study in our future research.

aCKNOWLEDGEmENT
The present study was a part of the project TR 31043, funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia.