Eff ects of fungicides and biofungicides on Rhizoctonia solani , a pathogen of pepper

SUMMARY In vitro and in vivo sensitivity of Rhizoctonia solani to several commercial fungicides and biofungicides was studied. An isolate of R. solani, derived from diseased pepper plants originating from a greenhouse in Knjaževac, Serbia, was used. The highest eﬃ cacy in R. solani control under greenhouse conditions was achieved by iprodione (95.80%, compared to control), although diﬀ erences in the eﬀ ectiveness of iprodione, tea tree oil, azoxystrobin and thiophanate-methyl were not statistically signiﬁ cant. The isolate was sensitive to all tested products in vitro. The obtained EC 50 s were: 0.43 mg/l for iprodione, 1.84 mg/l for thiophanate-methyl, 13.84 mg/l for prochloraz, 430.37 mg/l for ﬂ uopyram, 596.60 mg/l for azoxystrobin, and 496.79 mg/l for tea tree oil.


INTRODUCTION
Agricultural production in Serbia is central to the country's economy. It is also considered to be an engine for rural development. According to the 2012 Serbian Agriculture Census, solanaceous vegetable crops (potato, tomato, pepper and eggplant) were grown on more than 35 000 ha in that year with pepper, grown on 7 661 ha, being the most important in economic terms. Pepper production is severely aff ected by soilborne disease outbreaks that occur almost every year both in open fi elds and in protected cultivation. One of the most important soil-borne diseases is Rhizoctonia root and collar rot, caused by Rhizoctonia solani Kiihn species complex (teleomorph: Th anatephorus cucumeris (A.B. Frank) Donk 1956 (Ogoshi, 1987). Under favorable weather conditions (prolonged periods in cool, moist conditions), this pathogen causes root and crown rot, aerial blight of leaves, stems and fruit of pepper plants, as well as damping-off of seedlings. For instance, Rini and Sulochana (2006) reported dumping-off of 33.2 and 40.2 % of pepper seedlings in protected and open fi eld cultivation, respectively. Even though diseased plants oft en produce an abundance of secondary roots above rotted taproot, wilting and death of plants scattered throughout a fi eld are the most eyecatching above-ground symptoms of Rhizoctonia root rot (Coa et al., 2004). Additionally, the fungus has a tremendous capacity for saprotrophic growth and can survive in soil indefi nitely in the absence of a host plant.
Similar to other soil-borne diseases, Rhizoctonia root rot is oft en more diffi cult to control than diseases that aff ect above-ground plant parts because they are very diffi cult to detect and identify before serious damage has already occurred (Pritchard, 2011). To control serious disease outbreaks, conventional synthetic fungicides and fumigants are applied at regular intervals before crop establishment or throughout the growing season. However, there are evident issues with synthetic fungicides, including ecological disturbance, human health hazard, damage to aquatic ecosystems, reduction of benefi cial microorganisms in soil and even ozone layer depletion (UNEP, 1994). With increasing environmental constraints, alternatives to broadspectrum fungicides and fumigants are being developed and put into use. However, these alternative diseasemanagement methods either have inconsistent results (Gerik & Hanson, 2011) or show less eff ective results than the phased-out methyl bromide that had been used for decades. Biological control by using antagonists is an ecofriendly and promising alternative to the use of chemicals. Th e application of Bacillus species, including Bacillus subtilis, is an example of biocontrol agents with antagonistic eff ects on plant pathogens coupled with plant growth promoting ability (Ryder et al., 1998;Whipps & Lumsden, 2001). Likewise, essential oils have been investigated for their wide spectrum of antimicrobial activity. Tea tree oil has a long history of use as a topical microbicide in human pharmacology (Markham, 1999;Carson et al., 2006).
In Serbia, only a few products have been registered for the control of soil-borne pathogens. However, none have been registered for the control of R. solani in pepper production during the growing season, although some fungicides in the dicarboximide, benzimidazole and triazole groups have been recommended Mijatović et al., 2007).
Th e objective of this study was to examine the possibility of Rhizoctonia root rot control in pepper by using conventional fungicides and biofungicides based on tea tree essential oil and B. subtilis. In vitro sensitivity tests were conducted in order to determine if there is any correlation between the effi cacy of pepper protection under greenhouse conditions and the sensitivity of a R. solani isolate to conventional fungicides and tea tree oil.

R. solani isolate
A R. solani isolate, derived from infected pepper plants from Knjaževac in Serbia, was chosen for a study that used a method described by Dhingra and Sinclair (1995). Th e isolate was purifi ed by the single hyphal tip method (Narayanasamy, 2001), identifi ed based on morphological characteristics and maintained on slants at 5°C in the Culture Collection of the Department for Phytomedicine and Environmental Protection, University of Novi Sad, Serbia. Prior to greenhouse experiments, the identity of the isolate was confi rmed based on morphological macroscopic and microscopic traits, and by sequencing of an amplicon obtained by polymerase chain reaction (PCR) using universal primer pair ITS1/ITS4 (White et al., 1990). Colony appearance and the presence of sclerotia were observed in 10-day old colonies growing on PDA (Sharma et al., 2005), while the hyphal branching pattern was observed microscopically using a compound microscope (Olympus, Japan).

Greenhouse potting experiment
Th e inoculum of R. solani for a potting experiment was prepared by transferring a mycelial disk, cut from the edge of a 5-day-old colony, into a 500 ml glass bottle containing 150 g sterilized barley grains, and incubated at 25°C for 21 days. Th en the inoculum was mixed thoroughly with sterile growth substrate (Floragard®, Germany) at the rate of 5% (Gaskill, 1968). Fiveweek-old pepper plants (cv. Novosadska babura) were transplanted into 10 cm × 5 cm pots fi lled with 400 ml of inoculated growth substrate and 60 ml of each fungicide/ biofungicide was added to pots at label rate. Plants inoculated and watered with 60 ml of sterile distilled water served as positive control (K-1). Uninoculated pepper plants, watered with 60 ml sterile distilled water, served as negative control (K-2). Th e pots were kept in a greenhouse (24±2°C) and watered regularly until fi nal evaluation. Th e degree of wilting was observed daily with a fi nal evaluation performed 22 days aft er inoculation by visual observation of symptoms and by measuring the height and fresh weight of plants. Infection was rated based on a 0-5 scale, where 0 = no symptoms, 1 = chlorosis of lower leaves, 2 = slight wilting with pronounced chlorosis, 3 = slight wilting and necrosis, 4 = pronounced wilting and necrosis, and 5 = death of plant (D'Ercole et al., 2000;EPPO, 1997). Th e experimental design was a complete randomized block with fi ve replicates per treatment and fi ve plants per replicate. Th e experiment was conducted twice. Disease severity (infection degree, ID) was calculated using the Townsend and Heuberger formula (Swiader et al., 2002): ID = (nv)100/NV where: n = degree of infection rated on a scale of 1-5, v = number of plants in a category, N = highest degree of infection rate, and V = total number of plants screened. Th e effi cacy was evaluated using Abbott's formula. Data were analyzed separately for each trial using ANOVA and the means were separated by Duncan's multiple range test.

In vitro sensitivity tests
Sensitivity of the isolate in vitro was determined in a radial growth assay on PDA medium as described by Leroux and Gredt (1972) and Löcher and Lorenz (1991). Based on preliminary concentrations of all investigated fungicides and tea tree oil, ranging from 0.10 to 1000 mg/l of active ingredient (a.i.), the following fi nal concentrations of the fungicides in medium were used: thiophanate-methyl 0.6, 1.25, 2.5, 5 and 10 mg/l; iprodione 0.3, 0.6, 1.25, 2.5 and 5 mg/l; prochloraz 1.56, 3.12, 6.25, 12.5, and 25 mg/l; fl uopyram 250, 350, 500, 700 and 1000 mg/l; azoxystrobin 10, 100, 500, and 1000 mg/l; tea tree oil 62.5, 125, 250, 500 and 1000 mg/l. Each fungicide-amended medium was made by adding a fungicide from appropriate dilution series prepared in sterile distilled water to the molten PDA medium (50°C). In the fungicide-free control media, sterile distilled water was added instead of fungicide dilutions. In order to inhibit an alternative respiratory pathway in the fungus that can interfere with the activity of azoxystrobin in vitro (Wood & Hollomon, 2003), salicylhydroxamic acid (SHAM), dissolved in ethanol, was added to azoxystrobin-amended and azoxystrobin-free media at a previously determined nontoxic concentration of 0.1 mg/l. Mycelial plugs (3 mm diameter) were cut from the edge of 5-day-old R. solani culture grown on PDA medium at 25°C and used for inoculation of the fungicide-amended and fungicide-free media. Th e experiment was conducted in three independent replications using two petri dishes containing three mycelial plugs each, per replicate. Aft er incubation for four days at 25°C, mycelial growth was measured. Th e growth on fungicide-amended media was presented as the percentage compared to the control. Since experimental conditions were identical in all replications, the obtained data were pulled together and fungicide concentrations that inhibited mycelial growth by 50% (EC50) and regression coeffi cients (b), expressing relative fungicide toxicity, were determined using probit analysis (Finney, 1971).

The isolate
Th e chosen isolate formed a round, fast-growing colony which was white as young and became partially brown colored with age ( Figure 1). Aft er 7-10 day incubation, small (0.5-1 mm in diameter), initially creamy and then light-brown superfi cial or partly immersed sclerotia were formed. Th e isolate exhibited a typical Rhizoctonia-like branching pattern with multinucleate hyphal cells able to anastomose with each other. Th e observed morphological features confi rmed that the isolate belonged to Rhizoctonia solani species complex as it had been previously determined. Th is was also confi rmed by the sequence of approx. 700 bp amplicon, obtained by using the universal primer pair ITS1/ITS4. Table 1 summarizes the results of the disease severity and effi cacy of the products applied aft er inoculation of pepper plants with R. solani. Under greenhouse conditions, the highest effi cacy in R. solani control was achieved by iprodione (95.80% compared to control), although diff erences in disease severity between treatments with iprodione, tea tree oil, azoxystrobin and thiophanate-methyl were not statistically signifi cant. Among the tested products, the lowest effi cacy of 47.4% was achieved by fl uopyram and the B. subtilis-based product. Table 2 summarizes the eff ects of tested products on the height and fresh weight of pepper plants inoculated prior to product application. Maximum height was achieved by plants treated with iprodione (5.96 cm), although the diff erence from treatments with B. subtilis, fl uopyram and prochloraz was not statistically signifi cant. Th e lowest plant height (3.40 cm) was observed aft er treatment with the tea tree oil product. Similarly, maximum fresh weight was recorded in plants treated with iprodion (1.56 g), while the other treatments were not statistically diff erent from the inoculated untreated control (11.34 g).

Greenhouse experiment
A moderate positive correlation between fungicide effi cacy and plant height (r = 0.60), and a weak positive correlation between fungicide effi cacy and plant fresh weight (r = 0.34) were found.

In vitro tests
Sensitivity of the R. solani isolate to the tested fungicides and tea tree oil in vitro is presented in Table 3. Th e EC 50 value of iprodione (0.43 mg/l) was the lowest compared to the other tested fungicides. Th e isolate was capable to grow well at 0.3 mg/l, while severe inhibiton was observed at 1.25 mg/l and higher concentations of iprodione. Azoxystrobin and fl uopyram exhibited the lowest toxicity of all conventional fungicides; their EC 50 values were 596.60 mg/l and 430.37 mg/l, respectively. Th e tee tree oil product severely inhibited isolate growth at 1000 mg/l, while its inhibitory eff ect was signifi cantly weaker at the lower studied concentrations. Th e EC 50 value of tea tree oil was 496.79 mg/l.

DISCUSSION
Th e results of the present study provide new information on the effi cacy of the Quinone outside Inhibitor (QoI) azoxystrobin in the control of Rhizoctonia root rot. In our experiments under greenhouse conditions, azoxystrobin provided a signifi cant reduction in disease severity of 89.50% compared to the control. However, it did not exibit high toxicity to the same R. solani strain in laboratory tests, where the EC 50 value was 596.60 mg/l despite the use of SHAM, a specifi c terminal oxidaze inhibitor that inhibits alternative respiration pathway which had been proven to interfere with the activity of strobilurins in vitro (Ziogas et al. 1997). As a QoI fungicide, azoxystrobin inhibits mitochondrial respiration by blocking electron transport. It binds at the quinol outer binding site of the cytochrome b-c1 complex, where ubiquinone (coenzyme Q10) would normally bind when carrying electrons to that protein. As a consequence, ATP production in fungi is prevented (Bartlett et al., 2002). Th e low toxicity of azoxystrobin found in our in vitro experiment using PDA medium could be either due to its mode od antifungal action or to the medium infl uence on its toxicity despite terminal oxidaze inhibition. Anyway, QoI fungicides constitute one of the most signifi cant classes of fungicides due to their broad-spectrum activity against major groups of plant pathogenic fungi, low application rates and some yield benefi ts (Bartlett et al., 2002). Taking into account the results of the glasshouse experiments in which azoxystrobin was highly eff ective against the same R. solani strain, further research is needed to determine whether in vitro sensitivity tests conducted on growth media should be used as a reliable information source for general conclusions, at least for the combination QoI fungicides-R. solani.
In our present study, iprodione was highly eff ective against R. solani on inoculated pepper plants (95.8% compared to control). It was also highly toxic to the R. solani isolate in vitro (EC 50 = 0.43 mg/l), suggesting that it could be eff ectively used against this pathogen. Csinos and Stephenson (1999) reported that iprodione showed good in vitro activity against R. solani cultures isolated from diseased tobacco plants. Furthermore, their fi eld studies suggested that iprodione reduced damage caused by R. solani, and had an excellent activity in reducing lesion development in naturally-infected seed beds of tobacco (Csinos & Stephenson, 1999).
In recent years, many studies have documented problems arising from the presence of pesticide residues in the environment, food and feed. Th is has led to restrictions and a reduction in the availability of some chemical fungicides previously used to control plant diseases and spoilage of their products used for food. Biological control of soil-borne plant pathogens by microorganisms has gained widespread acceptance as a potential tool in optimizing agricultural productivity.
Understanding the potencial use of an antagonist for biological control of a disease depends on answers to a series of questions regarding interactions of the host (crop), pathogen, and antagonist. Bacillus species, including Bacillus subtilis, are known for their antifungal properties, hence their importance in the biological control of a number of plant and animal diseases (Pandey & Palni, 1997;Ryder et al., 1998). Th e B. subtilis-based product used in our experiment was partially eff ective in controlling Rhizoctonia root rot.
Besides microorganisms, plant extracts and especially volatile essential oils from medicinal plants, have been reported to possess antimicrobial activity against a variety of food-borne, human and plant pathogens and pests (Isman, 2000;Kalemba & Kunicka, 2003;Burt, 2004). A wide variety of essential oils are known for antimicrobial properties and in many cases their activity is due to the presence of active monoterpene constituents. Tea tree oil has a long history of use as a topical microbicide in human pharmacology (Markham, 1999;Carson et al., 2006). Th e biofungicide based on tee tree oil that was tested under laboratory conditions in the current study exhibited low toxicity to the tested isolates of R. solani with EC 50 values of 496.79 mg/l. On the other hand, it exibited very high effi cacy in the greenhouse experiment, 90.50%, compared to control plants.
R. solani is an important pathogen of pepper in all its growth stages from seedlings to harvest. Knowledge of the sensitivity of this pathogen to commercial fungicides and alternative natural compounds is an important tool for the success of Rhizoctonia root rot management in fi elds with detected presence of R. solani. Th e present study revealed high effi cacy of the tea tree oil product tested (90.50%), suggesting that it could be used as a safe solution for application aft er the disease has been detected and the pathogen identifi ed.
of Bacillus isolated in China to suppress take-all and rhizoctonia root rot, and promote seedling growth of glasshouse-grown wheat in Australian soils. Soil Biology and Biochemistry, 31(1), 19-29.