Anthracnose caused by Colletotrichum lindemuthianum (Sacc & Magnus) is a major destructive disease of common bean (Phaseolus vulgaris L.). The seed borne fungi has high pathogenic variability with over 100 described strains (Campa et al., 2011). In Brazil, infected seeds may cause up to 100% losses in bean yield under favorable climatic conditions (Damasceno et al., 2007).

 

The most economically viable and effective strategy for disease control is by using bean cultivars resistant to fungi (Ishikawa et al., 2008). However, this kind of management is difficult to implement due to the high pathogenic variability of C. lindemuthianum (Ishikawa et al., 2012). Consequently, synthetic fungicides are widely used by farmers to control bean anthracnose (Gillard et al., 2012). Short term use of these products effectively helps the farmer achieve high yields. Thus, several research groups have sought cheaper and low-toxicity alternatives for controlling fungi pathogenic to plants. Essential oils (EOs) and potassium phosphite (PP) are potentially useful both for their direct toxicity for fungi, which inhibit mycelial growth and spore germination, as for induction of phytoalexins, which indicate the presence of elicitor compounds (Pereira et al., 2012).

 

This study assessed essential oils of Cymbopogon flexuosus (EOC), Vernonia polyanthes (EOV), and potassium phosphite (PP) to control of bean anthracnose caused by C. lindemuthianum.

Essential oil extraction

 

Leaves of C. flexuosus and V. polyanthes were collected in the season of winter on the month August in the city of Lavras. Essential oils were extracted from 1000 g fresh leaves through steam distillation for 90 min, using a Marconi MA480 stainless steel distiller. The aqueous phase was extracted with dichloromethane (3 × 25 ml). The organic phase was dried with anhydrous magnesium sulfate, filtered and the solvent evaporated until dryness.

 

 

Essential oil analysis

 

Analysis of chemical composition was performed according to Rosado et al. (2013). Oil quantitative analysis was performed using gas-phase chromatography coupled to a hydrogen flame ionization detector (GC-FID) on Agilent® 7890. A system equipped with HP-5 fused in the electron impact ionization mode (70 eV), injector split, capillary colum HP-5 (30 m × 0.25 mm × 0.25 μm). Temperature: injector = 220°C, collum = 60°C for 1.5 min, ramp = 3°C for min. Carrier gas He = 1 ml min^-1.

 

Retention indices (RI) have been obtained according to the method of Van den Dool and Dec Kratz (1963).

 

 

Analysis of mycelial growth inhibition in C. lindemuthianum

 

Pure culture of C. lindemuthianum, strain 65, isolate LV 175 was provided by the Laboratory of Plant Resistance, Department of Biology, UFLA. The single spore culture was obtained. In inhibition test of mycelial growth, the treatments were EOC and EOV at concentrations of 125, 250, 500, 1000, and 2000 ml L^-1, PP at a dose of 5 ml L^-1 and trifloxystrobin + fungicide at a dose of 0.75 ml L^-1. All treatments were added to potato dextrose Agar (PDA) medium containing Tween 20 (0.1%). A treatment containing Tween 20 (0.1%) was used to isolate its effect and treatment with sterile distilled water as negative control. After solidification, mycelial disks (8 mm) of C. lindemuthianum were placed in the center of petri dishes (90 mm) with the treatments described earlier and incubated at 25°C under photoperiod of 12 h.

 

Evaluations were performed daily until fungal colonies covered 2 thirds of the medium surface. Percentage of growth inhibition (PGI) was calculated using the formula: PGI = [(diameter of control - diameter of treatment) / diameter of control] × 100 for each treatment as compared to the control. The experiment was conducted in completely randomized design with six replicates, each plot consisting of a petri dish.

 

 

Evaluation of conidial germination

 

In this study, suspensions of C. lindemuthianum conidia growing at PDA medium in test tubes were used. Sterile distilled water (10 ml) was added into the test tubes and they were agitated. The suspension obtained was filtered with sterile gauze and its concentration was adjusted to 1.15 × 10⁵ conidia ml^-1 in a Neubauer chamber. One milliliter spore suspension and 1 ml of the treatments described earlier were spread over the surface of petri dishes (60 mm) with the water ágar medium at 2% with handles Drigalski. Then the plates were incubated at 25°C under a 12-h photoperiod. After 24 h, lactoglycerol was used for stopping the germination process.

 

Percentage of growth inhibition (PGI) was evaluated by counting the number of germinated and non-germinated conidia. PGI was obtained using the formula: PGI = [(number of germinated conidia control - number of conidia in the treatment) / number of conidia in the control] × 100 for each treatment as compared to the control. The experiment was conducted in completely randomized design with six replicates, each plot consisting of a petri dish.

 

 

Evaluation of treatments in anthracnose control

 

For evaluation of treatments in anthracnose control bean seedlings of cultivar Pearl was used. Twenty one days after sowing, the treatments were performed. 48 h after application, the plants were sprayed with a suspension of inoculum of 1 × 10⁶ and incubated in a humid chamber for 14 h. The experiment was conducted in a randomized block design with four replications. Each plot consisted of two pots containing three plants each.

 

Anthracnose severity was assessed every seven days through a rating scale proposed by Godoy et al. (2006). The area under the disease progress curve (AUDPC) was calculated for each treatment, according to Shaner and Finney (1977).

 

 

Enzymatic activity of peroxidase (POX) and phenylalanine ammonia lyase (PAL)

 

To determine the enzymatic activity of peroxidase (POX) and phenylalanine ammonia-lyase (PAL), the most promising treatments were selected. Distilled water was used as negative control. Bean plants at the stage V3/V4 were sprayed after 7 days and were inoculated as previously described. The experiment was a randomized complete block design with three replications and the portion composed of three plants.

 

Bean leaf samples were collected on days 1, 3, 6, 8, 10 and 13 after spraying treatments. The samples were stored at -80°C for later analysis.

 

To determine enzymatic 0.2 g of leaf tissue with 1% polyvinylpyrrolidone and liquid N₂ were ground in a mortar. It was homogenized in 1.3 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM phenylmethylsulfonyl fluoride, and was centrifuged at 11,000 ×g for 30 min at 4°C and the supernatant was used for enzymatic determination.

 

The POX activity was determined by oxidation of guaiacol according to the method of Kar and Mishra (1976). Ninety microliters of potassium phosphate buffer 100 mM (pH 7.0), 30 µl of guaiacol 40 mM and 25 µl of H₂O₂ 125 mM were added to 10½ extract. Absorbance was measured at 420 nm in ELISA reader every 10 s for 2 min after adding the sample to the mixture. Molar extinction coefficient of 1.24 mM cm^-1 was used to calculate the POX activity (Maehly and Chance, 1955), which was expressed in mM purpurogallin produced min^-1 mg^-1 protein.

 

The PAL activity was measured by adding 40 μ to a mixture containing 110 µl of 100 mM Tris-HCl (pH 8.8) and 50 µl of 100 mM L-phenylalanine. The reaction mixture was incubated in  the  ELISA reader at 37°C for 10 min. The absorbance of derivatives of trans-cinnamic acid was measured in a spectrophotometer at 280 nm and the molar extinction coefficient of 5000 was used mM^-1 cm^-1 (Zucker, 1965) for calculating the PAL activity, which was expressed in mM min^-1-mg protein. The protein concentration in each sample was determined according to the colorimetric method described by Bradford (1976).

 

 

Analysis in scanning electron microscope

 

Pearl bean cultivar at V3/V4 stage was sprayed with the most promising treatments:  OEV and OEC 1000 µl L^-1 and PP 5 µl L^-1. As a negative control, sterile distilled water was sprayed. Two days after application, seven of the third trifoliate leaves were collected and packaged in plastic trays with the bottom of the tray covered with foam rubber and two sheets germitest wetted with distilled water.

 

Points were marked and 25 µl of suspension with 1.5×10³ spores of C. lindemuthianum ml^-1 was deposited. After inoculation, the trays were covered with clear plastic and placed in a growth chamber at 25°C until the end of collection.

 

Samples were collected after 4, 8, 16, and 48 h of inoculation, using 5 mm circular cuts with a scalpel inoculated into each point. The samples were placed Karnovsky's fixative at pH 7.2 and stored at 4°C for 24 h. After this period, the samples were placed in 0.05 M cacodylate tampon and washed three times for 10 min and transferred to osmium tetroxide and 1.0% water for 1 h, washed three times with distilled water.

Then the samples were dehydrated in acetone series (25, 50, 70, 90, and 100% three times) and brought to the critical point dryer Balzers CPD 030 for replacing acetone by CO₂. The samples were mounted on stubs and coated with gold using the Balzers SCD 050 evaporator for observation in a scanning electron microscope LEO EVO 40. The images were digitally generated 20 kV to a 10 mm working distance, then the images were worked in Corel Draw Photo Paint 12.

 

 

Statistical analysis

 

Data were subjected to analysis of variance, and mean values were compared by the Scott Knott test at 5% probability for qualitative factors, while regression analysis was applied for quantitative factors.

Yields and chemical composition

 

Chemical composition of essential oils of C. flexuosus and V. polyanthes are as shown in Table 1. Oil yields were 1.27 and 0.02% (v/w), respectively for C. flexuosus and V. polyanthes.

 

 

Twenty compounds were identified in the essential oil extracted from C. flexuosus leaves, representing 97.7% of total constituents. Twenty two compounds were identified in V. polyanthes leaf essential oil, representing 97.6% of the total constituents.

 

The composition of C. flexuosus essential oil is much simpler and mainly composed by monoterpenes (96.4%) in contrast to V. polyanthes essential oil, which is primarily composed of sesquiterpenes (94.3%).

 

 

Evaluation of mycelial growth inhibition and conidial germination in C. lindemuthianum

 

Essential oils of C. flexuosus and V. polyanthes inhibited mycelial growth and conidial germination in C. lindemuthianum. Oil of C. flexuosus was more efficient, and reduced colony diameter in C. lindemuthianum starting from the lowest concentration (125 μl L^-1). Total suppression of fungus mycelial growth occurred in concentrations higher than 615 μl^-1 of EOC (Figure 1). The concentration capable of inhibiting mycelial growth was 50% in 162 μl L^-1 and 1918 μl L^-1 in EOC and EOV, respectively.

 

 

EOC was more efficient for percentage of conidia germination inhibition, and concentration of 689 µl L^-1 totally inhibited C. lindemuthianum germination (Figure 1C). The concentration capable of inhibiting at least 50% conidial germination was estimated in 220 and 850 µl L^-1 in EOC and EOV, respectively.

 

Table 2 compares the effect of essential oil (EO) doses, showing higher antifungal activity with potassium phosphite (PP), fungicide (F), and EOC at 1000 µl L^-1. The highest antifungal activity occurred with F and EOC, which completely inhibited mycelial growth and fungal germination.

 

 

 

Essential oils and potassium phosphite in control of bean anthracnose in greenhouse

 

In greenhouse, essential oils of C. flexuosus and V. polyanthes reduced the area under the curve of progress of anthracnose (AUCPA). EOC provided greater reduction (57.2%) as compared to the control in concentration 1450 μl L^-1 whereas EOV provided 37.6% as compared to the control in concentration 1320 μl L^-1 (Figure 2).

 

 

 Treatments with potassium phosphite and fungicide reduced AUCPA by 60.4 and 62.1%, respectively, compared to control. There was no statistical difference between PP, F and EOC at 1000 μ L^-1.

 

 

Studies of germination and mycelial growth in vivo in C. lindemuthianum

 

Images of scanning electron microscopy showed no conidia germination in C. lindemuthianum in all treatments 4 h after inoculation. Samples collected 8 h after inoculation had no germination either, except for the treatment with distilled water. In this treatment, we observed conidia starting the germination process (Figure 3).

 

 

Bean leaves treated with distilled water 16 h after inoculation showed conidia in advanced stage of germination and mycelial growth on the surface of leaves. However, leaves treated with EOC and EOV showed  low conidia germination while the treatment with PP had no germination (Figure 3). Large differences were found 48 h after inoculation. Leaves treated with distilled water had clear mycelial growth and emission of numerous germ tubes near ribs in the abaxial surface. Conversely, leaves sprayed with essential oils showed low conidia germination and poorly developed mycelium. The treatment with PP had no conidia germination (Figure 3).

 

 

Biochemical analysis of induced resistance

 

For the enzymatic activities, only treatments with EOC and PP in concentration of 1000 µl L^-1 and 5 ml L^-1, respectively were selected.

 

In inoculated plants, peroxidase (POX) activity was significantly higher in treatment with PP at 8 and 13 days after spraying differing from the control treatment. However, at day 10, there was no significant difference between treatments. Essential oil of C. flexuosus provided higher peroxidase activity at day 8 compared to the control. Increase of peroxidase at day 8 in treatments with PP and EOC helped control the pathogen, as inoculation was performed at 7 days after spraying. Plants recognized the fungus and increased the level of POX.

 

In non-inoculated plants at 8 days after spraying, only PP differed from the other treatments (Table 3). With respect to collection period, there was statistical difference in inoculated plants since PP showed higher content of POX at day 13 compared to days 8 and10. Plants sprayed with EOC had greater activity at 10 and 13 days after spraying. There was a significant difference between inoculated and non-inoculated plants, and plants challenged with fungus C. lindemuthianum showed higher levels of POX.

 

 

POX activity at 1, 3, 6, 8, 10 and 13 days after spraying in non-inoculated plants (Table 4) suggests that spraying PP had an active role in potentiating and anticipating enzyme activity. This result demonstrates the role of PP in plant defense, since there was difference from the control treatment at 1, 3, 6 and 8 days after spraying. With respect to EOC, there was significant difference compared to control at 1, 3, 6 and 8 days after spraying.

 

 

Regarding the activity of the enzyme phenylalanine ammonia lyase (PAL), there was significant difference.  In plants inoculated and sprayed with PP, there was greater activity of PAL compared to plants sprayed with EOC and water to 8 and 10 d.a.p. (Table 5). Already at 13 d.a.p., plants sprayed with PP produced less PAL and not different from oil.

 

 

Considering PAL activity in non-inoculated plants at 1, 3, 6, 8, 10 and 13 days after spraying, PP was superior to other treatments at days 1, 6 and 10 (Table 6). Plants sprayed with EOC had greater PAL activity at days 3 and 13. There was no significant difference between treatments at day 8. Regarding time of collection, plants sprayed with PP and EOC showed higher PAL activity at day 3. The control treatment had increased PAL production at days 1, 3, 6 and 10.

The constituents of C. flexuoso in greater quantity were geranial (46.9%)  and  neral  (33.4%),  called  citral,  and comprised 80.3% of the essential oil. These results are in agreement with the literature (Adukwu et al., 2012).

 

The constituents of V. polyanthes is mainly composed by germacrene D (42.2%), bicyclogermacrene (17.2%), β-caryophyllene (13.6%) and α-humulene (7.8%), repre-senting 80.8% of total oil. These results are in agreement with the literature (Maia et al., 2010).

 

Fungicide and fungistatic effect were found when mycelium discs with 100% PIMG were taken from the culture medium at the end of growth analysis and sub-cultured to PDA plates. Only PP had fungistatic activity, whereas in the other treatments, the effect was fungicidal. This probably resulted from the high volatility of C. flexuosus essential oil, which killed fungal cells in the upper part of the mycelium disc that comes not in contact with culture medium. 

 

Anaruma et al. (2010)determined the activity of 28 essential oils from medicinal plants against Colletotrichum gloeosporioides. Four species (C. flexuosus, Cymbopogon citratus, Coriandrum sativum and Lippia alba) showed better antifungal activity. Citral is the main component of essential oils of most species of Cymbopogon species, and many authors attribute to this compound the control of plant pathogenic fungi. Valencia et al. (2011)found antifungal effect of essential oil of C. citratus on C. gloeosporioides. Alzate (2009)demonstrated that citral completely inhibited mycelial growth and sporulation of C. acutatum outperforming Mancozeb fungicide. According to Bakkali et al. (2008), as these oxygenated monoterpenes are hydrophobic they will probably want to move towards the aqueous phase of membrane structures. Accumulation of essential oil constituents in the lipid bilayer of the cytoplasmic membrane makes it permeable, thus promoting dissipation of the proton motive force. It also reduces ATP pool, internal pH and electric potential, causing loss of ions such as potassium and phosphate. Thus, damage leads to impairment of membrane functions.

 

According to Rasooli et al. (2006), when in direct contact with microorganisms, these substances cause permeability of cell membranes and leakage of their contents. Furthermore, terpene alcohols were identified in EOC composition, such as geraniol and linalool (Table 1).

Few studies have reported antimicrobial activity of species of Vernonia spp. Essential oil of V. polyanthes presented lower antifungal activity than C. flexuosus oil (Figure 1B and D). These results demonstrate that micro-organisms differ  in  their  resistance  to  certain  essential oils, showing specific reaction according to the chemical constitution of each oil.

 

Maia et al. (2010) evaluated essential oils of Vernonia braziliana and Vernonia remotiflora in Gram-negative and Gram-positive bacteria. The antimicrobial activity of these oils is related to the high amount of sesquiterpenes such as those found in this study: germacrene-D, bicyclo-germacrene, β-caryophyllene, and α-humulene (Table 1).

 

Montanari et al. (2011) attributed antimicrobial activity to sesquiterpene due to inhibition of breathing capacity and increased cell membrane permeability, as well as rupture of membrane integrity which results in leakage of K + ions and consequent loss of chemiosmosis control. Oils of Lantana camara and Aloysia virgata, rich in germacrene-D, showed moderate antibacterial activity against Gram-positive bacteria (B. cereus and S. aureus) and oil of A. virgata was active against Gram-negative bacteria (Costa et al., 2009).

 

Essential oil of C. citratus reduced the severity of anthracnose in passion fruit caused by C. gloeosporioides, with no significant difference in relation to Procloraz fungicide (Anaruna et al., 2010) or Hibiscus rosa-sinensis (Valencia et al., 2011).

 

Some authors attribute the partial control of other anthracnoses to citral. However, it is necessary to con-duct experiments testing the isolated effect of neral and geranial. Garcia et al. (2008)reported that citral 1% was responsible for 70% control of anthracnose in papaya and 60% of anthracnose in banana. Essential oils rich in terpenes cause damage to lipids and proteins and break down cell walls and membranes, which results in cell lysis. In eukaryotic cells, essential oils destabilize the mitochondrial membrane and cause damage to plasma membrane proteins (Bakkali et al., 2008).

 

Direct   effectiveness   of   phosphites  against  fungi  is mainly related to phosphite ion, which seems to have a direct effect on the pathogen (Pereira et al., 2012). Furthermore, phosphites can also reduce sporulation of microorganisms, thereby causing reduction of pathogen inoculum potential (Silva et al., 2012).

 

The results of scanning electron microscopy in this study corroborate the results found in conidia germination in vitro, which had inhibition of germination with EOC, PP and EOV. Other authors have used SEM to demonstrate antifungal activity of essential oils (Pereira et al., 2011)treated leaves of coffee with Cinnamomum zeylanicum and C. citratus oils in control of Cercospora coffeicola. Through SEM observations, the authors found reduction in germination and mycelial size, with plasmolysis occurring in some conidia.

 

Induction of resistance in essential oils (EOs) may be related to their chemical constitution. It is known that EOs have various organic substances capable of inducing POX activity, such as terpene hydrocarbons, terpene alcohols, and simple alcohols, aldehydes, ketones, phenols, esters, ethers, oxides, peroxides, furans, organic acids, lactones, coumarins, and sulfur-containing compounds (Dewick, 2002). Thus, by chromatography data (Table 1), the presence of some of these substances which probably contributed to the increased levels of POX in bean tissues were confirmed. Pereira et al. (2012)found increased POX enzyme in coffee plants sprayed with essential oil of citronella, which resulted in decreased Cercospora leaf spot.

 

According to Daniel and Guest (2006), after treatment with phosphite there is accumulation of phenolic compounds in cells, formation of cytoplasmic aggregates and phenols around the infected cells and rapid increase in production of reactive oxygen species, followed by hypersensitivity reactions.

 

The positive relationship between POX activity of plant resistance to disease has been reported in several studies. Thus, increased POX activity could explain the lower severity of bean anthracnose found in plants treated with PP and EOC in severity experiments. Martins et al. (2013), also studying Pérola cultivar found increased activity of POX in plants treated with rhizobacteria and inoculated with Curtobacterium flaccumfaciens pv. flaccumfaciens. The authors also found decreased severity of disease at levels 42 to 76%.

 

Campos et al. (2004)reported that POX activity was significantly higher in bean plants treated with salicylic acid before and after inoculation of C. lindemuthiaum, with positive correlation between increased POX activity and anthracnose resistance. This resistance is related to POX ability to produce free radicals toxic to the pathogen in oxidative burst and to participate in lignin synthesis for strengthening cell wall. In addition, POX produces signal molecules such as H₂O₂, which can lead to expression of genes related to other resistance mechanisms (Hsu and Kao, 2003).

 

There was a significant difference regarding the activity of enzyme phenylalanine ammonia-lyase (PAL). Inoculated plants sprayed with PP had greater activity of PAL compared to plants sprayed with EOC and water at 8 and 10 days after spraying (Table 5). At day 13, plants sprayed with PP produced less PAL and did not differ from oil. This was possibly due to increase in PAL in early periods post inoculation (at 8 and 10 days after spraying), which caused a metabolic cost to the plant on the last evaluation day. This same effect occurred with plants treated with water, that is, PAL activity decreased considerably on the last day after inoculation (day 13). However, enzyme activity was  lower  than  the  activity of elicitors PP and EOC, which resulted in increased severity of anthracnose in plants treated with water. Similar to PP treatment, inoculated plants sprayed with EOC showed higher PAL activity in all periods. This increase can mean that the entire phenylpropanoid pathway was altered, that is, mechanisms such as synthesis of lignin, phenolic compounds, quinones and others may have been potentialized by the products used in the experiment.

 

Non-inoculated plants showed no statistical difference between products at 8 and 13 days after spraying. Only at day 10, plants sprayed with PP had higher PAL activity. Thus, it is evident that PAL peak occurred at day 10. The highest PAL activity in plants treated with PP and essential oils has already been reported by other authors. Sellamuthu et al. (2013)found PAL to increase in avocado fruits with application of essential oil of thyme. Martins et al. (2013)reported increase in PAL levels in the second phenological stage of bean plants Pérola cultivar treated with growth promoting rhizobacteria. The authors also reported decreased severity of wilt of C. flaccumfaciens pv. flaccumfaciens in common bean by 76%, and increased dry weight of shoots and roots of treated plants. Campos et al. (2003)reported that bean plants Pérola cultivar treated with salicylic acid had higher PAL activity than plants treated with water, and plants challenged with the pathogen C. lindemuthiahum showed higher PAL activity than non-inoculated plants.

 

PAL catalyzes the deamination reaction of L-phenylalanine to trans-Cinnamic acid. This process is the first step in the phenylpropanoid pathway which is highly important for the production of plant defense compounds against pathogens (Mandal et al., 2009).

 

Thus, essential oil of C. flexuosus and potassium phosphite could be an alternative for the management of bean anthracnose, because in addition to significant induction of defense enzymes such as POX and PAL, these products also presented antifungal proprieties. However, more studies need to be performed to assess the efficiency of these products in anthracnose control in field conditions, doses to be used and toxicology to man and the environment, as well as the adequacy of essential oil production and oil formulation.

To the National Council for Scientific and Technological Development (CNPq), Coordination Support for Improvement of Higher Education Personnel (CAPES) and Foundation for Research Support of the State of Minas Gerais (FAPEMIG) for granting the scholarship and financial support.

The author(s) have not declared any conflict of interests.