Antibacterial activity of extracts of three aromatic plants from Burkina Faso against rice pathogen , Xanthomanas oryzae

Plant extracts can have powerful antibacterial activity and may be used as biological control against important plant pathogens. This study assessed effect of extracts of Cymbopogon citratus, Eucalyptus camaldulensis and Mentha piperita against two pathovars of Xanthomonas oryzae attacking rice. Both in vitro and in vivo approaches were used for different strains of X. oryzae originating from Burkina Faso. The three essential oils (EOs) have antibacterial activity in vitro test (agar diffusion method), but, EO from C. citratus at 1:5 dilution (v/v) resulted in the highest inhibition (over 30 mm of inhibition zone) against X. oryzae pv. oryzae and X. oryzae pv. oryzicola. However, they reduced significantly rice seed germination and induced herbicide effects on rice leaves. On the other hand, aqueous extracts from C. citratus stimulated bacterial growth, while extracts from E. camaldulensis had an inhibitory effect (28 mm of inhibition zone at 3:10 dilution, w/v). Therefore, the characterization of active compounds will determine one or more compound(s) involved in the antibacterial activity.


INTRODUCTION
Rice (Oryza sativa) is the third most important cereal crop in the world following wheat and maize with an estimated production of 500 million metric tons in 2015 (www.fao.org).It is a staple food in developing countries and its consumption is steadily increasing.In Burkina Faso, the average per capita consumption of rice is about 18 kg per year.However, rice production faces several important constraints including rice diseases.Providing healthy rice seeds is a requirement to increase rice productivity in order to answer the challenge of feeding fast growing populations.Indeed, several seed-borne bacterial pathogens of rice were described, and among those, Xanthomonas oryzae causes important yield losses.Bacterial leaf blight (BLB) and bacterial leaf streak (BLS) are two important diseases due to X. oryzae pv.oryzae (Xoo) and X. oryzae pv.oryzicola (Xoc), respectively; they significantly reduce global rice production in the tropical areas.BLB is present in tropical *Corresponding author.E-mail: wonniissa@yahoo.fr.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License and temperate areas wherever rice is grown.The yields losses induced by BLB under irrigation scheme can reach about 20 to 80% depending on the rice variety and climatic conditions (Ou, 1985).However, BLS is mostly confined to tropical and subtropical areas in Asia, Africa and Australia (Ou, 1985) and can cause yield losses of up to 30% (Gonzalez et al., 2007).BLS was first reported in Burkina Faso in 2009 by Wonni et al. (2011).The disease is present in all major rice production areas and it is becoming a threat to rice production (Wonni et al., 2014).
Seeds are primary sources of inoculum for both bacterial diseases (Ou, 1985;Mew et al., 1993;Agarwal et al., 1994).Xoo and Xoc are quarantine pathogens in the USA and in some countries where these pathogens are endemic in order to limit the introduction of new virulent strains.
To avoid the dissemination of bacterial diseases by the means of infected seeds, Agarwal et al. (1994) suggested seeds disinfection with bactericides and heat.However, chemical treatments are expensive for the small rice producers of developing countries and harmful for environment and human health.Also, the thermotherapy needs a device that can maintain a constant temperature duration of treatment.The lack of seeds treatment against X. oryzae, and consequently the exchange of contaminated seeds, are likely involved in the rapid range expansion of BLB and BLS.
C. citratus or lemongrass is a perennial tropical grass; it is resistant to different temperatures and can grow in all climates.The essential oil from C. citratus, besides having antimicrobial effects, has been shown to have anti-inflammatory, analgesic and antipyretic properties (Kpoviessi et al., 2014;Vázquez-Briones et al., 2015).Mentha piperita L., a medicinally important plant belonging to the family Lamiaceae and commonly known as peppermint is a hybrid of Mentha spicata L. and Mentha aquatic.The extracts of Mentha piperita are found to possess antibacterial, antiviral and antifungal activities (Singh et al., 2016;Ilboudo et al., 2016).The leaves of Eucalyptus camaldulensis are usually used for various purposes, including treatment of infections.All these plants are grown traditionally on small or large areas everywhere in Burkina Faso, so interesting candidates for biological control of bacterial diseases.
The present study aimed at assessing antibacterial activities of extracts from three aromatic plants (C.citratus, E. camaldulensis and M. pipera) on X. oryzae pv.oryzae and X. oryzae pv.oryzicola strains in vitro and to assess their potential antibacterial effect on rice seed germination and rice plant development.

Bacterial cultures
The authors used two strains of X. oryzae originating from Burkina Faso: BAI3 (X.oryzae pv.oryzae) isolated from rice plants with BLB in 2003 by Gonzalez et al. (2007) and BAI105 (X.oryzae pv.oryzicola) isolated from BLS symptoms in 2012 by Wonni et al. (2014).These strains belong to major group representing the genetic diversity in West Africa (Poulin et al., 2015).These strains from INERA-IRD collection were stored at -80°C and recovered on Peptone, Sucrose, Agar (PSA) medium for use.

Vegetal material source
The leaves of C. citratus, E. camaldulensis and M. piperita were collected in the station of environmental and agricultural research of Farako-Ba located 10 km from the city of Bobo Dioulasso (11°9'56.97''N;4°18'19.80''W) in Burkina Faso.After collection, all the others tests were achieved in the laboratory.

Preparation of aqueous extracts for phytochemicals analysis
Leaves were dried at 40°C and milled to obtain a fine powder using a vegetable blender and then stored in airtight bottles.100 g of fined powder of each plant was suspended in 100 ml of distilled water and kept under room temperature for 24 h.Then, it was filtrated through muslin cloth and centrifuged at 5000 rpm for 15 min.The supernatant was collected under vacuum using a millipore membrane of 0.20 µm.The aqueous extract obtained was stored at 4°C for further use.Dilutions of 1:10; 2:10 and 3:10 were used for antibacterial activity tests.

Preparation of essential oils for phytochemicals tests
Essential oils (EOs) from C. citratus, E. camaldulensis and M. piperita were obtained from Phytofla laboratory located in Banfora (10°38'26.09''N;4°45'33.15''W), a town located in the west of Burkina Faso.These EOs were extracted from leaves by the hydrodistillation method.The emulsion of EOs and 0.1% water-agar solution were prepared following Remmal et al. (1993) method.Each EOs was mixed at the ratio of 1:5, 1:10 and 1:20 (v/v).

In vitro antibacterial assay
The modified method of Nguefack et al. (2005) was used to study the antibacterial activity of plants extracts.15 µl of bacterial suspension concentrated at 10 8 CFU was inoculated in 4.5 ml of PSA liquid medium at 75%.The mix was homogenized and poured in solid PSA medium containing plates.
A volume of 10 µl of each ratio of emulsion oils and aqueous extract respectively was placed equidistantly onto Xoo and Xoc spread medium followed by incubation at 28°C.Water-agar was used as the control treatment.The inhibition zones were measured after 96 h of incubation.Each treatment was replicated three times, with three plates per replication.Three independent assays were performed with similar results.

In vivo antibacterial assay
The EOs from C. citratus and E. camaldulensis respectively found   to be highly and moderately effective in suppressing the growth of Xoo and Xoc strains in vitro, were tested for their antibacterial activity on seed germination and their effects on rice leaves.

Effect of EOs on rice seeds germination
The seeds were treated with an emulsion of EOs from C. citratus and E. camaldulensis, and with agar-water at the following ratios: 1:5, 1:10, 1:20 and 1:25.100 µl of each EO mixture was applied on 400 rice seeds according to Adegoke and Odela (1996) method.The treated seeds were placed at room temperature for 24 h before germination test.One control with water-agar solution without EOs was used.Then, the seeds treated were tested using the blotter papers method.Four replicates of 100 seeds for each EO were rolled in blotter papers and placed in polyethylene bags then incubated at 28-30°C under a cycle of 12 h light/12 h darkness.The rate of seeds germination was assessed at 3, 5 and 7 days after incubation.The experiment was repeated twice.

Effect of EOs on rice leaves
The effect of EOs on leaves was tested for efficient concentration of C. citratus and E. camaldulensis at the concentration found to have antibacterial effect in vitro.Three EOs emulsions at the ratio of 1:5; 1:10 and 1:20 were sprayed on both sides of each leaf from thirty days old plants.Five leaves were treated with each EOs and three replicates were done.Data were collected on the leaves 48 h after treatment.

Statistical analysis
The data on diameter of inhibition area and the germination rate of rice seeds treated, induced by the essential oils were assessed by analysis of variance (ANOVA), and treatment means were compared by Duncan's multiple range test.Statistical significance was set at P<0.05 and the analyses were performed with IBM SPSS Statistics Base 20 software.

Efficiency of essential oils
All tested EOs exhibited considerable antibacterial activity against both pathovars of X. oryzae (Table 1).EOs from C. citratus was the most effective in inhibition of Xoo and Xoc growth than E. camaldulensis and M. piperita.The inhibition area of Xoc at 1:5 ratio was to 34 mm for OE from C. citratus (Figure 1) against 12.67 and 10.22 mm OEs from E. camaldulensis and M. piperita respectively.

Efficiency of aqueous extracts
Bacterial growth was affected by the aqueous extract of E. camaldulensis only with 19.67 and 15 mm mean diameter of inhibition zone, respectively for Xoc and Xoo after 5 days incubation at 1:10 dilution (data not shown).
The inhibition effect increases with the high concentration of aqueous extracts.In comparison with the control, aqueous extract from C. citratus stimulates the growth of X. oryae strains at the level of the deposit point of the drop of aqueous extracts (Figure 2).

Effect of EOs on rice leaves
The three efficient concentrations of EOs from C. citratus  and E. camaldulensis used to treat rice leaves in vitro induce partial burns or complete desiccation of the leaves (Figure 3).

Effect of EOs on rice seeds germination
The rate of seeds germination varied according to EOs and the incubation duration.The seeds treated with Eos from E. camaldulensis and C. citratus at the ratio of 1:5 and 1:10 reduced significantly seed germination (≤70%) as compared to the control.However, at the concentration of 1:20, the percentage of germination was similar to the control (Figure 4).

DISCUSSION
EOs from C. citratus was found to be highly effective in inhibiting the growth of two pathovars of X. oryzae.Moderate activity was recorded from Eos of E. camaldulensis and M. piperita.The data showed that efficiency of each EOs was similar for both pathovars of X. oryzae.Considering aqueous leaf extracts, it was found that only E. camaldulensis inhibited Xoo and Xoc growth, while C. citratus stimulated bacterial growth as compared to the control.The efficiency of extracts from the three aromatic plants on phytopathogenic bacteria was previously reported (Nguefack et al., 2005;Paret et al., 2010;Lucas et al., 2012).In addition, several plant extracts are known to possess antibacterial activity.Govindappa et al. (2011) have reported antibacterial activity of aqueous extract of Adathoda vasica on Xoo.Nguefack et al. (2005) demonstrated the efficiency of EOs from Ocimum gratissimum and Thymus vulgaris on Xoc NCPPB 1632 R1 strain.In contrast, they found that Xoo NCPPB 2446R11 strain was not sensitive to EO from C. citratus at 1:10 dilution which is not in conformity with the present study results.
The results showed that seeds treatment with the EOs from C. citratus and E. camaldulensis reduces seeds germination rate, except when applied at a low concentration.Also, these EOs cause various symptoms ranging from burns to drying, when applied on the leaves.The secondary metabolites contained in the EOs of these two aromatic plants could explain the reduction of seeds germination rate, burns and leaves drying that was observed following the treatment and may confer them allelopathic and herbicide effects.Indeed, Gargouri et al. (2014) reported that C. citratus and E. camaldulensis exhibited inhibitory effects on associated vegetation.It is known also that the use of citrals-rich EOs induces the inhibition of weed seeds germination.For instance, the citrals, major component of the EO from C. citratus showed phytotoxicity effects against Sinapis arvensis and Phalaris canariensis even at low concentration.
During this study, the antibacterial activity of the EOs from C. citratus, E. camaldulensis and M. piperita against both pathovars of X. oryzae was demonstrated.These plants are an alternative to the use of pesticides and have the advantage of protecting human health and the environment.However, these results reveal that the effective concentrations of these Eos reduce seeds germination and induce herbicide effects on rice leaves.
This study shows the identification of molecules responsible for the antibacterial activity and their purification in order to test their effects on seeds contaminated with Xoo and Xoc.Also, the components of aqueous extracts from E. camaldulensis that stimulates Xo strains growth could be used to enrich medium culture.
different letters are significantly different (P< 0.05) based on Duncan's multiple range test.

Figure 1 .
Figure 1.Inhibition zone (34 mm of diameter) of the growth of X. oryzae pv.oryzae induced by essential oil from C. citratus at the ratio 1:5.

Figure 2 .
Figure 2. Stimulation of the growth of Xanthomonas oryzae pv.oryzicola strain in deposit area of aqueous extracts of Cymbopogon citratus.

Figure 3 .
Figure 3.Effect of essential oils of Cymbopogon citratus and Eucalyptus camaldulensis on rice leaves.

Figure 4 .
Figure 4. Germination rate of Azecuna seeds treated with essential oil from Eucalyptus camaldulensis and Cymbopogon citratus.The germination rate was evaluated at 3, 5 and 7 days after incubation (DAI).Different letters denote a significant difference between essential oil at 1:5, 1:10 and 1:20 dilution.

Table 1 .
Mean diameter (mm ±SE) of inhibition zone of Xanthomonas oryzae strains with essential oils from Cymbopogon citratus, Eucalyptus camaldulensis and Mentha piperita.