Citrus bacterial canker (CBC) caused by Xanthomonas citri pv. citri (Xcc) represents the most devastating bacterial disease on citrus production  through  the  world (Salifou and Bounou, 2020). The disease causes defoliation, fruit cankers, a premature fall of fruit and a general withering of  trees  (Gottwald  et al., 2002; CABI, 2019). There are no highly efficient strategies for controlling CBC disease. In Burkina Faso, CBC was reported on all commercial citrus cultivars in the provinces of Comoé, Houet and Kénédougou (Juhasz et al., 2013). In endemic areas, the quarantine and eradication are the major means of control against Xcc (OEPP/CABI, 1990). However, the use of windbreaks, destruction of infected branches and spraying of copper-based products during periods of susceptibility, can reduce the severity of the disease (Civerolo, 1984; Behlau, 2021).Indeed, copper-based products, despite their effectiveness (Stall et al., 1980; Idrissou-Touré et al., 2017), have an adverse effect on human and environmental health (Villeneuve, 2008; Damien et al., 2017). In addition, their repetitive and uncontrolled use causes resistance phenomenon in pathogens.

 

In order to prevent major damage caused by CBC, alternative less harmful tools have been extensively studied in recent years (Oliveira et al., 2021; Raveau, 2020). For example, several studies have shown that many plants from the West African flora have an enormous biocidal potential (toxic, repellent, anti-appetizing) against a broad spectrum of pests. They can be used in the form of aqueous extracts or essential oils (Saha et al., 2013; Zombré et al., 2015). In Burkina Faso, despite the  high  prevalence  of  CBC  and  the  resulting crop losses, no means of control, including the use of copper-based products, have been deployed. Wonni et al. (2016) showed that aqueous extract and essential oil of Eucalyptus camaldulensis have an antibacterial activity in vitro test against both pathovars of Xanthomonas oryzae. The results of Rabbee et al. (2021) showed that ethyl acetate extract from Bacillus velezensis, have antibacterial activity against Xcc wildtype strains, in contrast to Bacillus amyloliquefaciens strains which had no antibacterial activity.

 

In order to promote an effective control of Xcc, the present study aimed to evaluate in vitro and in vivo, the antibacterial activity of some chemical, biological substances, plant aqueous extracts, and essential oils.

 

Substances tested

 

Five types of substances were tested. They were (i) Chemicals; sulphate and hydroxide copper, (ii) plant aqueous extracts; of Azadirachta indica (A.) JUSS, Cymbopogon citratus (D.C.) STAFF, and E. camadulensis DEHNH, (iii) essential oils;  Ocimum gratissimum LINNE, (iv) biological substance derived from B. amyloliquefaciens supplied by BAYER company and (v) formulations based on essential oils that were Neco 50EC and Proraly 50EC from Côte d’Ivoire.

 

Preparation of aqueous plant extracts

 

The aqueous extracts of leaves of A. indica (A.) JUSS., C. citratus (D.C.) STAFF and E. camadulensis DEHNH, were prepared according to the method described by Guédé-Guina et al. (1996).

 

The leaves were collected at the Farako-Ba station with coordinates Longitude 4°20’ West, Latitude 11°6' North and an altitude of 405 m. The leaves were firstly dried in the shade for two weeks, and then powdered using a mortar. The powders were macerated in the proportion of 1 g of powder/1 ml of distilled water and the suspension was kept for 24 h. The mixture was then, centrifuged at 4000 rpm for 10 min and the supernatant was sterilized in order to remove contaminants. The final substrate was stored in the refrigerator for its use.

 

Concentration range  

 

Six concentrations per substance were used in this study. They were prepared by the method of double dilution in according to a geometric progression of ½ reason (Toty et al., 2013) from a maximal concentration (C1) to a minimal concentration (C6) (Table 1).

 

 

Efficiency test in vitro

 

It was performed using the standard disc or antibiogram method (Abo-Elyousr and Asran, 2009). Bacterial suspension of 40 μl at 10⁸ CFU/ml was spread uniformly over the entire surface of the LPGA solid medium (LPGA for 1 L: Yeast 7 g, Peptone 7 g, Glucose 7 g, Agar 18 g) contained in Petri dishes. After drying, sterile blotting paper discs of 6 mm diameters were placed and inoculated with 10 μl of each substance. A negative control was inoculated with sterile distilled water. Petri dishes were firstly incubated for 1 h at room temperature, and then at 30°C in inverted position into incubator. After 72 h incubation, the diameters of inhibition (ID) zones were measured.

 

The efficacy of each substance was determined based on the criteria of Ponce et al. (2003) as follows: (i) ineffective: ID <8 mm, (ii) effective: 9 mm < ID <14 mm; (iii) very effective: 15 mm <ID <19 mm, and (iv) highly effective: ID > 20 mm.

 

Determination of antibacterial parameters

 

It was carried out using the technique of microdilution in nutrient liquid. This technique consisted of inoculate in a standardized inoculum, and decrease concentrations of the tested substances. For this, a series of seven test tubes, each containing 3 ml of LPGA nutrient liquid, were inoculated with 50 μl of bacteria inoculum at 10⁸ colonies/ml, and then 50 μl of each substance according to the concentrations defined in Table 1 except for the control tube. The tubes were placed at 28°C under shaking for 24 h, and then at 28°C for 72 h in a bacteriological incubator. Three parameters were assessed in the following.

 

Bacterial growth (T_survey)

 

It was determined to measure the optical density (DO) of each tube using spectrophotometer before and after incubation and to calculate with the formula used by Moroh et al. (2008).

 

T_survey (x) = (T_i -T_f) / (T_it –T_ft),

 

where T_i: initial DO; T_f: final DO; T_it: initial DO of control without substance; T_ft: final DO of control without substance.

 

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

 

They were determined to assess the bactericidal or bacteriostatic effect (Berche et al., 1991) of each substance by calculating the MBC/MIC ratio as follows:

 

(1) bactericidal if MBC/MIC ≤ 2;

(2) bacteriostatic if MBC/MIC > 2

 

In vivo test

 

Young plants of the tangelo species with leaves infected by CBC, from nurseries in the city of Orodara, were used under greenhouse conditions, to test the effective concentrations of the substances which were found to be effective in vitro. Therefore, copper hydroxide and sulfate (25 and 12.5 mg/ml), HE of O. gratissimum (5 and 10 µl/ml), the aqueous extract of E. camadulensis (50 and 100 mg/ml), and B. amyloliquefaciens formulation (30 and 60 µl/ml) were tested every 15 days.

Before foliar treatment with the substances, we counted (i) the number of symptomatic leaves per plant, (ii) the proportion of canker on each symptomatic leaf per plant, and (iii) the number of symptomatic leaves of the plant. These data were used to calculate the foliar incidence and severity of CBC according to the following formulae defined respectively by Idroussou-Touré et al. (2017) and Mayee and Datar  (1986).

 

Disease incidence (%) = (NSL/NTL) × 100

 

where NSL: the number of symptomatic leaves; NTL: the number of total leaves.

 

The severity was evaluated using the scale of Lakshmi et al. (2011).

Severity = Addition of numerical notation × 100 / Number of examined unit × Maximal note.

 

Statistical analysis

 

The analysis of variance and Tukey's test were carried out with the Minitab18 software in order to compare the effectiveness of the substances used at different doses in vitro and in vivo, but also to compare the variations between MIC and BMC.

Sensitivity of the Xcc strain to the tested substances

 

All the tested substances showed an inhibitory effect at the different concentrations used, with a very remarkable effect of copper hydroxide at 12.5 mg/ml, inducing 13.3 ± 1.6 mm of inhibition diameter (Table 2). In general, the inhibition diameter was proportional to the concentration of each tested substance. In fact, the highest inhibition   diameters   were    obtained    with   the maximum concentrations (C1).

 

 

Efficiency of the different tested concentrations

 

Tables 3 to 6 show the efficiency of the antibacterial activity of each substance in terms of the tested concentrations. The products based on copper were efficient from the concentration of 3.12 mg/ml for copper hydroxide and 6.25 mg/ml for copper sulphate (Table 3). The antibacterial activities of Proraly 50EC and Neco 50EC were efficient, respectively from 25 and 50 µl/ml concentrations (Table 4). Below these doses, they became inefficient. Furthermore, the antibacterial activity of the formulation based on B. amyloliquefaciens was efficient to the concentration of 6.25 µl/ml, whereas the essential oil of O. gratissimum was efficient from 1.5 µl/ml (Table 5). The efficiency of plant aqueous extracts was very variable according to the vegetal species. So, the aqueous extracts of C. citratus and  E. camadulensis  were  efficient to the concentration of 50 mg/ml, whereas that of A. indica was efficient from 100 mg/ml (Table 6).

 

 

 

Survival rate of Xcc

 

The survival rates of Xcc at the different tested concentrations were inversely proportional to the increase of the concentration of each substance (Table 7). Indeed, the lowest rate of survival (-9.2±3.26%) was registered with O. gratissimum at 5 µl/ml. However, the highest rate of survival (124.32±31.54%) was registered with the aqueous extract of A. indica at 6.25 mg/ml.

 

Characteristics of the tested substances

 

The different values of Minimal Inhibitory Concentrations (MIC) and Minimal Bactericidal Concentrations (MBC) are shown in Table 8. Referring to survival rates, MBC/MIC ratios ≤ 2 were obtained with the copper sulphate, essential oil of O. gratissimum and aqueous extract of E. camadulensis confirming their antibacterial properties. However, we were unable to determine the MIC and MBC of B. amyloliquefaciens, A. indica, C. citratus and Proraly 50 EC due to their opacity.

 

 

Effect of treatments on the incidence and severity of citrus bacterial canker

 

The results showed that the averages of incidence and severity of CBC significantly differed among the treated substances. We noted a significant decrease of the incidence and severity on the treated plants compared to untreated controls, which incidence progressed from 41.32 to 56.12% after 120 days of observation (Figures 2 and 3). The C2 concentrations tested were the most effective. Analysis showed that the strongest disease reduction rates were obtained with copper hydroxide and sulfate at 12.5 mg/ml. However, with O. gratissimum at 10 µl/ml, we recorded a reduction in disease without significant difference with chemical products.

 

Through this study, we evaluated the antibacterial activity of the diverse substances in vitro and in vivo on the growth of X. citri pv. citri. The two chemical products based on copper at 25 mg/ml, were the most active with the high inhibition diameters. Furthermore, the aqueous extract of A. indica, C. citratus and E. camadulensis and essential oils of O. gratissimum were each active inducing an inhibition diameter superior to 10 mm at the maximal tested concentrations. The lowest inhibition diameters were obtained with Neco 50EC and Proraly 50EC.

According to MBC/MIC ratios of copper hydroxide (12.5 mg/ml), copper sulphate (25 mg/ml), O. gratissimum and E. camadulensis (50 mg/ml), the results showed that these substances have antibacterial properties.

 

For in vivo tests, the results showed that chemical products based on copper and the essential oil of O. gratissimum  were the most effective, when they were used in foliar treatment each two week intervals.

 

In fact, the antibacterial action of copper hydroxide and copper sulphate in vitro and in vivo has been demonstrated by several studies (Girard, 2004; Idrissou-Touré et al., 2020; Behlau et al., 2021). However, some authors claimed that the use of copper can induce resistance to pathogens (Giraud et al., 2007; Villeneuve, 2008; Damien et al., 2017), create environmental pollution and copper accumulation in soils (Alva et al., 1995).

 

To attenuate the harmful effect of copper, some authors recommended its use in combination with mancozeb (Roberts et al., 2008; Fayette et al., 2012) or other biological components such as B. subtilis (Provost et al., 2012).

 

Studies have shown that the essential oil of O. gratissimum contains components such as thymol over 43%, gamma-terpinene 18.77% and para-cymene 6.77% (Soro et al., 2011), which would give it a bactericidal activity. Thus, thymol was one of the components of essential oils that are the most active against pathogens (Ajjouri et al., 2008).

 

The results confirmed those finding of Kpodekon et al. (2013) that demonstrated that the MICs and CMBs of O. gratissimum would be respectively between 6.10^-3 and 144.10^-3 mg/ml. In addition, Zombré et al. (2015) have shown that O. gratissimum was effective against bacterial blight of cashew and mango caused by X. citri pv. mangiferaindica. Saha et al. (2013) have also reported the antibacterial activity of O. gratissimum against Gram negative bacteria.

 

The effectiveness of aqueous plant extracts of A. indica, C. citratus and E. camadulensis has been proven in numerous studies, especially against fungi of the Fusarium genus (Dao, 2013; Tiendrebeogo, 2011).

 

As for the results obtained in the study with the biopesticide, the component of B. amyloliquefaciens would be the active molecule. Chen et al. (2009) reported that B. amyloliquefaciens produces various antibiotics which confer antibacterial activity. Roberts et al. (2008) and Ibrahim et al. (2016) have shown its efficacy on numerous species of Xanthomonas, Pseudomonas and Erwinia. Ye et al. (2016) reported that in vivo treatment with the combination of copper products and B. subtilis formulations reduced the incidence of CBC by only 19%, whereas untreated plants had 43%.

All the substances tested showed antibacterial properties against Xcc and their efficacy changes with concentration. The copper products were the most effective followed by the essential oil of O. gratissimum, the aqueous extracts of E. camadulensis and the substance formulation of B. amyloliquefaciens respectively. These substances were induced reductions of the incidence and severity of CBC, when applied at certain concentrations at two weeks intervals. Therefore, they can be tested in the field to determine their effectiveness.

The authors have not declared any conflict of interests.