Abstract

Mango bacterial black spot is an emerging disease in Côte d'Ivoire and the damage caused is becoming more and more significant. This study aims at diagnosing bacterial black spot incidence and severity level in regions of intensive mango production. Surveys were carried out during the vegetative stage of mango trees from July to August 2019 and the fruiting stage from March to April 2020 in 50 orchards in the Poro and Tchologo regions. In each orchard, 5 mango trees regularly dispersed over an area of 0.5 ha were assessed. On each tree, three (3) twigs per cardinal point were selected then the leaves counted and the leaf area covered by bacterial black spot symptoms determined. At fruiting stage, bacterial black spot severity on 40 fruits per selected tree, at the rate of 10 fruits per cardinal point was assessed. Bacterial black spot symptoms were observed in all orchards and the bacterium, Xanthomonas campestris pv. Mangiferaeindicae, was systematically isolated basing on symptoms taken from the leaves and fruits collected. Statistical analysis of the mean incidence and severity per town revealed a variability of 83.2% up to 90% on the leaves and 41.42% up to 55% on the fruits regarding incidence. As for severity, the highest values, 10.58% and 5.23% on leaves and fruits respectively were recorded in the department of Tafiéré. Mango bacterial black spot is found in orchards located in the Poro and Tchologo regions. Incidence and severity are strongly related to the humidity conditions of the production area.

1. Introduction

In Côte d’Ivoire, mango is produced throughout the territory, particularly in the northern zone of the country (savannah zone) where climatic conditions are favorable for good quality fruit production ¹. The mango sector is today a very important component in the agricultural economy of savannah zones ¹. The country produces 180000 tons of mango and exports around 42000 tons to the European market ². The mango orchards ensuring this production are however subjected to strong parasitic pressures, in particular those of fruit flies, fungi and phytopathogenic bacteria ³. Mango bacterial black spot disease, which has recently appeared in Côte d'Ivoire ⁴, is now the most damaging phytopathogenic constraint in mango production ⁵. The pathogen responsible for this disease is a bacterium of the genus Xanthomonas sp. which causes cankers and purulent wounds on twigs and fruits and can cause plant death ⁶. It causes severe defoliation, early fruit drop and deterioration of their quality, thus affecting export possibilities ⁷. This bacterium poses a serious threat to mango cultivation with losses of up to 50% in untreated orchards ⁸. In Côte d'Ivoire, this disease is managed through preventive control measures such as the identification and systematic elimination of symptomatic organs. For curative control, only copper-based products are used. With regard to mango plant production mode, the use and exchange of plant material between producers, studies on bacterial black spot penetration level are necessary in order to prevent the spread of this disease. Thus, the spatial distribution of bacterial black spot and the assessment of its incidence in mango orchards in the intensive production zone of northern Côte d'Ivoire are all preliminary work to be carried out. This study therefore focused on assessing bacterial black spot incidence and severity on mango trees of the Kent variety in the Poro and Tchologo regions in northern Côte d'Ivoire.

2. Material and Methods

The study was conducted in three departments (Korhogo, Ferkessédougou and Tafiré) located in the Poro and Tchologo regions (Figure 1). These regions are characterized by a Sudanese-type climate with two distinct seasons. A long dry season from November to April and a rainy season from May to October ⁹. The annual average temperature varies between 26°C and 27.5°C. The average annual rainfall is 1000 - 1400 mm ⁹.

The orchards visited were mainly composed of mango trees of the Kent variety. Fruits and leaves were diagnosed regarding mango bacterial black spot symptoms.

Fifty (50) mango tree orchards (Kent var.) spread over the two regions were surveyed. Twenty-five (25) plantations were visited in the Poro and Tchologo regions, respectively. For the Tchologo region, 21 and 4 orchards were taken into account in the departments of Ferkéssédougou and Tafiéré, respectively 0.5 hectare was prospected per orchard during mango tree vegetative stage (July-August 2019) and fruiting stage (March-April 2020). Bacterial black spot incidence and severity were the two epidemiological parameters assessed. This assessment concerned the leaves and fruits (Figure 2 and Figure 3). In order to assess disease severity on the leaves in each orchard, 05 mango trees regularly scattered over a surface area of 0.5 ha were selected. On each tree, three (3) twigs were selected per cardinal point (north, south, east and west). Leaves were counted on the last growth unit of each selected twig. The percentage of leaf area covered by bacterial black spot symptoms was determined according to the scale described in Table 1 ¹⁰. At fruiting stage, bacterial black spot severity on 40 fruits per selected tree, at the rate of 10 fruits per cardinal point was assessed according to the scale of ¹⁰.

The disease severity index for each mango tree assessed per orchard was calculated using the Mckinney formula ¹¹

Where:

Is: Severity index;

xi: score or index of the severity scale ranging from 0 to 5;

ni: number of leaves with index xi;

N: total number of leaves or fruits observed;

Z: highest score;

The ratio of the number of leaves or fruits assessed, showing bacterial black spot symptoms and all the leaves or fruits assessed, made it possible to determine disease incidence.

IM = Disease incidence

Ni = Number of infected leaves or fruits showing bacterial black spot symptoms

Nt = Total number of leaves or fruits assessed.

Five (5) samples of symptomatic leaves per tree at vegetative stage were collected for isolation and characterization of the bacteria responsible for the damage.

Pathogen isolation was carried out according to the method of ¹². It consisted in sterilizing the fragments of leaves taken, with black spots surrounded by yellow halo and located on the secondary veins. These fragments were soaked in 70% ethanol for 30 seconds, then in 1% sodium hypochlorite for 1 minute, then rinsed three times in sterilized distilled water. The sterilized fragments were individually aseptically dilacerated in a few drops of sterile water, then left to macerate for 10 to 15 minutes at 28°C. 50 μl of the suspension obtained was spread by parallel streaks in three sectors on a Petri dish containing the YPGA (Yeast extract Peptone Glucose Agar) medium.

The YPGA culture medium (18 g of Agar, 7 g of Glucose, 7 g of yeast and 7 g of peptone for 1 liter of this medium) was obtained by autoclaving the different constituents at 121°C under a pressure of 1 bar for 15 minutes. The Petri dishes were then incubated on the reverse side at a temperature of 28°C. Bacterial colonies revealed after 72 hours of incubation following the spreading of streaked leaf macerates on the YPGA culture medium were purified. Bacterial colonies were taken using a Pasteur pipette, then transferred to new YPGA medium. The purified strains were taken and then placed in 2-ml Eppendoff tubes containing a 50% glycerol nutrient solution. This mixture that was homogenized using a vortex was stored in the freezer at -80°C

For the macroscopic study of the bacterial strains obtained, the shape, size, color, appearance and smell were the main characteristics observed.

The microscopic study was done after the cells were stained. A bacterial colony growing on the YPGA medium was taken, then spread on a slide containing 60 μl of sterile distilled water. The bacterial suspension was dried under the flame, for staining, Crystal violet, PVP Iodine (LUGOL), a drop of ethanol, and Fuchsin were successively added. Cell morphology, size, clustering and staining observed under a microscope were used for classifying bacterial isolates.

The incidence and severity data obtained were subjected to analysis of variance (ANOVA) with STATISCA 7.1 software. When the mean values showed significant differences, the Newman-Keuls test at 5% threshold was used for their separation.

3. Results

The bacterial isolates obtained during isolation appeared in the form of scintillating ivory white colonies, with regular outline, convex, smooth and apigmented, non-smelly (Figure 4A). Under microscopy, the cells were in the form of small rod-shaped bacilli grouped together in clusters whose walls were pink stained (Figure 4B). This stain is characteristic of gram negative bacteria.

Mango bacterial black spot symptoms were observed in all orchards surveyed in the Poro and Tchologo regions. The mean disease incidence values on leaves and on fruits showed no significant difference between the 3 departments in the 2 areas of study (Figure 5). However, it was more significant on the leaves than on the fruits. On the leaves, it varied from 83.2% in the department of Korhogo to 90% in the department of Tafiré. On fruits, the mean incidence of bacterial black spot was 44%, 41.42% and 55% in Korhogo, Ferkessédougou and Tafiré, respectively. In terms of severity, the mean values were significantly different for leaves as well as fruits (Figure 6). The highest severity values for these two organs were recorded in the department of Tafiéré, 10.58% on leaves and 5.23% on fruits.

4. Discussion

The characteristic symptoms of bacterial black spot were demonstrated on the leaves, fruits and twigs of mango trees in all the areas surveyed. These symptoms appeared as translucent angular black spots with a yellow halo. These symptoms were similar to those reported in Burkina Faso in 2017 ¹³. In addition, ¹⁴ reported similar symptoms in Ferkéssédougou and Korhogo which would be responsible for 11% of yield losses.

The bacteria, Xanthomonas campestris pv. mangifeareindicae was systematically isolated basing on symptoms taken from leaves and fruits collected in the two regions concerned by this study. Bacterial strain identification was made on the basis of cultural, morphological and biochemical specificities. The isolates were apigmented. The colonies were convex in shape with regular outline, ivory white in color. The appearance of Xanthomonas campestris pv. mangifereindicae colonies on YPGA medium made them easily distinguishable from other bacteria with white pigment. These media produce convex, round, creamy white, mucoid colonies with a regular colony margin ¹⁵. The main strains of Xanthomonas campestris pv. mangifereindicae showed typical characteristics associated with the genus Xanthomonas except for the production of Xanthmonadine. Many strains produce unpigmented colonies when grown on Nutrient agar medium ⁷, but in recent years a few strains producing yellow pigmentation have been isolated from mangoes in Brazil, Florida, South Africa and Réunion ⁷. The Gram stain test revealed rod-shaped bacilli with pink stained walls, which is the proof that the bacteria are Gram negative. These characteristics of the bacteria have been described as a reliable diagnosis method for Xanthomonas campestris pv. mangifereindicae bacteria ¹⁶. These results are also in agreement with those of ¹⁷ which characterized the same bacteria on cassava organs showing symptoms of cassava bacterial blight.

Data on bacterial black spot incidence and severity on leaves and fruits showed their variability from one region to another. The town of Tafiré had the highest incidence and severity on leaves and fruits. This could be attributed to the combined effects of environmental factors such as rain, storms and wind of this region ¹⁸. The Tafiéré area recorded an annual rainfall of 1094 mm of rain/year higher than that of Korhogo and Ferkessédougou of 951.4 and 1040 mm of rain/year, respectively ¹⁹.

The spread of bacterial black spot is amplified when the rains are associated with strong winds (≥ 8m/s) and dissemination distances then depend on wind speeds. A moderate storm with average hourly winds of 10 to 15 m/s has been shown to allow the pathogen to spread for at least 250 m ²⁰.

The work of ²¹ also showed the influence of environmental conditions on the evolution of bacterial black spot severity. ²² also showed that cassava bacterial blight thrives best in periods of high temperature and humidity (above 23°C, with RH greater than 80%). It spreads quickly when weather conditions (humidity and heat) are favorable ²³.

5. Conclusion

Mango bacterial black spot is found in all intensive mango production regions in Côte d’Ivoire. This production intended for export is increasingly threatened by Xanthomonas Campestris pv. Mangiferaeindica, pathogen responsible for bacterial black spot. The mean incidence values on fruits that reached 55% show that mango bacterial black spot can become the major constraint to mango production in Côte d’Ivoire. Its dissemination and severity are accentuated by the high humidity conditions in the towns located in the southern part of the mango production zone. This study must be expanded to the entire mango production zone in Côte d'Ivoire so as to better map the disease and design appropriate means of control for sustainable management of this disease.

Acknowledgements

We express our thanks to the mango interprofession of Côte d'Ivoire and the owners of the orchards of the Poro and Tchologo regions who allowed access to their different orchards for the collection of information on mango bacterial black spot.

References