Enhancement of mango fruit preservation by using antimicrobial properties of Bacillus subtilis GA1

Pesticides are used to eradicate negative impacts of pests on food crops. Fungi are main microorganisms that affect quality. Some bacteria, Pseudomonas F19, Bacillus subtilis GA1 have been studied for their biotechnological and phytopathogenic properties. The aim of this work is to study in phenotypic way the microorganisms associated to healthy and altered mango fruits and find out B. subtilis GA1 aptitude for inhibition of growth of those spoil mango. B. subtilis GA1, recognized for its properties was tested directly in vitro and in vivo. Colletotrichum sp. and Aspergillus sp. were naturally associated to mango. Colletotrichum caused greatest alteration with lesion length of 54.2 ± 1.92 mm after three days. In vitro inhibitory activity test of strain GA1 showed a strong inhibition of Colletotrichum at 72.78 ± 2.90%. In same time, test of in vivo inhibitory activity has reduced mango spoilage at 89.29 ± 3.84%. Subjects: Environment & Agriculture; Food Science & Technology; Engineering & Technology


PUBLIC INTEREST STATEMENT
Because of their potential source of nutrients, fruits consumption is recommended for people well being. For very long time, preservation of agricultural products and mainly food crops against pests has been made through the use of chemical substances. However, it has been demonstrated that these products are sources of environmental pollution and public health problems. They are responsible for acute or chronic contaminations at the consumers' level. Because of harmful effects of pesticides, consumers are demanding food products with good sanitary qualities. In the same time, it was found that certain microorganisms such as Pseudomonas and Bacillus produce metabolites which are safe for consumer's health and the environment. These microorganisms are used as biopesticides to prevent fruits alteration.
This study aims to highlight the ability of certain microorganisms to preserve mango fruits against alteration due to microorganisms such as Colletotrichum sp., Aspergillus sp. and Candida sp.

Introduction
Mango (Mangifera indica L.) is considered as the most popular fruits usually eaten by millions of people in the tropical area especially in the developed countries. The annual production of this fruit is more than 100,000 tons in Côte d'Ivoire. Most of this production is exported (Kouassi, 2012). The most produced mango varieties are Kent, Keitt and they offer logistical advantages which make them the third largest supplier markets (Gerbaud, 2008;Moulin, 2007). However, the exported fruits are damaged by several fungi such as Colletotrichum gloeosporioides responsible of the anthracnose, the major constraint of production and exportation of fresh mangoes fruits in Côte d'Ivoire (Akem, 2006). This disease is considered as the most devastating of mango fruits. Anthracnose deteriorates fruits quality and also causes severe post harvest losses (Lakshmi, Reddy, & Pascal, 2011). The fruit can be infected in the field through lenticels or wounds during harvest, transportation or storage activities. The symptoms of Anthracnose on green infected fruits remain latent and invisible. The symptoms are most conspicuous and important on ripened or overipened fruits (Silva & Michereff, 2013). Infected mangoes offer rounded brown to black lesions with an indefinite border on their skin. Different lesion sizes can coalesce and cover extensive area of the fruit skin. In a case of severe infection, the fungi can invade the pulp and reach the stone. These necroses affect the commercial value of the fruits and induce income losses at the producers level and all the actors involved in the mango chain value.
Application of synthetic fungicides is the major approach for the control of fruits diseases. It has been reported that hot benomyl dips controls anthracnose on harvested mango fruits (Kim, Brecht, & Talcott, 2007). Also, using fungicides at high concentrations over a long time can result in pathogen resistance (Tian, Liu, Zhang, & Meng, 2010). The utilisation of chemical pesticides can also be source of public health problems. Additionally to that, public concern about fungicide residues. This brinks researchers to find new technologies for fitting against fruits diseases. Some of Gram positive bacteria, such as Bacillus subtilis GA1, are able to stop inhibition growth of spoilage microorganisms responsible of fruits quality deterioration. They are able to produce under certain conditions, antibacterial or antifungal metabolites that reduce growth and development of undesirable microorganisms.
The main objective of this study was to find out B. subtilis GA1 aptitude to stop or reduce growth and development of microorganisms responsible of mango fruits alteration in order to enhance mango fruit conservation. So, microorganisms that naturally associated to altered or inaltered mango fruits are isolated and B. subtilis GA1 biological activity are tested on them.

Biological materials
One hundred and three samples of altered or unaltered mango fruits, Keith variety, were collected from fruit markets of Abidjan Plateau, Cote d'Ivoire. A purified and well identified B. subtilis strain GA1, from the collection of Walloon Center of Industrial Biology (CWBI), University of Liege Gembloux Agrobio Tech (Belgium) was used for different tests.
Isolates of microorganisms got from healthy and altered mango fruits, Keith variety were used for the different tests. Mangoes were obtained from a commercial warehouse in Abidjan-Plateau, Côte d'Ivoire.

Pathogen isolation
One hundred and three fruits were washed with sterilized water before removing fragments of fruit from margins of decayed and healthy tissues with a sterile scalpel (Djossou et al., 2011). The fragments were placed on Petri dishes containing PDA culture medium (Becton, Dickson and Company, Le pont de Claix, France) supplemented with chloramphenicol in order to prevent bacterial development. The Petri dishes were incubated at 27°C during 5to 7 days. Isolates of microorganisms were identified by macroscopic and microscopic examination based on identification keys (Botton et al., 1990;Guiraud, 1998;Mathur & Kongstal, 2003).

Microbial inocula preparation for in vitro and in vivo assays
Bacterial endospore suspensions used in biocontrol experiments were prepared from 72 h old cultures of B. subtilis GA1 grown at 30°C in agitated flasks (105 rpm) in 500 ml of 863 media described by Jacques et al. (1999). Cultures were centrifuged at 35,000 rpm for 20 min and the biomass pellet was washed three times in physiological water (9‰). The supernatant was collected in sterile tube and was filtered with a sterile membrane Millipore of 0.2 μm. Fungi concentration was determined by plate count on PDA medium. They were then resuspended in a sterile distilled water to obtain the final desired concentration of 10 5 CFU/ml.

Pathogenicity assay
Twenty-five healthy fruits without lesion had their surfaces disinfected by immersion during 5 min in 1% sodium hypochlorite solution.
They were rinsed three times with sterile distilled water. Wells measuring 3 mm wide and 5 mm deep were created with a sterile cork borer (Regnier, du Plooy, Combrinck, & Botha, 2008). Fruits were treated with fungi isolated (yeast or mould) by adding 50 μl of cell containing 10 5 CFU/ml in the wells. Control sample were inoculated with 50 μl of sterile distilled water.
Inoculated fruits were stored at 25°C room temperature during 5 days. The fruits that develop microorganisms are recovered and microorganisms are identified as described previously.
Each treatment was achieved 5 times. The lesion diameter (LD) were measured by using a ruler.

In vitro antagonism activity developed by B. subtilis GA1
B. subtilisGA1 was tested for its ability to inhibit the growth of various fungi plant pathogens in Petri dishes containing PDA medium. Two sterile disks of 6 mm has been laid to 3 cm of Petri dishes center. One dish carried out B. subtilis GA1 and the other one carried out the fungi pathogens. A second Petri dish contained only the fungi pathogens. 2 μl of microbial suspension were dropped on every sterile disk. The radii of fungal growth in both the control and dual culture plates were measured 3 days after incubation. The rate inhibition was defined as the difference between the distance of the growth in the dual culture plate (r in centimeters) and the fungal growth radius (R in centimeters) of the control plate, where ∆R = R − r and the percentage inhibition of fungal growth was calculated using the following equation (Erdogan & Benlioglu, 2010).

In vivo antagonism activity developed by B. subtilis GA1
Sixty-four healthy and freshly harvested mature mango fruits were carefully selected on the basis of absence of any disease or wounding symptoms. The fruits were washed with soapy water and rinsed 3 times with sterile distilled water. Then, fruits were surface sterilized by swabbing with 70% ethanol solution. 6 mm wide and 3 mm deep wells were then artificially created with a sterile cork borer. Fruits were treated with B. subtilis GA1, the antagonist bacteria by adding 50 μl of cell (10 5 CFU/ml) or 25 μl of its supernatant 24 h prior to pathogen challenge. Infection with fungal pathogens were realized in all cases 24 h later, by adding the same volume of a microbial suspension prepared as described above in order to introduce 10 5 (10 5 CFU/ml) conidia per site (Toure, Ongena, Jacques, Guiro, & Thonart, 2004). Two other treatments were also carried out. The first one is a control test regarding fruits that were only inoculated with 50 μl of sterile distilled water and the second one is a control disease test based on a none treated fruits with the antagonist bacteria but inoculated with the pathogens. Disease incidence was evaluated 3-7 days after pathogen challenge. It based on the diameter of spreading lesions developed around infected sites. Results were expressed as percentage disease reduction by using the following relation: where K is the wound diameter (6 mm) and D t and D e are lesion diameters measured on disease controls and on treated fruits, respectively (Toure et al., 2004).

Statistical assay
The software R version i386 3.2.2 for Windows was used for statistical analyses. The homogeneity of variances was tested by ANOVA and data from experiments with the same set-up were pooled for analysis when interaction between experiment and treatment was not significant at p = 05. Means from the different treatments were compared by the method of Tukey (least significant difference at α = 0.05).

Isolated fungi from tested mango fruits
Colletotrichum sp., Aspergillus sp. and Candida sp. were the main pathogens isolated from mango fruits.
Colletotrichum sp. was the most important pathogen isolated both healthy and spoiled fruits at 100% level. Aspergillus sp. and Candida sp. are also associated to healthy and spoiled fruits at a respective level of 70 and 30 (%). All these three fungi are involved in mango fruits deterioration (Table 1).

Pathogenicity assay
Pathogenicity tests carried out on mango fruits with each of the isolated fungi reveal fruits alteration after 3 days (Figure 1). Table 2. The most important alteration was observed on fruits artificially inoculated with Colletotrichum sp. The diameter of the lesion was estimated at 54.2 ± 1.92 b mm. However, the alteration length of fruits inonculated with a combination of Aspergillus sp. and Colletotrichum sp. was estimated at 55.6 ± 3.50 b mm and the one with the total pathogens was estimated at 61 ± 3.16 d mm. Mango artificially inoculated with Candida sp., is almost alterated (Figure 1(a)). Alteration from Aspergillus sp. was blackened ( Figure  1(b)). The alteration of fruits inoculated with Candida sp. and Aspergillus sp. started 3 days after artificial inoculation and the alteration of those inoculated with Colletotrichum sp. showed signs of alteration from the 2nd day with a total alteration 5 days later (Figure 1(c)). The lesion alteration diameters (Table 2) are different according to microorganisms with p < 0.05. Fruits spoilage was important with Colletotrichum sp. with a lesion diameter of 54.2 ± 1.92 mm, followed by Aspergillus sp. with 38 ± 1.58 mm of lesion diameter. The greatest lesion diameter of 61 ± 3.16 mm was  obtained when the inoculation was carried out with the combination of all identified fungi. It is following by inoculation with combination of Colletotrichum sp. and Aspergillus sp. with a lesion diameter of 55.6 ± 3.50 mm (Figure 2).

Assessment of inhibition of bacterial growth
Ability of Strain GA1 inhibition or reduce the growth of Colletotrichum sp., Aspergillus sp. and Candida sp. is shown in Table 3. The inhibition level runs from 71.50 ± 1.66 to 75.79 ± 1.32% with an intermediate value estimated at 74.54 ± 0.94% respectively for Candida sp., Aspergillus sp. and Colletotrichum sp.
In vivo treatments of fruits treated with strain GA1 cell were not alterated 5 days after pathogen inoculation (Figure 3).
Inhibition of Colletotrichum sp., Candida sp. and Aspergillus sp. is observed respectively at level of 92.48 ± 1.74%, 90.45 ± 2.57 and of 84.60 ± 1.80% after testing with Bacillus subtilus GA1. After 10 days, treated fruits with strain GA1 cells showed a rate mean inhibition value of 89.29 ± 3.84% for    Rispail (2008) and N'guettia, Kouassi, and Kouakou (2014). Post-harvest mango infection could be also influenced by orchards, market sanitation and transportation (N'guettia et al., 2014). That might favored the accumulation of inoculums from one crop to another during the market cycle. This contribute to enhance the rate infection.
The pathogenicity of isolated germs was confirmed by the deterioration of fruits quality and necrosis observed on fruits skin. That observation is in agreement with Onyeani, Osunlaja, Owuru, and Sosanya (2012) who have proved that these microorganisms are responsible of mangos infection in Asia. The degree of pathogenicity and virulence varies from one organism to another. This result corroborate with that of Kouame et al. (2011). Also, Onyeani and Oguntade (2015) demonstrated the ability of C. gloeosporioides to necrosis strawberry fruits inoculated with it. Strain GA1 aptitude to protect mango fruits against Colletotrichum sp., Apergillus sp. and Candida sp. spoilage is confirmed by those of Toure et al. (2004) andN'guettia et al. (2014). In fact, fruits treated with vegetative cells or supernatant from strain GA1 allowed an effective control of the pathogens. Supernatant from strain GA1 also provided a strong protective effect that was similar to the one observed with live cells. This suggested the production of metabolites by strain GA1 that are involved in the growth inhibition of pathogens. One of these metabolites could be the lipopeptides which were studied by Toure et al. (2004) on apple. Study achieved by Ongena (2014) showed also the antifungal properties of these lipopetides.

Conclusion
It has been demonstrated that Colletotrichum sp., Aspergillus sp., Candida sp. are phytopathogenic microorganisms naturally associated to healthy mango fruits and are involved in mango fruits alteration. However, vegetative cells and supernatant obtained from biopesticides such as B. subtilis GA1 could contribute to prevent agricultural products and environment against alteration. They could be efficient in the biocontrols of these phytopathogenic microorganisms.
Research of wide spectre of bacteria susceptible to be used as biopesticides should be carried out in order to reduce the use of pesticides and their negatives impacts on the environment, food crops quality and on public health.