# New bacterial agents to limit Colletotrichum gloeosporioides development on mango

Ahmed Taïbi, Jean-Christophe Meile, Hugo Dieudonné, Lise Korsten, Marc Chillet, Fabienne Remize

► To cite this version:

Ahmed Taïbi, Jean-Christophe Meile, Hugo Dieudonné, Lise Korsten, Marc Chillet, et al.. New bacte-rial agents to limit Colletotrichum gloeosporioides development on mango. Advances in Microbiology, 2020, 10 (12), pp.691-712. 10.4236/aim.2020.1012050 . hal-04667788

HAL Id: hal-04667788

https://hal.science/hal-04667788v1

Submitted on 22 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# New Bacterial Agents to Limit *Colletotrichum gloeosporioides* Development on Mango

Ahmed Taïbi<fnref n="1" />,<fnref n="2" />, Jean-Christophe Meile<fnref n="1" />,<fnref n="2" />*, Hugo Dieudonné<fnref n="2" />,<fnref n="3" />, Lise Korsten<fnref n="4" />, Marc Chillet<fnref n="1" />,<fnref n="2" />, Fabienne Remize<fnref n="2" />,<fnref n="3" /> ID

**How to cite this paper:** Taïbi, A., Meile, J.-C., Dieudonné, H., Korsten, L., Chillet, M. and Remize, F. (2020) New Bacterial Agents to Limit *Colletotrichum gloeosporioides* Development on Mango. *Advances in Microbiology*, 10, 691-712.

https://doi.org/10.4236/aim.2020.1012050

Received: November 5, 2020

Accepted: December 26, 2020

Published: December 29, 2020

Copyright © 2020 by author(s) and
Scientific Research Publishing Inc.
This work is licensed under the Creative
Commons Attribution International
License (CC BY 4.0).

http://creativecommons.org/licenses/by/4.0/

<figure><img src="image_2.png" /></figure>

Open Access

## Abstract

Mango anthracnose disease forms typical irregular-shaped black necrotic spots on the fruit peel of mature fruit and is caused by *Colletotrichum gloeosporioides*. In order to improve the disease control with a limited use of fungicides, new microbial agents able to limit the growth of the pathogen were searched in the indigenous natural flora of mango surface. In order to find a suitable biocontrol agent, a screening was applied to 305 epiphytic bacteria isolated from the carposphere of 17 mango cultivars sampled from eight locations on Reunion Island. The screening approach involved a first step based on the ability of the isolates to form a biofilm, to grow under fruit storage conditions, and to interfere with the development of *C. gloeosporioides*. In a second step, the capability of selected isolates to limit *C. gloeosporioides* in vitro mycelial growth and conidia germination was assessed and species identified. The most effective bacteria belonged to the *Enterobacter*, *Pantoea*, *Kosakonia* and *Leuconostoc* genera, but for some of them, their safe use has to be demonstrated. Efficacy *in vivo*, performed on wounded mature mango fruit, was limited, probably because of the wounding inoculation strategy favoring the pathogen. Future biocontrol treatments should focus on preharvest applications to enhance the protective benefit.

## Keywords

Anthracnose, Epiphytic Bacteria, Antagonism

## 1. Introduction

Mango occupies a very important place in culinary traditions and is of economic

<footnote n="1">¹CIRAD UMR QualiSud F-97410 Saint Pierre, Réunion, France</footnote>

<footnote n="2">²QualiSud, Univ Montpellier, InstitutAgro, Avignon Université, Univ de La Réunion, Montpellier, France</footnote>

<footnote n="3">³Univ de La Réunion, UMR QualiSud, Saint Pierre, France</footnote>

<footnote n="4">⁴Department of Plant and Soil Science, Agricultural Sciences Building, University of Pretoria, Pretoria, South Africa
Email: *meile@cirad.fr</footnote>
importance in some tropical and subtropical countries. Mango production currently ranks seventh in global fruit production with 50.65 million tons in 2017 [1]. For commercial purpose, the high-end fruit should be free of external damage, decay, bruises, and latex or sap injury.

Postharvest diseases such as anthracnose reduce fruit commercial quality and lead to significant losses. Black spots mostly develop during post-harvest stages and lead to fruit decay. Mango anthracnose is caused by *Colletotrichum gloeosporioides* fungal species and remains the major mango postharvest disease in Reunion Island [2]. Mature fruit affected by anthracnose develop sunken, prominent, dark brown to black decay spots before or after picking. The development of fungal mycelia under the mango skin leads to the necrosis of the epidermal cells, which makes the fruit unmarketable. The disease symptoms only appear after fruit ripening, although mango contamination occurs by splashing conidia during the rainy period and is followed by an appressorial (quiescent form) phase which lasts until favourable germination conditions [3].

*C. gloeosporioides sensu lato* is responsible for fruit diseases, referred to as “anthracnose”, on many other tropical fruits including banana (*Musa* spp.) [4] [5] [6], avocado (*Persea americana*) [7] [8], papaya (*Carica papaya*) [9] [10] guava (*Psidium guajava*) [11] [12], passion fruit (*Passiflora* spp.) [13] [14], dragon fruit (*Hylocereus undatus*) [15] and others [5] [6] [16] [17].

Despite the high efficacy of commercial fungicides against *C. gloeosporioides*, the increasing emergence of fungicide-resistant isolates has been reported [18] [19] [20] [21]. Therefore, several fungicides have been withdrawn from the market due to pathogen resistance. Moreover, fungicides are responsible for environmental and public health hazards [22]. On the other hand, there is a growing public demand for organically produced crops and recent European regulations impose the decrease the Maximal Residue Levels (MRL) [23]. Therefore, there is a need for the development of new technologies and methods that are alternative to synthetic fungicides for better fruit postharvest disease control.

Among alternative approaches to fungicides, biological control of postharvest diseases of mango by microbial antagonists has been under investigation for decades. Both bacteria and fungi have been isolated and characterized as potential biocontrol agents for fruit post-harvest diseases. They can inhibit the proliferation of pathogens both *in vitro* and *in vivo*. Efficiency and mode of action of these antagonists against *Colletotrichum* species complex have been particularly investigated [22] [23].

Despite extensive research on anthracnose biocontrol, only few commercial products are available and used. This is due to a lack of efficacy of biocontrol agents when it comes to large scale *in vivo* trials [22] [23] [24]. The inability of biocontrol agents to colonize fruit surface has been hypothesized. In most cases, postharvest disease biocontrol agents are applied after harvest. They have then to compete with the natural epiphytic microflora to colonize fruit surface. One solution to limit this effect, beside adapting the biocontrol agent to fruit surface
conditions, would be to select microflora from the same environment, thereby
limiting adaptation requirement to a different niche. Efficient biocontrol agents
have to persist in a wide range of environmental conditions [25] [26] [27].

Most fungal biocontrol agents screened against mango anthracnose were iso-
lated from mango environment, either orchard soil [28] [29] [30] or fruit. The
yeasts *Debaryomyces nepalensis* [30] and *Metschnikowia pulcherrima* [31] [32]
reduced the severity of mango anthracnose symptoms. The yeast *Cryptococcus
laurentii* was isolated from mango surface and its antagonistic activity relies on
several mechanisms such as competition for nutrients and space [33]. *Mey-
erzyma caribbica* also isolated from mango fruit showed competition for space,
competition for nutrients, parasitism and lytic enzyme production [33]. The
mechanism of action of *Trichoderma asperellum* against the phytopathogen was
parasitism, through the production of glucanase, cellulase and chitinase [34].

Contrarily to fungal biocontrol agents, bacteria selected to biocontrol mango
anthracnose come from a wide range of environments: plant isolate collections
[35], soil [36], green banana surface [37] or mango leaf [38] [39]. The spore-forming
bacteria *Bacillus thuringiensis*, *Bacillus pumilus* [36], *Bacillus licheniformis* [40]
[41] and *Bacillus amyloliquefaciens* [37] produce antifungal metabolites which
mediate a direct *in vitro* inhibition of phytopathogen growth, whereas *Pseudo-
monas fluorescens* induces plant defences, especially by production of fungal cell
wall lytic enzymes [35].

Bacterial biocontrol agents present several advantages comparatively to fungi
[42]. They are easier to grow and many of them, especially lactic acid bacteria
(LAB), cope with food safety regulation criteria [43]. In addition, LAB do not
form spores and can easily be inactivated during fruit processing. Therefore, the
first objective of this study was to isolate potential biocontrol agents, particularly
targeting LAB, from the surface of mature mango locally harvested, to investi-
gate the *in vitro* interactions of mango epiphytic bacteria with *C. gloeo-
sporioides*, and to assay the *in vivo* activity in order to evaluate the impact on
disease development on fruit.

2. Materials and Methods

**2.1. Isolation of Bacteria and Culture Conditions**

Mangoes were harvested from eight locations of Reunion Island, over a period of
10 weeks (Table 1). Reunion Island is characterized by a tropical climate, with
average annual temperature of 25°C and ferrallitic soils [44]. Annual rainfall
largely varies depending on the location: from 500 - 1000 mm for West locations
like Saint Paul, Grand Fond and Savana, to 1250 - 2000 mm for Bassin Plat and
2000 - 3000 mm for East locations like Cambuston, Quartier Français and Sainte
Suzanne [45]. Mature mangoes, from 17 cultivars, were selected and manipu-
lated without direct hand contact. Bacteria were collected from the fruit surface
with a sterile cotton swab soaked in 10 mL of buffered peptone water [46]. By
using a sterile pipette, the peptone water was collected and poured into a sterile
<table><caption>**Table 1.** Mango sampling and bacteria isolation. Both number and name of isolates are indicated depending on the medium of isolation, either MRS at 30°C or NM at 12°C.</caption><thead><tr><th>Location</th><th>Cultivar</th><th>Sampling date</th><th>Number of MRS isolates</th><th>MRS isolate names</th><th>Number of NM isolates</th><th>NM isolate names</th></tr></thead><tbody><tr><td>Saint Paul - organic</td><td>Cogshall</td><td>12/11/15</td><td>1</td><td>H001</td><td>5</td><td>H002 to H006</td></tr><tr><td>Saint Paul - organic</td><td>Cogshall</td><td>02/12/15</td><td>7</td><td>H008, H009, H014 to H018</td><td>5</td><td>H020 to H024</td></tr><tr><td>Saint Paul - organic</td><td>Cogshall derived</td><td>02/12/15</td><td>1</td><td>H010</td><td>0</td><td></td></tr><tr><td>Saint Paul - organic</td><td>David Haden</td><td>02/12/15</td><td>2</td><td>H012, H013</td><td>1</td><td>H025</td></tr><tr><td>Saint Paul - organic</td><td>José derived</td><td>02/12/15</td><td>2</td><td>H011, H019</td><td>1</td><td>H026</td></tr><tr><td>Bassin Plat</td><td>Cogshall</td><td>11/01/16</td><td>0</td><td></td><td>2</td><td>H027, H028</td></tr><tr><td>Bassin Plat</td><td>Irwin</td><td>11/01/16</td><td>4</td><td>H306, H309 to H311</td><td>2</td><td>H029, H296</td></tr><tr><td>Bassin Plat</td><td>Kensington Pride</td><td>11/01/16</td><td>0</td><td></td><td>3</td><td>H030, H031, H032</td></tr><tr><td>Bassin Plat</td><td>Tommy Atkins</td><td>11/01/16</td><td>1</td><td>H037</td><td>4</td><td>H033 to H036</td></tr><tr><td>Grand Fond</td><td>Kent</td><td>15/01/16</td><td>2</td><td>H050, H302</td><td>14</td><td>H086 to H098, H292, H293</td></tr><tr><td>Grand Fond</td><td>Nam DokMaï</td><td>15/01/16</td><td>8</td><td>H053 to H056, H059 to H062</td><td>9</td><td>H109 to H117</td></tr><tr><td>Grand Fond</td><td>Tommy Atkins</td><td>15/01/16</td><td>4</td><td>H057, H303 to H305</td><td>12</td><td>H099 to H108, H294, H295</td></tr><tr><td>Bassin Plat</td><td>Cogshall</td><td>18/01/16</td><td>5</td><td>H080 to H084</td><td>8</td><td>H174 to H181</td></tr><tr><td>Bassin Plat</td><td>Irwin</td><td>18/01/16</td><td>4</td><td>H067 to H070</td><td>12</td><td>H132 to H143</td></tr><tr><td>Bassin Plat</td><td>Kensington Pride</td><td>18/01/16</td><td>8</td><td>H058, H063 to H066, H071, H307, H308</td><td>17</td><td>H118 to H131, H297 to H299</td></tr><tr><td>Bassin Plat</td><td>Nam DokMaï</td><td>18/01/16</td><td>10</td><td>H051, H052, H072 to H079</td><td>17</td><td>H144 to H160</td></tr><tr><td>Bassin Plat</td><td>Tommy Atkins</td><td>18/01/16</td><td>12</td><td>H040 to H049, H038, H039</td><td>13</td><td>H161 to H173</td></tr><tr><td>Grand Fond</td><td>Heidi</td><td>23/01/16</td><td>0</td><td></td><td>23</td><td>H182 to H204</td></tr><tr><td>Saint Paul</td><td>Auguste</td><td>29/01/16</td><td>0</td><td></td><td>7</td><td>H205 to H211</td></tr><tr><td>Saint Paul</td><td>Heidi</td><td>29/01/16</td><td>0</td><td></td><td>3</td><td>H212 to H214</td></tr><tr><td>Saint Paul</td><td>José</td><td>29/01/16</td><td>1</td><td>H085</td><td>13</td><td>H219 to H231</td></tr><tr><td>Cambuston</td><td>José</td><td>08/02/16</td><td>7</td><td>H247 to H253</td><td>11</td><td>H271 to H281</td></tr><tr><td>Quartier Français</td><td>José</td><td>08/02/16</td><td>11</td><td>H236 to H246</td><td>7</td><td>H264 to H270</td></tr><tr><td>Sainte Suzanne</td><td>José</td><td>08/02/16</td><td>3</td><td>H254 to H256</td><td>2</td><td>H282, H283</td></tr><tr><td>Savana</td><td>José</td><td>08/02/16</td><td>4</td><td>H232 to H235</td><td>7</td><td>H257 to H263</td></tr><tr><td>Saint Paul - organic</td><td>Caro</td><td>17/02/16</td><td>1</td><td>H300</td><td>3</td><td>H284 to H286</td></tr></tbody></table>
Continued

<table><tr><td>Saint Paul - organic</td><td>Cécile</td><td>17/02/16</td><td>0</td><td>1</td><td>H287</td></tr><tr><td>Saint Paul - organic</td><td>Pierrefontaine</td><td>17/02/16</td><td>0</td><td>2</td><td>H288, H289</td></tr><tr><td>Saint Paul - organic</td><td>Ticroix</td><td>17/02/16</td><td>0</td><td>1</td><td>H290</td></tr><tr><td>Saint Paul - organic</td><td>Valencia</td><td>17/02/16</td><td>1</td><td>H301</td><td>H291</td></tr></table>

tube. The microbial solution obtained was streaked over MRS (de Man, Rogosa and Sharpe) agar and Nutritive Medium (NM) plates and placed at 30°C for 72 h and at 12°C for 10 days, respectively. Colonies with different aspects were isolated on the same growth medium after microscopy examination. Isolated strains were stored at -80°C in 20% glycerol.

*Bacillus subtilis* AvoGreen was used as a reference biocontrol strain [38].

Before use, bacteria were cultivated in broth, either MRS or NM depending on the isolation medium, at 25°C during 72 h in an incubator with agitation at 100 rpm.

## 2.2. Colletotrichum spp. Cultivation

The MUCL 43868 strain of *C. gloeosporioides* (Penzig) from the pathogen collection of the Catholic University of Leuven (Leuven, Belgium) was used and cultivated on potato dextrose agar (PDA) medium at 27.5°C. This strain was isolated from Mexican mangoes by GL Hennebert [47]. Strains CG Aust Mango 3-3 and CG Avocado 23-703 were obtained from the collection of Pr. Korsten.

An inoculum of *C. gloeosporioides* was grown on PDA plates over 10 days at 27°C. Conidia were collected by pouring 10 mL of sterilized buffered peptone water on the plates and recovered by pipetting and adjusted to a final concentration of 10⁵ conidia per mL using a Malassez cell.

## 2.3. Growth at Different Temperatures and Biofilm Formation

In a 96-well microplate, 180 µL of isolation medium broth and 20 µL of a 72 h bacterial culture were deposited. Each bacterial culture was loaded into 3 wells. Controls corresponded to 200 µL of isolation medium broth, distributed in 3 wells. Six batches of microplates were prepared and placed in incubator, with a lid on, at 12°C, 25°C, 30°C, 37°C and 42°C, over 72 h without agitation. The absorbance was read for each microplate three times a day until 72 h of incubation with the microplate reader. For each time point, the plate was shaken for 15s, then the reading was performed at a wavelength of 600 nm and the collected OD was the mean of 10 reads of the same well. For each well, the growth curve was plotted over time and maximal growth rate (µmax) was determined from OD slope over the growth phase. The optimal growth temperature was defined as the
temperature leading to the highest µmax.

After 72 h, each microplate incubated at 25°C was emptied and rinsed in clear water, then 125 µL of a 0.1% of crystal violet solution was added in each well. After 10 min, the content of the wells was dropped off, rinsed again with clear water and then dried at open air. A volume of 200 µL of 30% acetic acid solution was added and 125 µL were taken off and poured in a new microplate. The microplate was read on a 550 - 680 nm wavelength range on the microplate reader and the wavelength rendering the maximal absorbance (590 nm) was chosen for the exploitation of the results. Biofilm strength is given by the ratio of absorbance between the bacteria and the control wells.

**2.4. Identification of Isolates**

DNA extraction from bacterial strains was performed using the InstaGene Matrix commercial kit (Bio-Rad Laboratories, Hercules, CA, USA) [48]. For the amplification of the 16S rDNA region, the DNA primer pair was used: FD1-mod 5'-3': AGAGTTTGATCHTGGCTCAG and RD1-mod 5'-3':

GGMTACCTTGTTACGAYTTC [49]. The reaction volume was composed of 5 µl of purified DNA and 45 µl of a mixed solution composed of: 10 µl 5 × PCR buffer (Green Go Taq), 1 µl dNTP mixture (10 mM), 4 µl MgCl₂ (25 mM), 1.0 µl of each primers (FD1-mod and RD1-mod at 1 µM), 0.25 µl enzyme Go Taq DNA polymerase (5 U/µL) and qs of water. PCR amplification was carried out using Applied Biosystems Veriti™ Thermal Cycler. The thermal cycling program was: 3 min at 94°C, followed by 35 cycles of [40 sec at 94°C, 40 sec at 55°C, 60 sec at 72°C], and further for a time extension of 10 min at 72°C. The quality of the amplification products was analysed on 2% TAE agarose gels after electrophoresis for 90 min at 110 V and staining with ethidium bromide.

**2.5. In Vitro Colletotrichum Inhibition Assay**

**2.5.1. Plate Assay for Mycelial Growth Inhibition**

To assay mycelial growth inhibition, *C. gloeosporioides* MUCL 43868, CG Aust
Mango 3-3 and CG Avocado 23-703 strains were used. Mycelium (0.5 mm × 0.5
mm) was spotted in the middle of a plate containing PDA medium and incu-
bated for 21 days at 30°C. Bacterial isolates were grown on nutrient agar (NA)
incubated at 30°C for 48 h. A suspension was created by gently scraping the
bacterial lawn from NA. The OD at 600 nm of the suspension was adjusted to 1
unit (per mL) as assessed with PowerWave™ microplate spectrophotometer
(BioTek). A volume of 100 µL was used for inoculation.

Two mycelium pieces were deposited on opposite sides of the PDA plates. The bacterial strains were inoculated as a central streak 48 h after inoculation of the fungus. Plates were incubated for 10 - 15 days at 30°C, until the complete colonization of the plate surface by the fungus in the control plate (without bacteria). Thereafter, diameters of mycelium were measured to calculate a percentage of radial inhibition. Percentage of inhibition was calculated from the diameter of
*Colletotrichum* colony in the presence of an isolate compared to the diameter without any inhibitor (0% of inhibition).

## 2.5.2. Inhibition of the Germination of Conidia

A spore suspension of MUCL 43868 strain of *C. gloeosporioides* was prepared by placing 10 mL of peptone water (or sterile distilled water) in the Petri dish containing a 15-day fungus culture. After filtration, the concentration of spores was adjusted to 5 log spores/mL using a Malassez cell.

Conidia germination inhibitory bacterial activity was evaluated by microscopy on special sterile slides. The first step was the deposit of 100 µL of melted PDA medium on the slides, followed by drying under a laminar flow hood for 1 hour. To carry out the test, 10 µL of spore suspension were deposited on the slide and 10 µL of each bacterial strain were added. The slide was then incubated at 27°C inside a Petri dish lined with a double layer of moistened filter paper. Each treatment was repeated twice. The control corresponded to the suspension of spores in the absence of bacteria.

A conidiospore was considered to have germinated when a germinating tube of at least half the length of the conidiospore was observed under optical microscope. The qualitative data for the inhibition of germination of *C. gloeosporioides* MUCL 43868 were described as; (+): the germination of less than 25% of conidia was inhibited; (++): the germination of 25% - 50% of conidia was inhibited; (+++): the germination of 50% - 75% of conidia was inhibited; (++++): more than 75% of conidia germination was inhibited. Negative control showed no inhibition of conidia germination which corresponded to a germination rate close to 100%.

## 2.6. In Vivo Examination of the Severity of Anthracnose

The °C. gloeosporioides strain MUCL 43868 was cultivated in Petri dishes for 21 days on PDA solid medium at 27.5°C in the dark.

Mangoes cv. José were harvested and treated on the same day. A batch of 36 mangoes cv. José collected from the same orchard and with same maturity level (yellow point) was used. Fruit with no disease symptoms were selected, and the surface to be inoculated was washed with 70% ethanol and air-dried at room temperature. Subsequently, fruit inoculation for curative treatment was performed according to [50] by uniformly wounding (a cross: 2 mm deep and 10 mm wide) a relatively flat area in the middle of the fruit with a sterilized cork-borer and inoculating it with 20 µL of a spore suspension of *C. gloeosporioides* (10⁵ spores/mL). To ensure that anthracnose development was due to MUCL 43868, the peels from non-inoculated and inoculated fruits were placed on PDA amended with chloramphenicol and left for 8 days at 25°C. The identification of the re-isolated fungi was based on morphological criteria. After fungal inoculation, fruits were maintained at room temperature for 24 h (21°C, 85% relative humidity).

Bacteria *Enterobacter* sp. H222 and *Leuconostoc mesenteroides* H255 were
separately grown in nutrient broth for 3 days at 27.5°C in a nutrient broth. Two suspensions corresponding to OD at 600 nm of 0.5 and 1.0 were prepared for each isolate.

From all 36 mangoes inoculated with *C. gloeosporioides*, six fruits were sub-merged in distilled water, to be used as non-treated positive controls. Three treatments were used to assess the impact of bacteria on anthracnose devel-opment. The thirty remaining mangoes were separated into three batches of 10 fruits corresponding to [H222], [H255], and [H222 + H255] treatments. Pre-inoculated mangoes were immersed in each bacterial suspension for two minutes. Fruit were stored at 20°C with high relative humidity. After 10 days of storage, black spots on the surface of mangoes were measured using a digital caliper.

The experiment was repeated twice with independent batches of mango.

## 2.7. Statistical Analysis

XLSTATsoftware (Addinsoft, Paris, France) was used for statistical analysis. K-means clustering and hierarchical clustering analysis (HCA) were performed with maximal growth rate, optimal growth temperature, ability to form biofilm and inhibition of mycelial growth as variables and isolates as observations. Variables were centred and normalized, and Euclidian distances were used. For k-means clustering, trace (W), i.e. pooled SSCP matrix, was used as classification criterion. For HCA, Ward aggregation method was used. Dendrogram was built through the Ward's minimal distance algorithm. To compare inhibition of fun-gal growth, ANOVA was used with the REGWQ test and the bilateral Dunnet's test was used for *in vivo* assay.

# 3. Results and Discussion

## 3.1. Isolation and Phenotypic Characterization of Bacteria

Bacteria were collected from 17 mango cultivars grown in eight locations on Reunion Island in order to cover the widest diversity of epiphytic mango bacte-ria available locally.

As mangoes are generally stored under refrigerated conditions, isolation of bacteria was performed from NM incubated at 12°C to select psychrotrophic bacteria. Colonies of LAB were also recovered as these bacteria have a long his-tory of use in food and some of them are already used to preserve foods. From mango surface, 305 bacteria were isolated: 99 were isolated from MRS, and 206 from NM (Table 1).

The most appropriate candidates for biocontrol of mango anthracnose were then selected without prejudice of the bacterial species and through a fun-nel-shaped approach.

The maximal growth rate, the optimal temperature for growth, the ability to form biofilms and the level of inhibition of *C. gloeosporioides* MUCL 43868 mycelial growth were determined for the 305 isolates. Two classification methods,
k-means and HCA, were applied, resulting in similar results. Isolates were clus-
tered into six classes, leading to an inter-classes' variance of 79% of the total
variance (Table 2 and Figure 1). This high inter-classes' variance value implies
that classes were clearly defined and well differentiated.

The most homogeneous class (class 6), i.e. with the lowest intra-class variance,
gathered 24 isolates, characterized by low optimal growth temperature and low
inhibition level. Class 5 included the highest number of isolates (133) and was
also characterized by a low inhibition level of mycelial growth. Class 4, which
gathered 18 isolates, harboured the same characteristics, but with the highest
ability to form biofilm. Classes 1, 2 and 3 were the most interesting regarding the
inhibition of mycelial growth, with an inhibition level of the gravity centre of
18.3%, 25.4% and 17.0% respectively. Those three classes differed by the number
of isolates, class 1 being the largest. Class 1 was characterized by the highest
μmax and optimal growth temperature. Class 2 was characterized by the highest
mycelial growth inhibition level, an intermediate optimal growth temperature of
28.0°C and the lowest ability to form biofilm. Class 3 gathered psychrophilic
bacteria, with the highest ability to form biofilms. Among the three interesting
classes, inhibition level was the lowest in class 3.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Dendrogram of 305 isolates classified into six classes by hierarchical clustering analysis (HCA) based on the variables "maximal growth rate", "optimal growth temperature", "ability to form biofilm" and "inhibition of mycelial growth".</figcaption></figure>
<table><caption>**Table 2.** Characteristics of classes of isolates, according to maximal growth rate (µmax), optimal growth temperature (°C), biofilm strength and inhibition activity (%) against *Colletotrichum gloeosporioides* MUCL 43868 hyphal growth.</caption><thead><tr><th>Class</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th></tr></thead><tbody><tr><td>Number of isolates</td><td>73</td><td>28</td><td>29</td><td>18</td><td>133</td><td>24</td></tr><tr><td>Intra-class variance</td><td>14.1</td><td>19.7</td><td>15.5</td><td>28.2</td><td>23.9</td><td>0.7</td></tr><tr><td>Mean distance to gravity centre</td><td>3.6</td><td>3.7</td><td>3.1</td><td>4.8</td><td>4.1</td><td>0.7</td></tr><tr><td>Gravity centre µmax (h-1)</td><td>0.36</td><td>0.10</td><td>0.22</td><td>0.31</td><td>0.13</td><td>0.10</td></tr><tr><td>Gravity centre optimal growth temperature (°C)</td><td>34.3</td><td>28.0</td><td>12.0</td><td>35.4</td><td>30.1</td><td>12.0</td></tr><tr><td>Gravity centre biofilm strength</td><td>2.3</td><td>1.8</td><td>9.1</td><td>13.7</td><td>3.1</td><td>2.2</td></tr><tr><td>Gravity centre inhibition level (%)</td><td>18.3</td><td>25.4</td><td>17.0</td><td>11.5</td><td>12.1</td><td>11.6</td></tr></tbody></table>

## 3.2. Selection and Identification of Inhibitors of *Colletotrichum* Development

From the three classes showing the highest inhibition ability, 26 isolates were selected and identified by sequencing of the chromosomal region encoding 16S rRNA (Table 3). From the first class, 11 isolates were selected, representing 15% of the class size, whereas 12 isolates (43%) were selected from class 2 and 3 (10%) from class 3. No isolates from class 4 were selected in spite of the highest ability to form biofilm which is considered as an advantage to help the antagonist to colonize fruit surface [26], because of lower ability to inhibit the fungal pathogen.

Among those, 19 isolates were identified as Enterobacterales: eight belonged to the genus *Enterobacter* and seven were identified as *Kosakonia cowanii*. The other genera were *Pantoea* and *Serratia*. Besides, one isolate was identified as a *Microbacteriaceae*, *Curtobacterium luteum*, and two *Staphylococcus* species were represented. More interestingly because of their safety for food use, three isolates were identified as *L. mesenteroides* and one as *Gluconobacter* sp.

Most of the species hereby identified were previously described as part of plant microbiomes. *Enterobacter*, *Pantoea* and *Curtobacterium* were identified from salad leaves [51]. *Enterobacter*, *Pantoea*, *Leuconostoc* and *Curtobacterium* were identified on tomato fruit or leaves surface [52] [53] [54]. The same genera, plus *Staphylococcus* and *Gluconobacter* were identified on mango tree leaves [55]. *Gluconobacter* was identified on grape surface, but *Sphingomonas* was the most abundant genus in this niche [56]. Interestingly, *Kosakonia cowanii* was here for the first time reported as isolated from carposphere. *K. cowanii* is the type species of *Kosakonia*, and was isolated from clinical and environmental samples, especially soil and trees [57].

*Serratia marcescens* is essentially studied for its role in nosocomial infections and for chitinase production [58] [59]. For these reasons, isolates from the present work were not further studied. Four of the *Enterobacter* isolates belong to the *Enterobacter cloacae* complex, which gathers 12 species which cannot be easily differentiated from 16S rRNA coding region sequence [60] [61]. Many
<table><caption>**Table 3.** Isolate identification and inhibition of *C. gloeosporioides* MUCL 43868 hyphal growth (%) and conidia germination.</caption><thead><tr><th>Class</th><th>Isolates</th><th>Identification</th><th>Hyphal growth inhibition, %<fnref n="1" /></th><th>Conidia germination inhibition²</th></tr></thead><tbody><tr><td>3</td><td>H028</td><td>Enterobacter sp.</td><td>31.7 ±</td><td>9.9 +++</td></tr><tr><td>2</td><td>H061</td><td>Enterobacter sp.</td><td>32.4 ±</td><td>23.9 +</td></tr><tr><td>2</td><td>H068</td><td>Enterobacter sp.</td><td>27.7 ±</td><td>6.1 +++</td></tr><tr><td>1</td><td>H221</td><td>Enterobacter sp.</td><td>25.9 ±</td><td>4.0 +</td></tr><tr><td>1</td><td>H222</td><td>Enterobacter cloacae complex</td><td>37.4 ±</td><td>7.8 +++</td></tr><tr><td>1</td><td>H223</td><td>E. cloacae complex</td><td>26.7 ±</td><td>6.6 +++</td></tr><tr><td>2</td><td>H232</td><td>E. cloacae complex</td><td>23.0 ±</td><td>4.8 +++</td></tr><tr><td>1</td><td>H267</td><td>E. cloacae complex</td><td>22.0 ±</td><td>12.8 +</td></tr><tr><td>1</td><td>H219</td><td>Kosakonia cowanii</td><td>52.0 ±</td><td>21.2 +</td></tr><tr><td>2</td><td>H185</td><td>K. cowanii</td><td>43.3 ±</td><td>18.1 +</td></tr><tr><td>2</td><td>H182</td><td>K. cowanii</td><td>33.9 ±</td><td>9.0 ++</td></tr><tr><td>2</td><td>H184</td><td>K. cowanii</td><td>32.4 ±</td><td>7.1 ++</td></tr><tr><td>2</td><td>H192</td><td>K. cowanii</td><td>30.5 ±</td><td>3.1 ++</td></tr><tr><td>2</td><td>H191</td><td>K. cowanii</td><td>29.4 ±</td><td>5.0 +++</td></tr><tr><td>2</td><td>H188</td><td>K. cowanii</td><td>27.3 ±</td><td>6.3 ++</td></tr><tr><td>2</td><td>H186</td><td>Pantoea dispersa</td><td>30.2 ±</td><td>2.8 ++</td></tr><tr><td>1</td><td>H272</td><td>Pantoea sp.</td><td>24.9 ±</td><td>0.8 +</td></tr><tr><td>3</td><td>H129</td><td>Serratia sp.</td><td>24.2 ±</td><td>5.1 +</td></tr><tr><td>2</td><td>H177</td><td>Serratia marcescens</td><td>22.7 ±</td><td>6.9 ++</td></tr><tr><td>3</td><td>H117</td><td>Curtobacterium sp.</td><td>23.3 ±</td><td>11.6 +</td></tr><tr><td>1</td><td>H311</td><td>Gluconobacter sp.</td><td>20.4 ±</td><td>6.1 +</td></tr><tr><td>1</td><td>H270</td><td>Leuconostoc mesenteroides</td><td>21.1 ±</td><td>4.7 +</td></tr><tr><td>1</td><td>H255</td><td>L. mesenteroides</td><td>20.6 ±</td><td>8.8 +++</td></tr><tr><td>1</td><td>H305</td><td>L. mesenteroides</td><td>16.8 ±</td><td>6.1 +</td></tr><tr><td>2</td><td>H268</td><td>Staphylococcus fleurettii</td><td>14.5 ±</td><td>14.3 +</td></tr><tr><td>1</td><td>H300</td><td>Staphylococcus hominis</td><td>27.5 ±</td><td>11.5 +</td></tr></tbody></table>

isolates from this complex originate from clinical samples [61] [62] [63], and their multidrug-resistance capacity raises concerns about their pathogenicity and virulence. However, many other isolates come from plant, possibly plant pathogens and isolates from this complex might play a role in biocontrol [64]. The *Pantoea* genus is widely distributed in nature and many species are described as epiphytes, endophytes or plant pathogens [65]. Many isolates, some being from the species *Pantoea dispersa*, have been used as biocontrol agents against post-harvest rots of fruit, onion or sweet potato [66]-[71]. *Pantoea agglomerans* strain CPA-2 is an effective biocontrol agent (BCA) against the major postharv-

<footnote n="1">¹mean ± standard deviation; ²(+): the germination of less than 25% of conidia was inhibited; (++): the germination of 25% - 50% of conidia was inhibited; (+++): the germination of 50% - 75% of conidia was inhibited; (+++): more than 75% of conidia germination was inhibited.</footnote>
est pathogens present on pome and citrus fruits [72] [73] [74] [75]. *K. cowanii* was classified in this new genus in 2013 from *Enterobacter* and based on genetic polymorphism analysis [55] [76]. This species can act as a plant growth promoter, especially for sugar cane [77].

*Curtobacterium* was found as an endophytic bacterium of many plants including rambutan fruit [78] [79] [80]. It has been studied as a biocontrol agent for fungal brown rot of plum [27].

Among other LAB, *L. mesenteroides* is described for its antifungal activities and potential use for biocontrol [81] [82]. This bacterium is commonly found in fermented foods, especially from vegetables and fruit [83] [84].

Mycelial growth inhibition was in the range 20% - 40% for most isolates, the highest inhibition being observed for two *Enterobacter* isolates, two *Kosakonia* isolates and *P. dispersa*. Complementary to hyphal growth inhibition, isolates were tested for their ability to inhibit MUCL 43868 conidia germination (Table 3 and Figure 2). The results were listed in four classes according to isolate capability to inhibit germination, from low to strong inhibition [<25%, 25% - 50%, 50% - 75% and >75% of conidia inhibited]. Contrarily, conidia germination

<figure><img src="image_4.png" /><figcaption>**Figure 2.** *In vitro* slide test of inhibition of conidial germination of *C. gloeosporioides* MUCL 43868. The device used, germination of conidia under control condition, and germination of conidia in presence of bacteria isolates H182, H185, H186, H222, H232, H255 and H270 are shown. A magnification time of 400 was used.</figcaption></figure>
inhibition was clearly more marked for *Enterobacter* and *Leuconostoc* (Table 3). Six isolates inhibited the germination of more than 75% of conidia: five *Enterobacter* isolates (H028, H068, H232, H222, H223), and *L. mesenteroides* H255. The *K. cowanii* H191 isolate inhibited the germination of 50% - 75% of conidia. Six isolates, four from *K. cowanii* (H182, H184, H192, H188), *P. dispersa* H186 and *Serratia marcescens* H177 inhibited 25% - 50% of conidia. The other isolates inhibited the germination of <25% of conidia.

From these results, isolates from species exhibiting good inhibition ability were selected to further assay inhibition capacity: *Kosakonia* H182 and H191, *Pantoea* H186, *Enterobacter* H222 and H232, and *Leuconostoc* H255. *L. mesenteroides* H270 was added to this pool of isolates as it has a safe use status as a LAB. *B. subtilis* AvoGreen was used as a reference. The eight isolates were tested against the hyphal growth of two other strains of *C. gloeosporioides*, one from South Africa collected from avocado and the other one from Australian mango (Table 4). Most of the isolates showed a hyphal growth inhibition activity in the range 19.3% to 24.9%. The isolate *Kosakonia* H182 exhibited a lower inhibition activity (10.0%) on the strain CG Avocado 23-703. AvoGreen and *Enterobacter* H222 showed inhibition activities above 30.4% against the two fungal strains, and *Kosakonia* H191 a 34.9% inhibition of hyphal growth of strain CG Aust Mango 3-3.

Whatever the fungal strain assayed, *Enterobacter* H222 was clearly the most efficient.

### 3.3. In Vivo Activity

An *in vivo* assay was performed on a local variety of mango (cv. José), purposely injured and inoculated with fugal conidia. Selected bacteria, *Enterobacter* H222 and *Leuconostoc* H255, were added 24 h later and mangoes were stored at 20°C.

<table><caption>**Table 4.** Inhibition activity (%) of *C. gloeosporioides* CG Aust Mango 3-3 and CG Avocado 23-703 hyphal growth. Different letter in a column indicates a significant difference (p-value < 0.001).</caption><thead><tr><th rowspan="2">Isolate</th><th colspan="3">CG Aust Mango 3-3</th><th colspan="2">CG Avocado 23-703</th></tr><tr><th colspan="5">Hyphal growth, %<fnref n="1" /></th></tr></thead><tbody><tr><td><i>Enterobacter</i> H222</td><td>43.6</td><td>±</td><td>12.3 c</td><td>30.5</td><td>±</td><td>0.8 c</td></tr><tr><td><i>Enterobacter</i> H232</td><td>23.8</td><td>±</td><td>4.3 ab</td><td>23.2</td><td>±</td><td>1.4 bc</td></tr><tr><td><i>Kosakonia</i> H182</td><td>24.9</td><td>±</td><td>6.1 ab</td><td>10.0</td><td>±</td><td>8.1 a</td></tr><tr><td><i>Kosakonia</i> H191</td><td>34.9</td><td>±</td><td>3.9 bc</td><td>23.7</td><td>±</td><td>3.8 bc</td></tr><tr><td><i>Pantoea</i> H186</td><td>20.2</td><td>±</td><td>3.2 a</td><td>20.2</td><td>±</td><td>2.1 b</td></tr><tr><td><i>Leuconostoc</i> H255</td><td>20.3</td><td>±</td><td>2.5 a</td><td>20.3</td><td>±</td><td>2.8 b</td></tr><tr><td><i>Leuconostoc</i> H270</td><td>21.7</td><td>±</td><td>1.2 ab</td><td>19.3</td><td>±</td><td>3.0 b</td></tr><tr><td>AvoGreen</td><td>33.2</td><td>±</td><td>3.0 abc</td><td>30.4</td><td>±</td><td>3.3 c</td></tr></tbody></table>

<footnote n="1">¹mean ± standard deviation.</footnote>
Symptom diameter observed after 10 days was slightly lower for bacteria treated spots, especially with isolate H222 (21.1 mm ± 0.9 mm for H222 versus 23.4 mm ± 1.3 mm for the control condition), but the significance was low (p-value = 0.141) (Figure 3). Combination of the two isolates did not enhance the observed effect.

Conditions applied before addition of bacteria, i.e. wounding of mature fruit and incubation with *C. gloeosporioides* for 24 h, are drastic as they strongly favour fungal development and activate fruit defence mechanisms.

Moreover, the influence of temperature is a crucial parameter for microbiome, as well as the maturity level of mango [85]. *Enterobacter* H222 was isolated at 12°C but its optimal growth temperature is 27°C. *L. mesenteroides* can also grow at low temperatures [86] [87] and optimal growth temperature of isolate H255 is 30°C. It is thus likely that a different effect would have been observed with less mature fruit stored at 12°C.

Eventually, a pre-harvest application of fruit or application on unripe fruit and optimization of the bacterial population to spray on fruit surface should be considered. A more accurate identification and a careful examination of H222 isolate should be performed prior to consider any further development of bio-control product in order to assess the safety of the use.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Severity of anthracnose caused by *C. gloeosporioides* MUCL 43868 in wounded cv. José mangoes treated with H222 [*E. cloacae* complex], H255 [*L. mesenteroides*], and H222 + H255 [*E. cloacae* complex and *L. mesenteroides*] and stored 10 days at 20°C. Severity is expressed as diameter of symptom (mm) on mango.</figcaption></figure>

**4. Conclusion**

Isolation of 305 epiphytic bacteria from mango surface and identification of 26 isolates showed the presence of species previously observed on other fruit or leaves. Moreover, isolation of *Kosakonia* from carposphere was not previously described.
Fruit post-harvest biocontrol efficacy relies on the ability of biocontrol strains to survive, develop, colonize and exhibit an anti-fungal activity on fruit surface. The relationship between the efficacy and storage temperature should be more deeply investigated, as well as are combination of treatments, in order to increase *in vivo* efficacy. The safe use of isolates has to be particularly watched out.

## Acknowledgements

Part of this work was funded by a bilateral France-South Africa program (Campus France), PROTEA 33895VF. Part of this work was funded by the Cirad DP COSAQ (https://cosaq.cirad.fr/) agronomical research program funded by a grant from European Community (FEDER-working-program), the Regional Council of Réunion Island and CIRAD.

## Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

## References

[1] Statista (2017) Mango Production Worldwide. https://www.statista.com/statistics/577951/world-mango-production/

[2] Chillet, M., Minier, J., Hoarau, M. and Meile, J.-C. (2019) Potential Use of Thymol to Control Anthracnose Development in Mango. *European Journal of Plant Pathology*, **155**, 943-952. https://doi.org/10.1007/s10658-019-01825-9

[3] Simmonds, J.H. (1969) Studies in the Latent Phase of *Colletotrichum* Species Causing Rots in Tropical Fruits. *Queensland Journal of Agricultural Sciences*, **20**, 373-424.

[4] Intan Sakinah, M.A., Suzianti, I.V. and Latiffah, Z. (2014) Phenotypic and Molecular Characterization of *Colletotrichum* Species Associated with Anthracnose of Banana (*Musa* spp) in Malaysia. *Genetics and Molecular Research*, **13**, 3627-3637. https://doi.org/10.4238/2014.May.9.5

[5] Jeffries, P., Dodd, J.C., Jeger, M.J. and Plumbley, R.A. (1990) The Biology and Control of *Colletotrichum* Species on Tropical Fruit Crops. *Plant Pathology*, **39**, 343-366. https://doi.org/10.1111/j.1365-3059.1990.tb02512.x

[6] Weir, B.S.S., Johnston, P.R.R. and Damm, U. (2012) The *Colletotrichum gloeosporioides* Species Complex. *Studies in Mycology*, **73**, 115-180. https://doi.org/10.3114/sim0011

[7] Freeman, S., Katan, T. and Shabi, E. (1996) Characterization of *Colletotrichum gloeosporioides* Isolates from Avocado and Almond Fruits with Molecular and Pathogenicity Tests. *Applied and Environmental Microbiology*, **62**, 1014-1020. https://doi.org/10.1128/AEM.62.3.1014-1020.1996

[8] Sanders, G.M. and Korsten, L. (2003) Comparison of Cross Inoculation Potential of South African Avocado and Mango Isolates of *Colletotrichum gloeosporioides*. *Microbiological Research*, **158**, 143-150. https://doi.org/10.1078/0944-5013-00186

[9] Bautista-Baños, S., Sivakumar, D., Bello-Pérez, A., Villanueva-Arce, R. and Hernández-López, M. (2013) A Review of the Management Alternatives for Controlling Fungi on Papaya Fruit during the Postharvest Supply Chain. *Crop Protec-
tion, **49**, 8-20. https://doi.org/10.1016/j.cropro.2013.02.011

[10] Dickman, M.B., Patil, S.S. and Kolattukudy, P.E. (1982) Purification, Characterization and Role in Infection of an Extracellular Cutinolytic Enzyme from *Colletotrichum gloeosporioides* Penz. on *Carica papaya* L. *Physiological Plant Pathology*, **20**, 333-344, IN11-IN12, 345-347. https://doi.org/10.1016/0048-4059(82)90058-3

[11] Moraes, S.R.G., Tanaka, F.A.O. and Massola Jr., N.S. (2013) Histopathology of *Colletotrichum gloeosporioides* on Guava Fruits (*Psidium guajava* L.). *Revista Brasileira de Fruticultura*, **35**, 657-664. https://doi.org/10.1590/S0100-29452013000200039

[12] Moraes, S.R.G., Escanferla, M.E. and Massola, N.S. (2015) Prepenetration and Penetration of *Colletotrichum gloeosporioides* into Guava Fruit (*Psidium guajava* L.): Effects of Temperature, Wetness Period and Fruit Age. *Journal of Phytopathology*, **163**, 149-159. https://doi.org/10.1111/jph.12294

[13] Anaruma, N.D., Schmidt, F.L., Duarte, M.C.T., Figueira, G.M., Delarmelina, C., Benato, E.A., et al. (2010) Control of *Colletotrichum gloeosporioides* (Penz.) Sacc. in Yellow Passion Fruit Using *Cymbopogon citratus* Essential Oil. *Brazilian Journal of Microbiology*, **41**, 66-73. https://doi.org/10.1590/S1517-83822010000100012

[14] da Silva, A.C., Sales, N.L.P., de Araújo, A.V. and Caldeira Júnior, C.F. (2009) *In Viro* Effect of Plant Compounds on the Fungus *Colletotrichum gloeosporioides* Penz. Isolated from Passion Fruit. *Ciencia e Agrotecnologia*, **33**, 1853-1860. https://doi.org/10.1590/S1413-70542009000700026

[15] Nguyen, T.H.P., Säll, T., Bryngelsson, T. and Liljeroth, E. (2009) Variation among *Colletotrichum gloeosporioides* Isolates from Infected Coffee Berries at Different Locations in Vietnam. *Plant Pathology*, **58**, 898-909. https://doi.org/10.1111/j.1365-3059.2009.02085.x

[16] Phoulivong, S., Cai, L., Chen, H., McKenzie, E.H.C., Abdelsalam, K., Chukeatirote, E., et al. (2010) *Colletotrichum gloeosporioides* Is Not a Common Pathogen on Tropical Fruits. *Fungal Diversity*, **44**, 33-43. https://doi.org/10.1007/s13225-010-0046-0

[17] Sangchote, S. (1997) Postharvest Diseases of Tropical Fruits. *Proceedings of an International Workshop*, No. 80, 4-9.

[18] Chung, W.-H., Ishii, H., Nishimura, K., Fukaya, M., Yano, K. and Kajitani, Y. (2006) Fungicide Sensitivity and Phylogenetic Relationship of Anthracnose Fungi Isolated from Various Fruit Crops in Japan. *Plant Disease*, **90**, 506-512. https://doi.org/10.1094/PD-90-0506

[19] Kuo, K.-C. (2001) Sensitivity of Mango Anthracnose Pathogen, *Colletotrichum gloeosporioides*, to the Fungicide Prochloraz in Taiwan. *Proceedings of the National Science Council, Part B*, **25**, 174-179.

[20] Van Boxstael, S., Habib, I., Jacxsens, L., De Vocht, M., Baert, L., Van De Perre, E., et al. (2013) Food Safety Issues in Fresh Produce: Bacterial Pathogens, Viruses and Pesticide Residues Indicated as Major Concerns by Stakeholders in the Fresh Produce Chain. *Food Control*, **32**, 190-197. https://doi.org/10.1016/j.foodcont.2012.11.038

[21] Lin, T., Xu, X.F., Dai, D.J., Shi, H.J., Wang, H.D. and Zhang, C.Q. (2016) Differentiation in Development of Benzimidazole Resistance in *Colletotrichum gloeosporioides* Complex Populations from Strawberry and Grape Hosts. *Australasian Plant Pathology*, **45**, 241-249. https://doi.org/10.1007/s13313-016-0413-8

[22] Gupta, P.K. (2017) Chapter 37—Herbicides and Fungicides. In: Gupta, R.C., Ed., *Reproductive and Developmental Toxicology*, 2nd Edition, Academic Press, Cambridge, 657-679. https://doi.org/10.1016/B978-0-12-804239-7.00037-8
[23] European Union (2005) Regulation (EC) No. 396/2005 of the European Parliament and of the Council on Maximum Residue Levels of Pesticides in or on Food and Feed of Plant and Animal Origin and Amending Council Directive 91/414/EECText with EEA Relevance. European Union, Brussels.

[24] Droby, S., Wisniewski, M., Macarisin, D. and Wilson, C. (2009) Twenty Years of Postharvest Biocontrol Research: Is It Time for a New Paradigm? *Postharvest Biology and Technology*, **52**, 137-145.
https://doi.org/10.1016/j.postharvbio.2008.11.009

[25] Leneveu-Jenvrin, C., Charles, F., Barba, F.J. and Remize, F. (2019) Role of Biological Control Agents and Physical Treatments in Maintaining the Quality of Fresh and Minimally-Processed Fruit and Vegetables. *Critical Reviews in Food Science and Nutrition*, **60**, 2837-2855.

[26] Spadaro, D. and Droby, S. (2016) Development of Biocontrol Products for Post-harvest Diseases of Fruit: The Importance of Elucidating the Mechanisms of Action of Yeast Antagonists. *Trends in Food Science & Technology*, **47**, 39-49.
https://doi.org/10.1016/j.tifs.2015.11.003

[27] Janisiewicz, W.J. (2013) Biological Control of Postharvest Diseases: Hurdles, Successes and Prospects. *ISHS Acta Horticulturae*, **1001**, 273-284.
https://doi.org/10.17660/ActaHortic.2013.1001.31

[28] Janisiewicz, W.J. and Conway, W.S. (2010) Combining Biological Control with Physical and Chemical Treatments to Control Fruit Decay after Harvest. *Stewart Postharvest Review*, **6**, 1-6.

[29] Rungjindamai, N. (2016) Isolation and Evaluation of Biocontrol Agents in Controlling Anthracnose Disease of Mango in Thailand. *Journal of Plant Protection Research*, **56**, 306-311. https://doi.org/10.1515/jppr-2016-0034

[30] Luo, S., Wan, B., Feng, S. and Shao, Y. (2015) Biocontrol of Postharvest Anthracnose of Mango Fruit with *Debaryomyces nepalensis* and Effects on Storage Quality and Postharvest Physiology. *Journal of Food Science*, **80**, M2555-M2563.
https://doi.org/10.1111/1750-3841.13087

[31] Shao, Y.Z., Zeng, J.K., Tang, H., Zhou, Y. and Li, W. (2019) The Chemical Treatments Combined with Antagonistic Yeast Control Anthracnose and Maintain the Quality of Postharvest Mango Fruit. *Journal of Integrative Agriculture*, **18**, 1159-1169. https://doi.org/10.1016/S2095-3119(18)62128-8

[32] Tian, Y.Q., Li, W., Jiang, Z.T., Jing, M.M. and Shao, Y.Z. (2018) The Preservation Effect of *Metschnikowia pulcherrima* Yeast on Anthracnose of Postharvest Mango Fruits and the Possible Mechanism. *Food Science and Biotechnology*, **27**, 95-105.
https://doi.org/10.1007/s10068-017-0213-0

[33] Bautista-Rosales, P.U., Calderon-Santoyo, M., Servín-Villegas, R., Ochoa-Álvarez, N.A. and Ragazzo-Sánchez, J.A. (2013) Action Mechanisms of the Yeast *Meyerozyma caribbica* for the Control of the Phytopathogen *Colletotrichum gloeosporioides* in Mangoes. *Biological Control*, **65**, 293-301.
https://doi.org/10.1016/j.biocontrol.2013.03.010

[34] de los Santos-Villalobos, S., Guzmán-Ortiz, D.A., Gómez-Lim, M.A., Délano-Frier, J.P., De-Folter, S., Sánchez-García, P., et al. (2013) Potential Use of *Trichoderma asperellum* (Samuels, Liechfeldt et Nirenberg) T8a as a Biological Control Agent against Anthracnose in Mango (*Mangifera indica* L.). *Biological Control*, **64**, 37-44.
https://doi.org/10.1016/j.biocontrol.2013.03.010

[35] Vivekananthan, R., Ravi, M., Saravanakumar, D., Kumar, N., Prakasam, V. and Samiyappan, R. (2004) Microbially Induced Defense Related Proteins against Post-
harvest anthracnose Infection in Mango. *Crop Protection*, **23**, 1061-1067.
https://doi.org/10.1016/j.cropro.2004.03.014

[36] Zheng, M., Shi, J.Y., Shi, J., Wang, Q.G. and Li, Y.H. (2013) Antimicrobial Effects of Volatiles Produced by Two Antagonistic *Bacillus* Strains on the Anthracnose Pathogen in Postharvest Mangos. *Biological Control*, **65**, 200-206.
https://doi.org/10.1016/j.biocontrol.2013.02.004

[37] Alvindia, D.G. and Acda, M.A. (2015) The Antagonistic Effect and Mechanisms of *Bacillus amyloliquefaciens* DGA14 against Anthracnose in Mango cv. 'Carabao'. *Biocontrol Science and Technology*, **25**, 560-572.
https://doi.org/10.1080/09583157.2014.996738

[38] Korsten, L. and Bornman, C.H. (2004) Biological Control in Africa: Can It Provide a Sustainable Solution for Control of Fruit Diseases? *South African Journal of Botany*, **70**, 128-139. https://doi.org/10.1016/S0254-6299(15)30273-8

[39] Kefialew, Y. and Ayalew, A. (2008) Postharvest Biological Control of Anthracnose (*Colletotrichum gloeosporioides*) on Mango (*Mangifera indica*). *Postharvest Biology and Technology*, **50**, 8-11. https://doi.org/10.1016/j.postharvbio.2008.03.007

[40] Govender, V., Korsten, L. and Sivakumar, D. (2005) Semi-Commercial Evaluation of *Bacillus licheniformis* to Control Mango Postharvest Diseases in South Africa. *Postharvest Biology and Technology*, **38**, 57-65.
https://doi.org/10.1016/j.postharvbio.2005.04.005

[41] Silimela, M. and Korsten, L. (2007) Evaluation of Pre-Harvest *Bacillus licheniformis* Sprays to Control Mango Fruit Diseases. *Crop Protection*, **26**, 1474-1481.
https://doi.org/10.1016/j.cropro.2006.12.011

[42] Punja, Z.K. (1997) Comparative Efficacy of Bacteria, Fungi, and Yeasts as Biological Control Agents for Diseases of Vegetable Crops. *Canadian Journal of Plant Pathology*, **19**, 315-323. https://doi.org/10.1080/07060669709500531

[43] Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J.C., Gerds, M.L., Hammes, W.P., et al. (2012) Food Fermentations: Microorganisms with Technological Beneficial Use. *International Journal of Food Microbiology*, **154**, 87-97.
https://doi.org/10.1016/j.ijfoodmicro.2011.12.030

[44] Feder, F. (2013) Soil Map Update: Procedure and Problems Encountered for the Island of Réunion. *CATENA*, **110**, 215-224.
https://doi.org/10.1016/j.catena.2013.06.019

[45] Leneveu-Jenvrin, C., Quentin, B., Assemat, S., Hoarau, M., Meile, J.-C. and Remize, F. (2020) Changes of Quality of Minimally-Processed Pineapple (*Ananas comosus*, var. 'Queen Victoria') during Cold Storage: Fungi in the Leading Role. *Microorganisms*, **8**, 185. https://doi.org/10.3390/microorganisms8020185

[46] Shen, Y.M., Nie, J.Y., Dong, Y.F., Kuang, L.X., Li, Y.P. and Zhang, J.Y. (2018) Compositional Shifts in the Surface Fungal Communities of Apple Fruits during Cold Storage. *Postharvest Biology and Technology*, **144**, 55-62.
https://doi.org/10.1016/j.postharvbio.2018.05.005

[47] Prihastuti, H., Cai, L., Chen, H., McKenzie, E.H.C. and Hyde, K.D. (2009) Characterization of *Colletotrichum* Species Associated with Coffee Berries in Northern Thailand. *Fungal Diversity*, **39**, 89-109.

[48] Kim, C.S., Lee, C.H., Shin, J.S., Chung, Y.S. and Hyung, N.I. (1997) A Simple and Rapid Method for Isolation of High Quality Genomic DNA from Fruit Trees and Conifers Using PVP. *Nucleic Acids Research*, **25**, 1085-1086.
https://doi.org/10.1093/nar/25.5.1085

[49] Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S Ribosomal
DNA Amplification for Phylogenetic Study. *Journal of Bacteriology*, **173**, 697-703.
https://doi.org/10.1128/JB.173.2.697-703.1991

[50] Sellamuthu, P.S., Mafune, M., Sivakumar, D. and Soundy, P. (2013) Thyme Oil Vapour and Modified Atmosphere Packaging Reduce Anthracnose Incidence and Maintain Fruit Quality in Avocado. *Journal of the Science of Food and Agriculture*, **93**, 3024-3031. https://doi.org/10.1002/jsfa.6135

[51] Jackson, C.R., Randolph, K.C., Osborn, S.L. and Tyler, H.L. (2013) Culture Dependent and Independent Analysis of Bacterial Communities Associated with Commercial Salad Leaf Vegetables. *BMC Microbiology*, **13**, Article No. 274.
https://doi.org/10.1186/1471-2180-13-274

[52] Enya, J., Shinohara, H., Yoshida, S., Tsukiboshi, T., Negishi, H., Suyama, K., et al. (2007) Culturable Leaf-Associated Bacteria on Tomato Plants and Their Potential as Biological Control Agents. *Microbial Ecology*, **53**, 524-536.
https://doi.org/10.1007/s00248-006-9085-1

[53] Sajur, S.A.A., Saguir, F.M.M., de Nadra, M.C.M. and Manca de Nadra, M.C. (2007) Effect of Dominant Specie of Lactic Acid Bacteria from Tomato on Natural Microflora Development in Tomato Purée. *Food Control*, **18**, 594-600.
https://doi.org/10.1016/j.foodcont.2006.02.006

[54] Telias, A., White, J.R., Pahl, D.M., Ottesen, A.R. and Walsh, C.S. (2011) Bacterial Community Diversity and Variation in Spray Water Sources and the Tomato Fruit Surface. *BMC Microbiology*, **11**, Article No. 81.
https://doi.org/10.1186/1471-2180-11-81

[55] Jager, E.S., Wehner, F.C. and Korsten, L. (2001) Microbial Ecology of the Mango Phylloplane. *Microbial Ecology*, **42**, 201-207.
https://doi.org/10.1007/s002480000106

[56] Kecskeméti, E., Berkelmann-Löhnertz, B. and Reineke, A. (2016) Are Epiphytic Microbial Communities in the Carposphere of Ripening Grape Clusters (*Vitis vinifera* L.) Different between Conventional, Organic, and Biodynamic Grapes? *PLoS ONE*, **11**, e0160852. https://doi.org/10.1371/journal.pone.0160852

[57] Brady, C., Cleenwerck, I., Venter, S., Coutinho, T. and De Vos, P. (2013) Taxonomic Evaluation of the Genus *Enterobacter* Based on multilocus Sequence Analysis (MLSA): Proposal to Reclassify *E. nimipressuralis* and *E. amnigenus* into *Lelliottia* gen. nov. as *Lelliottia nimipressuralis* comb. nov. and *Lelliottia amnigena* comb. nov., Respectively, *E. gergoviae* and *E. pyrinus* into *Pluralibacter* gen. nov. as *Pluralibacter gergoviae* comb. nov. and *Pluralibacter pyrinus* comb. nov., respectively, *E. cowanii*, *E. radicincitans*, *E. oryzae* and *E. arachidis* into *Kosakonia* gen. nov. as *Kosakonia cowanii* comb. nov., *Kosakonia radicincitans* comb. nov., *Kosakonia oryzae* comb. nov. and *Kosakonia arachidis* comb. nov., respectively, and *E. turi-censis*, *E. helveticus* and *E. pulveris* into *Cronobacter* as *Cronobacter zurichensis* nom. nov., *Cronobacter helveticus* comb. nov. and *Cronobacter pulveris* comb. nov., Respectively, and Emended Description of the Genera *Enterobacter* and *Cronobacter*. *Systematic and Applied Microbiology*, **36**, 309-319.
https://doi.org/10.1016/j.syapm.2013.03.005

[58] Hejazi, A. and Falkiner, F.R. (1997) *Serratia marcescens*. *Journal of Medical Microbiology*, **46**, 903-912. https://doi.org/10.1099/00222615-46-11-903

[59] Robinson, C.J., Bohannan, B.J.M. and Young, V.B. (2010) From Structure to Function: The Ecology of Host-Associated Microbial Communities. *Microbiology and Molecular Biology Reviews*, **74**, 453-76. https://doi.org/10.1128/MMBR.00014-10

[60] Paauw, A., Caspers, M.P.M., Schuren, F.H.J., Leverstein-van Hall, M.A., Delétoile, A., Montijn, R.C., et al. (2008) Genomic Diversity within the *Enterobacter cloacae*
Complex. *PLoS ONE*, 3, e3018. https://doi.org/10.1371/journal.pone.0003018

[61] Davin-Regli, A., Lavigne, J.-P. and Pagès, J.-M. (2019) *Enterobacter* spp.: Update on Taxonomy, Clinical Aspects, and Emerging Antimicrobial Resistance. *Clinical Microbiology Reviews*, **32**, e00002-19. https://doi.org/10.1128/CMR.00002-19

[62] Brenner, D.J., McWhorter, A.C., Kai, A., Steigerwalt, A.G. and Farmer, J.J. (1986) *Enterobacter asburiae* sp. nov., a New Species Found in Clinical Specimens, and Reassignment of *Erwinia dissolvens* and *Erwinia nimipressuralis* to the Genus *enterobacter* as *Enterobacter dissolvens* comb. nov. and *Enterobacter nimipressuralis* comb. nov. *Journal of Clinical Microbiology*, **23**, 1114-1120.
https://doi.org/10.1128/JCM.23.6.1114-1120.1986

[63] Kosako, Y., Tamura, K., Sakazaki, R. and Miki, K. (1996) *Enterobacter kobei* sp. nov., a New Species of the Family *Enterobacteriaceae* Resembling *Enterobacter cloacae*. *Current Microbiology*, **33**, 261-265. https://doi.org/10.1007/s002849900110

[64] Kämpfer, P., McInroy, J.A. and Glaeser, S.P. (2015) *Enterobacter muelleri* sp. nov., Isolated from the Rhizosphere of *Zea mays*. *International Journal of Systematic and Evolutionary Microbiology*, **65**, 4093-4099. https://doi.org/10.1099/ijsem.0.000547

[65] Delétoile, A., Decré, D., Courant, S., Passet, V., Audo, J., Grimont, P., et al. (2009) Phylogeny and Identification of *Pantoea* Species and Typing of *Pantoea agglomerans* Strains by Multilocus Gene Sequencing. *Journal of Clinical Microbiology*, **47**, 300-310. https://doi.org/10.1128/JCM.01916-08

[66] Ait Bahadou, S., Ouijja, A., Karfach, A., Tahiri, A. and Lahlali, R. (2018) New Potential Bacterial Antagonists for the Biocontrol of Fire Blight Disease (*Erwinia amylovora*) in Morocco. *Microbial Pathogenesis*, **117**, 7-15.
https://doi.org/10.1016/j.micpath.2018.02.011

[67] Soto-Muñoz, L., Teixidó, N., Usall, J., Viñas, I. and Torres, R. (2014) Detection and Quantification by PCR Assay of the Biocontrol Agent *Pantoea agglomerans* CPA-2 on Apples. *International Journal of Food Microbiology*, **175**, 45-52.
https://doi.org/10.1016/j.ijfoodmicro.2014.01.014

[68] Trotel-Aziz, P., Couderchet, M., Biagianti, S. and Aziz, A. (2008) Characterization of New Bacterial Biocontrol Agents *Acinetobacter*, *Bacillus*, *Pantoea* and *Pseudomonas* spp. Mediating Grapevine Resistance against *Botrytis cinerea*. *Environmental and Experimental Botany*, **64**, 21-32.
https://doi.org/10.1016/j.envexpbot.2007.12.009

[69] Sadik, S., Mazouz, H., Benbouazza, A. and Achbani, E.H. (2016) Ecology of *Pantoea agglomerans* 2066-7 Strain: A Biological Control of Bacteria Onion Diseases. *Journal of Microbiology, Biotechnology and Food Sciences*, **5**, 612-616.
https://doi.org/10.15414/jmbfs.2016.5.6.612-616

[70] Smits, T.H.M., Duffy, B., Blom, J., Ishimaru, C.A. and Stockwell, V.O. (2019) Pantocin A, a Peptide-Derived Antibiotic Involved in Biological Control by Plant-Associated *Pantoea* Species. *Archives of Microbiology*, **201**, 713-722.
https://doi.org/10.1007/s00203-019-01647-7

[71] Jiang, L.M., Jeong, J.C., Lee, J.-S., Park, J.M., Yang, J.-W., Lee, M.H., et al. (2019) Potential of *Pantoea dispersa* as an Effective Biocontrol Agent for Black Rot in Sweet Potato. *Scientific Reports*, **9**, Article No. 16354.
https://doi.org/10.1038/s41598-019-52804-3

[72] Nunes, C., Usall, J., Teixidó, N. and Vias, I. (2001) Biological Control of Postharvest Pear Diseases Using a Bacterium, *Pantoea agglomerans* CPA-2. *International Journal of Food Microbiology*, **70**, 53-61.
https://doi.org/10.1016/S0168-1605(01)00523-2
[73] Nunes, C., Usall, J., Teixido, N., Fons, E. and Vinas, I. (2002) Post-Harvest Biological Control by *Pantoea agglomerans* (CPA-2) on Golden Delicious Apples. *Journal of Applied Microbiology*, **92**, 247-255.
https://doi.org/10.1046/j.1365-2672.2002.01524.x

[74] Poppe, L., Vanhoutte, S. and Höfte, M. (2003) Modes of Action of *Pantoea agglomerans* CPA-2, an Antagonist of Postharvest Pathogens on Fruits. *European Journal of Plant Pathology*, **109**, 963-973.
https://doi.org/10.1023/B:EJPP.0000003747.41051.9f

[75] Teixidó, N., Usall, J., Palou, L., Asensio, A., Nunes, C. and Viñas, I. (2001) Improving Control of Green and Blue Molds of Oranges by Combining *Pantoea agglomerans* (CPA-2) and Sodium Bicarbonate. *European Journal of Plant Pathology*, **107**, 685-694.
https://doi.org/10.1023/A:1011962121067

[76] Inoue, K., Sugiyama, K., Kosako, Y., Sakazaki, R. and Yamai, S. (2000) *Enterobacter cowanii* sp. nov., a New Species of the Family *Enterobacteriaceae*. *Current Microbiology*, **41**, 417-420.
https://doi.org/10.1007/s002840010160

[77] Khan, A.A.H. (2019) Plant-Bacterial Association and Their Role as Growth Promoters and Biocontrol Agents. In: Sayyed, R.Z., Ed., *Plant Growth Promoting Rhizobacteria for Sustainable Stress Management: Rhizobacteria in Biotic Stress Management*, Vol. 13, Springer, Singapore, 389-419.
https://doi.org/10.1007/978-981-13-6986-5_16

[78] Baldan, E., Nigris, S., Populin, F., Zottini, M., Squartini, A. and Baldan, B. (2014) Identification of Culturable Bacterial Endophyte Community Isolated from Tissues of *Vitis vinifera* "Glera". *Plant Biosystems*, **148**, 508-516.
https://doi.org/10.1080/11263504.2014.916364

[79] de Pereira, G.V.M., Magalhães, K.T., Lorenzetii, E.R., Souza, T.P. and Schwan, R.F. (2012) A Multiphasic Approach for the Identification of Endophytic Bacterial in Strawberry Fruit and Their Potential for Plant Growth Promotion. *Microbial Ecology*, **63**, 405-417.
https://doi.org/10.1007/s00248-011-9919-3

[80] Suhandono, S., Kusumawardhani, M.K. and Aditiawati, P. (2016) Isolation and Molecular Identification of Endophytic Bacteria from Rambutan Fruits (*Nephelium lappaceum* L.) Cultivar Binjai. *HAYATI Journal of Biosciences*, **23**, 39-44.
https://doi.org/10.1016/j.hjb.2016.01.005

[81] Crowley, S., Mahony, J. and van Sinderen, D. (2013) Current Perspectives on Anti-fungal Lactic Acid Bacteria as Natural Bio-Preservatives. *Trends in Food Science & Technology*, **33**, 93-109.
https://doi.org/10.1016/j.tifs.2013.07.004

[82] Trias, R., Badosa, E., Montesinos, E. and Bañeras, L. (2008) Bioprotective *Leuconostoc* Strains against *Listeria monocytogenes* in Fresh Fruits and Vegetables. *International Journal of Food Microbiology*, **127**, 91-98.
https://doi.org/10.1016/j.ijfoodmicro.2008.06.011

[83] Tabanelli, G., Pasini, F., Riciputi, Y., Vannini, L., Gozzi, G., Balestra, F., et al. (2018) Fermented Nut-Based Vegan Food: Characterization of a Home Made Product and Scale-Up to an Industrial Pilot-Scale Production. *Journal of Food Science*, **83**, 711-722.
https://doi.org/10.1111/1750-3841.14036

[84] Swain, M.R., Anandharaj, M., Ray, R.C. and Parveen Rani, R. (2014) Fermented Fruits and Vegetables of Asia: A Potential Source of Probiotics. *Biotechnology Research International*, **2014**, Article ID: 250424.
https://doi.org/10.1155/2014/250424

[85] Diskin, S., Feygenberg, O., Maurer, D., Droby, S., Prusky, D. and Alkan, N. (2017) Microbiome Alterations Are Correlated with Occurrence of Postharvest Stem-End Rot in Mango Fruit. *Phytobiomes Journal*, **1**, 117-127.
https://doi.org/10.1094/PBIOMES-05-17-0022-R

[86] Fessard, A. and Remize, F. (2019) Genetic and Technological Characterization of Lactic Acid Bacteria Isolated from Tropically Grown Fruits and Vegetables. *International Journal of Food Microbiology, **301*, 61-72.
https://doi.org/10.1016/j.ijfoodmicro.2019.05.003

[87] Vos, P., Garrity, G., Jones, D., Krieg, N. and Ludwig, W. (2011) Bergey's Manual of Systematic Bacteriology: The Firmicutes. Vol. 3, Springer-Verlag, New York.