1. Introduction
Tropical and subtropical crops, like mango (
Mangifera indica L.), avocado (
Persea americana Mill.), dragon fruit (
Hylocereus undatus (Haw.) Britton & Rose), passion fruit (
Passiflora edulis Sims) and litchi (
Litchi chinensis Sonn.), are becoming more popular and are rapidly expanding in southern Italy (Sicily; Calabria; Apulia; and, lately, also Sardinia). In Sicily (southern Italy), mango crops play a leading role both for export and local consumption, finding the optimal conditions along the northern coast of the island between the provinces of Messina and Palermo until an altitude of 50–80 m above sea level. The quality and marketability of mango fruits are highly correlated with different factors, such as the maturity stage [
1,
2], harvesting method [
3], postharvest treatment, packaging, handling procedures, and mode of transport [
4]. Moreover, the susceptibility of mango fruits to some fungal diseases increases after harvesting and prolonged storage as a result of physiological changes in the fruit promoting pathogen development [
5].
The major fungal diseases of mango are caused by
Colletotrichum spp. [
6,
7,
8,
9] and Botryosphaeriaceae species [
10,
11]. Botryosphaeriaceae species are responsible for aerial symptoms, such as woody cankers, shoot blight, and dieback, as is also the case for other fruit tree hosts [
12], and, above all, stem-end rot or fruit rot worldwide. Fruit symptoms can be observed on immature fruit still attached to a plant or during storage at a packing house and in transit [
13,
14]. Many species have been reported in mango fruits in China, Malaysia, Brazil, India and Australia such as
L. theobromae,
L. crassispora,
L. egyptiacae,
L. hormozganensis,
L. iraniensis,
L. pseudotheobromae,
L. brasiliense and
L. mahajangana,
N. parvum and
N. ribis,
Fusicoccum aesculin,
Pseudofusicoccum violaceum,
P. adansoniae,
P. ardesiacum,
P. kimberleyense,
Neoscytalidium dimidiatum and
N. novaehollandiae,
Botryosphaeria scharifii,
B. dothidea and
B. ramosa,
Dothiorella dominicana, and
D. mangiferae [
11,
15,
16,
17,
18,
19].
In Italy, fungal species belonging to Botryosphaeriaceae represent the main threat for mango production. Numerous surveys conducted between 2010 and 2021 showed a decline and dieback in young plants and the blight of seedlings in nurseries caused by Botryosphaeriaceae [
20,
21]. However, mango fruit symptoms caused by this family have not been reported so far in Italy. Thus, a deep investigation is needed to ascertain the spread of Botryosphaeriaceae infections in fruit and to identify the main associated fungal species.
Many studies have reported some chemical methods to manage Botryosphaeriaceae diseases in fruit [
22,
23,
24,
25,
26,
27]. However, the lack of authorized chemical compounds on mango in Italy as well as the requirement to reduce the applications of synthetic compounds according to the European legislation has led us to evaluate eco-friendly alternative control strategies in organic agriculture. Biological control of fruit rot using microbial antagonists is one of the most promising alternatives. However, so far, a few studies have only reported the effects of antagonistic microorganisms, such as
Bacillus spp. and yeasts [
28,
29,
30], to control stem-end rots and decays caused by
N. parvum.
Considering the increased spread of mango fruit rot caused by Botryosphaeriaceae species observed in Sicily in the last two years and the limited data available about their biological control, the aims of this study were as follows: (i) to detect, within the Botryosphaeriaceae family, which species are mainly involved as causal agents of fruit rot in different mango fruits in the main representative Italian area of production (eastern Sicily, province of Catania) using morphological characteristics and TUB sequence data; (ii) to preliminarily evaluate the virulence of a representative subset of N. parvum isolates in mango fruit; (iii) to assess the performance of few biological products and potential antagonistic wild yeasts in controlling mango fruit rot caused by both the most and the least virulent isolates of N. parvum under postharvest conditions.
2. Materials and Methods
2.1. Field Sampling and Isolation
During 2020, many field surveys were conducted in four mango orchards located in eastern Sicily (province of Catania, Italy). Different mango fruits showing fruit rot were sampled from at least 10 plants of ‘Glenn’, ‘Kent’, ‘Irwin’, ‘Palmer’, ‘Brokaw 2’, and ‘Gomera 3’ accessions and were transferred to the laboratory of Plant Pathology at the Department of Agriculture, Food and Environment of the University of Catania for pathogen isolation.
To this aim, about 1000 small pieces (about 5 × 5 mm), taken from the margin of symptomatic fruit, were superficially sterilized for 60 s with 1.2% sodium hypochlorite (NaClO), rinsed in sterile deionized water (SDW), dried, and plated on potato dextrose agar (PDA, Lickson, Vicari, Italia) amended with 100 mg L−1 of streptomycin sulphate (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the plates were incubated in the dark at 25 ± 1 °C for seven days. Subsequently, mycelial plugs from the actively growing margins of young colonies were transferred onto new PDA dishes to create pure cultures. To obtain monoconidial cultures, single-hyphal or terminal tips were selected from resulting colonies and transferred into new PDA plates. A total of 220 fungal isolates were collected from different mango accessions. Among these, 41 isolates representative of each accession and orchard were selected on the basis of morpho-biometric features and were stored in the collection of the above-mentioned department before performing further analyses.
2.2. DNA Extraction and PCR
The genomic DNA of the representative subset of 41 isolates was extracted using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) after they were grown on PDA for seven days. Primers Bt2a and Bt2b [
31] and ITS5 and ITS4 [
32] were used to amplify up the partial beta tubulin locus (
tub2) and internal transcriber spacer region (ITS). According to the manufacturer’s instructions, each amplification was performed in a final volume of 25 μL using One Taq™ 2× Master Mix with Standard Buffer (BioLabs, Ipswich, MA, USA) guidelines for using an AG 22371 Eppendorf Mastercycler (Fisher Scientific, Hampton, NH, USA). The PCR cycle was as follows: 30 s at 94 °C for the first 30 cycles, 30 s at 94 °C for 35 cycles, 52 °C (tub2) for 1 min, 1 min at 68 °C, and 5 min at 68 °C. The PCR result was seen on 1% agarose gels at 90 V for 40 min, followed by purification, GelRed™ staining, and sequencing by Macrogen Inc. (Seoul, Republic of Korea). Sequences were visualized and edited using the software MEGA 11: Molecular Evolutionary Genetics Analysis across computing platforms [
33]. A BLASTn search of TUB locus was conducted in NCBI to determine the closest relatives.
2.3. Pathogenicity Tests
Pathogenicity proofs were carried out with one representative isolate of N. parvum and one of B. dothidea on three ripe and three unripe mango fruits. Healthy fruits were wounded at two points with a sterile micro-needle, and a mycelial plug with a diameter of 6 mm from a 10-day-old culture of each isolate was placed on each wound of the fruits. The control consisted of wounded and inoculated fruits with PDA plugs. Successively, inoculated mango fruits were placed in a growth chamber at 25 °C and 80% relative humidity. Disease incidence (DI) and symptom severity (SS) were evaluated 3 days after inoculation (dai). The DI value was referred to as the assessment of the percentage of positive inoculation points, whereas the SS was counted at each inoculation point, measuring the mean lesion length based on two perpendicular diameters.
2.4. An Assessment of the Virulence of Neofusicoccum parvum
To assess the virulence of N. parvum, forty isolates were inoculated, each on three unripe and three ripe mango fruits (i.e., 3 replicates) of cv Gomera 3, respectively. Comprehensively, two-hundred and forty fruits were employed for this experiment. The experiment was performed under the same conditions of the previous one and was repeated once.
2.5. Performance Evaluation of Biological Treatments Against Neofusicoccum parvum
Experiment I. A preliminary test to evaluate the effectiveness of biological treatments in reducing fruit rot caused by the most virulent MO1 and the least virulent MF36 isolate was performed. A total of nine products were tested on mango fruit cv Gomera 3. The tested products were six trade bioformulates, two antagonistic yeasts and a chemical standard. The yeast strains used herein belong to the Di3A (Department of Agriculture, Food and Environment, University of Catania, Italy) collection, and they were previously identified as
Wickerhamomyces anomalus WA-2 and
Pichia kluyveri PK-3 by sequencing the D1/D2 region of the 26S rRNA gene [
34]. Moreover, the selected strains have been shown to exhibit the killer phenotype and have a wide spectrum of both cellular and extracellular enzyme activities [
35]. Each yeast strain was routinely maintained at 4 °C on Petri dishes containing Yeast Extract Peptone Dextrose Agar (YPDA; yeast extract, 10 g; peptone, 20 g; dextrose, 20 g; agar, 20 g (Oxoid, Basingstoke, UK) per liter of distilled H
2O) and was refreshed on the same culture medium prior to use. The scheme of this preliminary experiment was conceived to compare biological treatments with an effective chemical standard (
Table 1). The treatments were performed 24 h before or at the same time as the pathogen inoculation. Five mango fruits per treatment were used for each timing, with two points of inoculation for each fruit. The surface of the fruits was disinfected with 70% ethanol, rinsed in SDW, and air-dried. After that, all mango fruits were sprayed with 100 mL of the product solution with the dosages indicated in
Table 1. The same procedure was followed to evaluate the antagonistic activity of
W. anomalus WA-2 and
P. kluyveri PK-3. Each yeast suspension was prepared in SDW by collecting cells grown in YPD for 48 h at 25 °C and by adjusting the concentration to 10
7 cells/mL. Successively, each fruit was inoculated with two plugs (6 mm in diameter) of the mycelium of each isolate and was placed in plastic containers and was incubated in a growth chamber with a 12 h photoperiod at 25 ± 1 °C. The mean lesion length (two perpendicular axes or diameters) was measured after 3 days of incubation, and the area of the relative ellipse was calculated according to the following formula: A = (α/2) × (β/2) × π, where A is the area of ellipse, α and β are the two perpendicular axes of ellipse, and π = 3.1415… After incubation, the amount of disease reduction was also calculated and referred to the Abbott Index, AI = [(T − C)/T)] × 100, where T represents the value detected in the untreated fruits, and C represents the value detected in the treated fruits.
Experiment II. Based on previous data, an additional experiment was designed that included only biological treatments, with the exception of the trade citrus essential oil (Prev-Am Plus). Their application was performed twice, 3 days before the inoculation and at the same time of the pathogen inoculation using the same isolates of N. parvum used in experiment 1 (MO1 and MF36). This experiment was performed twice and was conducted as described above for the previous one.
2.6. Statistical Analysis
Data regarding the preliminary virulence evaluation of 40 isolates of
N. parvum and the performance of biological treatments on mango fruit cv Gomera 3 were analyzed by using the Statistica Package Software (Vers. 10; Statsoft Inc., Tulsa, OK, USA). The arithmetic means of lesions were calculated by averaging the values for all replicates of each isolate or biological treatment. Initial analyses of the disease lesions on the fruits were performed by calculating associated F and
p values to evaluate whether the effects of single factors and interactions are significant. In the post hoc analyses, the mean values of the DI and SS parameters were subsequently separated by Fisher’s least significant difference test (
p = 0.05). For all experiments performed for biological performance evaluation. The percentage of disease reduction was also accounted for [
36].
4. Discussion
In this paper, we first identified the diversity within the Botryosphaeriaceae family that is responsible for fruit decay, i.e., among a subset of
N. parvum isolates, obtained from different mango fruits in one of the most representative Italian production areas (eastern Sicily, province of Catania) by using morphological characteristics and ITS and tub2 sequence data. Although it is well known that besides
N. parvum, other different genera and species (
Lasiodiplodia theobromae,
B. dothidea,
Diaporthe spp.) are involved in infections of the above-ground parts of mango [
20,
21], our findings clearly demonstrate that
N. parvum is the most prevalent species involved in mango fruit decay. Indeed, surveys on several accessions clearly show that
N. parvum is the species that is more frequently associated with fruit rot of mango in Italy, whereas
B. dothidea has rarely been recovered. This is not surprising, since other surveys have already confirmed that
N. parvum is widely spread in the Mediterranean basin, and it is able to infect other host species [
37,
38,
39,
40,
41], including avocado [
42].
Moreover, our findings show differences in virulence within an Italian subset of 40
N. parvum isolates from mango fruits, and this behavior variability strictly depended on the tested isolate and the maturity stage of the fruits. In detail, the data show a higher susceptibility of ripe fruits. Similarly, Baskarathevan et al. [
43] and Puig et al. [
44] detected virulence variability among isolates of
N. parvum tested in pathogenicity tests on green shoots of grapevines and cacao fruit, respectively.
Determining the main species responsible for fruit infections is the first step to developing efficacy control strategies. Since fungicide application is limited in the EU by restrictive regulations, and potential biological products to be used in pre- and postharvest conditions are not yet available on the market, the application of biological products or antagonistic microorganisms could represent an attractive alternative for the sustainable management of diseases on mango commodities.
Overall, this work reported on the performance of antagonistic microorganisms and chitosan in reducing the amount of postharvest fruit rot. Serenade™ and
W. anomalus WA-2 and
P. kluyveri PK-3 were revealed to be the most effective against the fruit SER of mango, reducing the amount of decay in all experiments, whereas T34 Biocontrol, Prevatect™, Remedier™, and Amylo-X™ were the least effective. This clearly shows that pathogen virulence is a crucial factor that can affect the performance of biological products, which in turns can also depend on antagonist microorganism-targeted phytopathogenic fungus interactions. The potential of yeasts as biological control agents has been reported previously by Carvalho Castro et al. [
45], who evaluated the efficacy of yeast in pre- and postharvest conditions on cultivar Tommy Atkins fruits, which were treated with yeasts different from the ones tested here before the inoculation with
N. parvum and
L. theobromae. In a more recent paper,
Saccharomyces spp. and
P. kudriavzeviii showed good activity in controlling postharvest fruit rot of mango [
30]. Otherwise, the performance of
W. anomalus WA-2 and
P. kluyveri PK-3 against
N. parvum has not been previously reported. Both
W. anomalus and
Pichia kluyveri are generally recognized as safe (GRAS) microorganisms and are not associated with human pathogenicity. Their safety profile and potential probiotic properties [
34], combined with their metabolic versatility, supports their application in food-related environments, including postharvest protection and fermentation processes [
46,
47,
48].
The antagonistic activity of the selected yeasts is likely attributed to their killer phenotype [
34], as has been reported for other microorganisms [
6,
49,
50]. In fact, several studies have shown that yeast strains with extracellular glucanase activity can inhibit a wide range of postharvest pathogens in different fruits and through various modes of application [
35,
51]. Moreover, besides the production of glucanase enzymes, several putative direct biocontrol mechanisms, such as the production of volatile compounds, the competition for space and nutrients, and wound colonization, could also be involved [
52]. Regarding the latter, the strong colonization ability of
W. anomalus may also contribute significantly to its biocontrol efficacy. As reported by Taïbi et al. [
53],
W. anomalus was able to dominate the fruit microbiota, replacing other yeast species after 14 days of mango storage. This capacity to establish and persist on the fruit surface reinforces its role as a competitive and resilient antagonist. In our study, this characteristic likely contributed to the observed biocontrol performance. Furthermore,
W. anomalus has also been shown to activate plant defense responses [
51,
54], thus demonstrating its ability to modulate key defense-related enzymes, such as peroxidase, which plays a central role in the induction of systemic resistance. As regards antagonistic bacteria,
Pseudomonas chlororaphis PCL1606 and
Bacillus velezensis UMAF6639 have provided encouraging results for disease control in mango and avocado fruits [
55].
Based on our data, the timing of treatment applications is another crucial aspect for improving the performance of these products. When applied 3 days before pathogen inoculation, the biological products and yeasts were more effective, while when applying one day before or at the same time as the pathogen inoculation, they can be ineffective. Some authors have previously reported on the in vivo and in vitro efficacy of four species of
Trichoderma spp. against stem-end rot caused by
L. theobromae and
N. parvum on avocado fruit [
56]. However, to our knowledge, these findings are the first data on the efficacy of
Trichoderma-based trade formulates against
N. parvum causing fruit rot of mango. Further studies should be performed for a large-scale evaluation of the effectiveness of these biological products against natural
N. parvum infections in field conditions and following postharvest storage conditions.