Effect of Epiphytic Bacteria from Citrus against Green Mold Post-Harvest Diseases of Citrus

Abstract

This study investigates the potential of epiphytic bacteria isolated from citrus fruits to control green mold post-harvest disease caused by Penicillium digitatum in Thompson Navel sweet oranges in the north of Iran. Twenty-three epiphytic bacteria were isolated and screened in vitro against P. digitatum, and 13 isolates with antagonistic activity were selected for further studies. Isolates B15 (Bacillus sp.), P17 (Pseudomonas sp.), and S13 (Streptomyces sp.) exhibited the most effective inhibitory effects on P. digitatum in vitro and in vivo. Among these, B15 showed the highest percentage of mycelium growth reduction and was further identified as B. cereus by 16S rRNA sequence analysis. Metabolic analysis of Bacillus sp. isolate B15 extract revealed the presence of the inhibiting antifungal substance Iturin A. The result showed that the application of epiphytic B. cereus decreased the percentage of green mold post-harvest diseases in orange fruit. This indicates their potential as an environmentally friendly alternative to chemical post-harvest control of orange diseases caused by P. digitatum.

1. Introduction

Citrus fruits are a highly popular fruit crop that is cultivated extensively around the world. They are grown in over 140 countries, and in 2020, the global production of citrus fruits will amount to approximately 158 million tons [1]. In 2016, Iran produced 4.1 million tons of citrus, making it the sixth biggest citrus producer in the world and contributing to 3.3% of the global citrus production [2]. The Thompson Navel sweet orange is grown in the northern regions of Iran [3]. During the transportation and storage of harvested fruits and vegetables, post-harvest diseases can cause significant damage [4]. Post-harvest losses, resulting from diseases and metabolic disorders in fruits, are a significant problem, leading to the waste of up to 30–50% of total production. Therefore, it is crucial to focus on preserving the quality and prolonging the shelf life of harvested produce through post-harvest technological innovations. This can be achieved through proper handling, treatment, storage, and transportation of the produce [5]. Postharvest disease caused by Penicillium is a major concern for the citrus fruit industry, leading to significant losses, ranging from 10% to as high as 90% in severe cases [6]. Among various postharvest pathogenic fungi, P. digitatum is widely recognized as responsible for causing up to 90% of the economic losses in agriculture, making it a significant concern [7].

Penicillium digitatum (green mold) and Penicillium italicum (blue mold) are significant citrus fruit post-harvest diseases with global economic impacts. These diseases are primarily managed using synthetic fungicides, such as imazalil, fludioxonil, pyrimethanil, and thiabendazole. However, the toxic nature of these chemicals has led to limitations on their prolonged and excessive use. As a result, there is a shift towards safer and more environmentally friendly alternatives for post-harvest citrus disease control [8] Among the fungal agents of post-harvest diseases that can affect citrus fruit, the damage caused by P. digitatum green mold is the most severe. This necrotic fungus enters the fruit through wounds and has a brief life cycle of three to five days. Despite its short lifespan, the fungus produces a vast number of conidial spores, which can start a new life cycle and cause further damage [9]. P. digitatum can enter and contaminate fruits through openings caused by various environmental factors, such as wind, hail, and insects, as well as during harvesting, transportation, and subsequent handling procedures [10].

The utilization of bacterial antagonistic strains is gaining popularity worldwide as an effective biological alternative. Extensive research has been conducted over the past 30 years on the use of microbial biocontrol agents to combat post-harvest disease, leading to the development of various biocontrol products that have been successfully commercialized [11]. In recent years, biocontrol strategies that reduce the reliance on chemical pesticides have gained significant popularity, leading to extensive research in this field. When compared to synthetic fungicides, compounds sourced from biological origins are generally less harmful to humans and more environmentally friendly. These qualities make them a highly appealing substitute for conventional chemical pesticides [12]. Utilizing natural epiphytic antagonistic microflora and exogenous microbes with antagonistic activity is an effective approach to mitigate postharvest damage caused by microbial pathogens [13]. Microbial antagonists, which are predominantly found on the surfaces of fruits and vegetables, are widely distributed, and naturally occurring. Kloeckera apiculata and Bacillus subtilis are antagonistic bacteria isolated from citrus roots and potatoes, respectively, and they have demonstrated their efficacy as biocontrol agents in effectively managing postharvest diseases [14,15]. Several bacterial strains have proven useful in controlling post-harvest pathogens through several modes of action. However, despite their efficacy, there is limited knowledge about the microbial communities present in harvested produce and their potential for use in post-harvest biocontrol research [12]. There are specific factors and mechanisms that influence the colonization and distribution of microbes on fruit surfaces. Understanding these can help us better comprehend the dynamics of fruit-associated microbial communities and their implications for fruit health. However, the complexity of microbiota makes it challenging to develop a consistently balanced structure and biocontrol effect. To predict potential microbial combinations for biocontrol against post-harvest disease by exploiting the structure of the epiphytic microbiota associated with healthy citrus fruit, more information on the microbiome is required [16].

This research aimed to collect native bacteria from Thompson Navel oranges and evaluate their ability to inhibit P. digitatum in a dual-culture antagonistic test, as well as investigate their potential to prevent fruit decay by green mold in vivo. Additionally, the identification of effective bacterial isolates at the level of phenotypic traits and PCR and bioactive compounds was studied.

2. Materials and Methods

2.1. A Sampling of Citrus Fruit

In January, fruit sampling was carried out in the northern region of Iran from two different markets. The oranges obtained from these markets were carefully selected for the study, as they were confirmed to be free of any chemical treatment. For this study, 10 fully grown Thompson Navel oranges that displayed no apparent signs of damage were chosen based on their consistent size and color. To obtain the epiphytic microbiota residing on the peel of the fruit, they were picked using sterile gloves and then enveloped in a specialized citrus polyurethane (PU) film. The fruits were then transported to the laboratory without delay for further analysis.

2.2. Collection of Epiphytic Bacteria on the Peel

The fruits were cleaned using sterilized water, and then 2 g of the surface from each sample were separated. These samples were ground in 20 mL of 0.05 M phosphate buffer (pH 6.5) by sterile thistle mortar. One hundred μL of each sample was transferred to a Petri dish containing Nutrient Agar medium (NA) (Merck, Darmstadt, Germany) supplemented with the 100 µg/mL antibiotic ampicillin and incubated at 28 °C. Single colonies were re-streaked on new NA plates at least five times to obtain a pure culture.

2.3. Fungal Pathogens

The isolate of Penicillium digitatum that was used in this investigation was obtained from the collection of fungal cultures at the Pathology Laboratory, situated in the Golestan Agricultural and Natural Resources Research and Education Center, Iran. This isolate was grown on Potato Dextrose Agar medium (PDA) (Merck, Darmstadt, Germany) and regularly transferred onto the surfaces of healthy citrus fruits (Thompson Navel oranges) that had been disinfected with 2% sodium hypochlorite to preserve their pathogenic properties. Following this, the strains were re-isolated to maintain their pathogenicity.

2.4. In Vitro Screening of Isolates

To assess the ability of isolated bacteria to inhibit the growth of P. digitatum, in vitro antagonism assays were conducted using the dual-culture method. A straight line of each bacterium was applied through the center of a 9 cm Petri plate containing PDA medium. A mycelium plug measuring 5 mm in diameter of P. digitatum was then placed around 2.5 cm from each side of the strain being tested. The plates were then incubated at a temperature of 25 °C for 7 days. The control petri plate was inoculated with green mold (P. digitatum). The percent inhibition of mycelium growth in comparison to the control was used to determine the antagonistic activity. Antifungal activity was computed using the following formula: I (%) = (R₁ − R₂)/R₁ × 100, where I represents the percentage of growth inhibition and R₁ is the mean growth of P. digitatum as a control. R₂ is the colony area of P. digitatum that has grown in the presence of isolated bacteria. Each chosen isolate was tested three times, with three replicates for every experiment [17].

2.5. Effect of Isolated Bacteria on Orange Fruits

The most efficient bacteria isolates, which were chosen based on their ability to inhibit the mycelial growth of P. digitatum in in vitro conditions, were also evaluated in vivo for their ability to reduce the severity of green mold disease. The inhibitory effect of the isolates was examined on citrus fruits that were infected with P. digitatum through wounds, according to the protocols described by [18]. The citrus fruit was immersed in a 2% sodium hypochlorite solution for 2 min and left to dry in the air. Next, the equatorial area of the fruit was disinfected with 75% alcohol. Using a needle, two holes were created on opposite sides of the equator, each measuring 3 mm in depth and 3 mm in width. This process was repeated three times for each treatment. For each wound, 30 µL of a cell suspension of the antagonist bacteria was applied at a concentration of 1 × 10⁸ CFU/mL. After 24 h, 20 µL of an aqueous suspension of P. digitatum (containing 10⁵ conidia/mL) was added to each wound. In the case of the control treatments, the wounds were treated only with 30 µL of sterile distilled water. After air-drying, the fruits were kept in a clear plastic bag that had been sterilized in an autoclave and stored at a temperature of 20 °C for a week. The disease reduction (DR) was recorded by calculation according to the formula provided by [19]:

DR (%) = (a − b)/a × 100

DR: the percentage of disease reduction, a: the mycelium growth diameter in the control fruit (cm), and b: the mycelium growth diameter in treated fruits (cm) from the place of inoculation.

2.6. Morphological and Biochemical Characterization

The macroscopic properties of the isolated bacterial colonies were evaluated using different criteria: colony morphology and gram staining were used to identify microscopic characteristics. Heat-fixing bacterial smear preparations were used to test endospore production. Biochemical tests, such as fluorescent pigmentation on Kings B agar, aerobic growth, positive reactions to oxidase and catalase tests, and negative hypersensitive reactions (HR) on tobacco and potato soft rot tests, were performed.

2.7. Molecular Identification of Effectively Isolated Bacteria

Total DNA from three bacterial isolates was prepared using a DNA extraction kit (SINA GEN Extraction Kit: Cat. No. DN8115C). The extracted DNA was used as a template for PCR amplification of the 16S rRNA gene using primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1390R (5′-GACGGGCGGTGTGTACAA-3′). PCR products were sent for sequencing to Macrogen Inc. company (Seoul, Republic of Korea) (www.macrogen.com. The sequence blast of these three bacteria on the NCBI website determined the level of their homology in comparison with the sequences deposited in GenBank (http://www.ncbi.nlm.nih.gov/BLAST).

2.8. Antifungal Activity of Isolated Bacteria Secondary Metabolites

The effective isolate B15 that exhibited the most antifungal properties was cultured in 250 mL of nutrient broth in a flask and incubated for one week at 32 °C in a shaking incubator (Jaltajhizco, GTSL90 series, Iran) with a speed of 100 rpm/min in Iran Islamic Azad university plant pathology lab. Then, the culture media were centrifuged in a refrigerated centrifuge (Hettich, Universal 320/320R, Germany) at 8000× g and 5 °C for 25 min. The resulting supernatant was separated, and its pH level was adjusted to 2 by adding hydrochloric acid. The suspension was centrifuged again at 12,000× g for 15 min at 4 °C in a refrigerated centrifuge, and the residual sediment containing antifungal metabolites was dissolved in 2–3 mL of methanol and centrifuged at 20,000× g for 10 min at 4 °C. The supernatant liquid contained antifungal metabolic products from methanolic extract [20].

2.9. Analysis of Antifungal Metabolites by HPLC

Twenty µL of the methanolic extract of the selected strain was injected into HPLC (Agilent Technologies 1200 series, USA) with a C18 column (250 mm× 4 mm) as the stationary phase. Acetonitrile: water (70:30) at 1 mL/min flow rate was used as the mobile phase. mL HPLC analysis was carried out at 240 nm wavelength, which was monitored for peak absorption using a photodiode array. Under standardized conditions, 20 microliters of sample and standard Iturin A were injected into HPLC. The detector response was assessed by means of peak regions after each run was performed three times [20,21]. Iturin A, a powerful lipopeptide with antifungal properties, is primarily synthesized by Bacillus sp. It consists of a cyclic heptapeptide and a β-amino fatty acid chain, ranging from 14 to 17 carbons [22]. In order to determine the type of antifungal metabolite, the results of the bacterial extract were compared with the standard Iturin A chromatogram (Sigma company).

2.10. Statistical Analysis

The results were statistically analyzed using the statistical program. All data were analyzed using analysis of variance (ANOVA), and the means were subjected to Duncan’s test (p < 0.05) was considered significant with three replications for each treatment.

3. Results

3.1. Screening of Bacteria for In Vitro Antagonistic Activity

Twenty-three bacteria were isolated from the fruit of Thompson Navel orange from the north of Iran and evaluated for their ability to inhibit P. digitatum isolate. Only 13 isolates showed significant antifungal activity, and the efficacy varied.

All 13 isolates from 23 bacteria which separated from the oranges peel have demonstrated antagonistic properties against P. digitatum in the dual culture test, and most of them showed significant differences in their effect on the percentage of inhibition growth of P. digitatum (

Table 1. In vitro, the percentage reduction of Penicillium digitatum mycelium growth by the selected bacteria.

Isolates	% Reduction of P. digitatum Mycelium Growth
B11	42 ± 1 f
B2	30 ± 0 h
B3	30 ± 0 h
B14	54 ± 0 e
B15	89 ± 1 a
B6	30 ± 1 h
S13	68 ± 0 c
S23	11 ± 1 j
S19	24 ± 0 i
S18	36 ± 1 g
P1	60 ± 1 d
P10	7 ± 1 k
P17	70 ± 0 b

According to Duncan’s multiple range test (p < 0.05), means (±SE) followed by various letters (a–k) are significantly different.

). There were no significant differences between B2, B3, and B6. Three isolates (B15, P17, and S13) were the most effective in inhibiting the growth of P. digitatum. The highest antifungal activity was shown by the isolated B15 (Bacillus sp.), which inhibited 89% of the mycelium growth of P. digitatum, and P10 showed the lowest inhibition on P. digitatum growth (7%).

3.2. Bioassay Effect of Isolated Bacteria on Orange Fruits

In this study, 13 bacterial isolates were tested for their ability to inhibit the growth of P. digitatum, the fungus responsible for green mold disease in citrus fruits. The disease reduction (DR) was recorded after one week, and the results showed that some isolates, such as B15 (Bacillus sp.), P17 (Pseudomonas sp.), and S13 (Streptomyces sp.), were highly effective in reducing the severity of the disease on fruits and most protective candidates. However, the S23 and P10 did not have an obvious inhibition effect on citrus green mold. The highest level of antagonistic activities (81%) was observed by Bacillus spp., particularly Bacillus sp. B15. The graph shows that selected epiphytic bacteria can significantly reduce disease incidence on orange fruit. Columns with different lowercase letters show that there are significant differences in the effect of the epiphytic isolates on inhibiting the growth of P. digitatum. The letter “a “ stands for the highest effect, and the letter “ j “for the lowest effect. The same lowercase letters for isolates B2, B3, and B6 show that there is no significant difference in their efficacy (Figure 1). The inhibitory effect of three effective isolates B15 (Bacillus sp.), P17(Pseudomonas sp.), and S13 (Streptomyces sp.) on P. digitatum growth of fruit is shown in Figure 2.

3.3. Phenotypic Characteristics and Pathogenicity of Bacteria Antagonists

Based on certain physiological and biochemical characteristics such as Gram reaction, fluorescent pigmentation on Kings B agar, cell morphology, heat test for endospore formation, aerobic growth, positive reaction to oxidase and catalase tests, and negative for hypersensitive reaction (HR) on tobacco and potato soft rot test, the isolates were tentatively identified in the genera Bacillus, Streptomyces, and Pseudomonas, and six isolates belonged to Bacillus sp., four isolates belonged to Streptomyces sp., and three isolates belonged to Pseudomonas sp. (

Table 2. Biochemical characteristics of the isolated bacteria from orange.

Characteristic	Bacillus (B11, B2, B3, B14, B15, B6)	Streptomyces (S13, S23, S19, S18)	Pseudomonas (P1, P10, P17)
Gram reaction	+	+	−
Fluorescent pigmentation on KB agar	Absent	Absent	Present
Cell morphology	Rod-shaped	Filamentous	Rod-shaped
Heat test for endospore formation	+	−	−
Aerobic growth	Yes	Yes	Yes
Oxidase test	+	−	+
Catalase test	+	+	+
HR on tobacco test	−	−	−
Potato soft rot test	−	−	−

).

3.4. Molecular Identification of Three Antagonistic Bacteria

The three selected bacteria produced a single sharp 1500 bp fragment. The PCR products were sent to Macrogen Inc. for sequencing. Sequencing was done in both directions of sense and antisense using 27F and 1390R primers. The molecular identification of the 16S rDNA sequence of Bacillus sp. (B15) was confirmed. The size of the PCR products, Bacillus sp. B15, Pseudomonas sp. P17, and Streptomyces sp. S13 isolates, measured 1500 bp with a ladder of 1 kb (Figure 3).

3.5. Determination of Antifungal Metabolites of B. cereus by Using HPLC

Antifungal metabolites of Bacillus cereus were analyzed, which was found the most effective isolated bacteria between three selected bacteria. Bacillus cereus produces a cyclic lipopeptide known as Iturin A, which exhibits potent antifungal activity against a wide range of fungi. After the purification of the effective isolate Bacillus cereus (B15), a new culture of the bacteria was prepared in the growth medium and then incubated in a shaking incubator at 30 °C for 72 h. The sample was extracted with methanol and injected into an HPLC system, and the results were compared with standard chromatograms.

The chromatogram of the Bacillus cereus (B15) isolate was compared with the standard chromatogram of Iturin A at a concentration of 1 mg/mL. The first peak appeared at 1.585 retention time, and the area under the curve was 53763.26 mAU, which is probably related to the antifungal compound Iturin A. The retention times of the peaks of B. cereus (B15) were compared with the selected standards of Iturin A, and it was determined that most of the unknown peaks had corresponding peaks in the standards, and the peaks were overlapping.

To identify the presence and abundance of these compounds in the sample, their retention times were compared with those of similar peaks in the standard chromatogram. Comparing the retention times of the peaks obtained from B. cereus extract with the standard sample emphasized the presence of the same compounds, Iturin A in isolated B. cereus confirms its antifungal activity (

Table 3. Comparison of the retention time range of similar peaks in the HPLC chromatogram of B. cereus (B15) extract with the standard.

Peak	Ret Time [min]	Area (mAU)	Height [mAU]
Bacillus cereus	1.585	26.53763	5.82007
Iturin A	1.412	26.89453	5.9432

).

4. Discussion

Screening of bacteria for in vitro antagonistic activity is an important step in identifying potential biocontrol agents for plant pathogens. In this study, only 13 out of 23 isolates from the fruit of Thompson Navel oranges demonstrated significant antifungal activity and inhibited the growth of P. digitatum in the dual culture test. These findings suggest that the isolated bacteria, particularly B15, P17, and S13, have the potential to be developed as biocontrol agents for the management of postharvest decay in citrus fruits. However, further studies are needed to fully identify the isolated strains and to evaluate their efficacy under field conditions.

Previously, other reports have shown that Bacillus amyloliquefaciens, B. pumilus, and B. subtilis were isolated from the peel of citrus and exhibited antifungal activity against P. digitatum and P. italicum by reducing the growth of mycelium [23]. The B. subtilis SK1-2 strain has been presented as a successful antagonist for the biocontrol of post-harvest rot in kiwifruit. It exhibited significant antagonistic activity against Botryosphaeria dothidea, Diaporthe actinidiae, and Botrytis cinerea in vitro cultures [24].

The B15 (Bacillus sp.) isolate selected in this study showed a high in vitro inhibitory activity on the mycelium growth of P. digitatum and proved to be effective in preventing the Penicillium green mold of orange. These results are according to previous research that confirms the successful use of Bacillus spp. in the management of numerous postharvest diseases of diverse fruits [25].

In a previous study, citrus treatment with several bacterial isolates (B. subtilis, B. pumilus, B. cereus, B. megaterium, and Agrobacterium radiobacter) was most efficient in preventing postharvest citrus fruit disease caused by P. digitatum [26]. Bacillus subtilis exhibits both a direct and indirect biocontrol mechanism to suppress disease caused by pathogens. The direct mechanism includes the synthesis of many secondary metabolites, hormones, cell wall-degrading enzymes, and antioxidants that assist the plant in its defense against pathogen attack [27].

A study suggested that the application of epiphytic Pseudomonas fluorescence can be an effective strategy for reducing the postharvest decay of tomatoes at room temperature [28]. A published study reported that the application of Streptomyces griseus to strawberries significantly reduced the incidence of gray mold post-harvest diseases caused by Botrytis cinerea [29].

These findings are consistent with previous studies that have demonstrated the efficacy of bacterial isolates in controlling fruit postharvest diseases. Bacillus sp., Pseudomonas sp., and Streptomyces sp. were most efficient in preventing postharvest fruit disease.

The isolated Bacillus B15 as the most effective bacteria had round colonies on NA and positive Gram staining. The indole production of Bacillus B15 and its oxidase test were negative, and these biochemical properties followed the properties of B. cereus, described by [30].

The production of cyclic lipopeptides by Bacillus species has gained significant attention in recent years due to their potent antifungal activities and their potential as biological control agents against fungal plant diseases. In this study, Bacillus cereus (B15) was found to produce Iturin A, a cyclic heptapeptide with potent antifungal activity against P. digitatum.

Several Bacillus strains contain these lipopeptides, which have been commercialized as biological control agents against fungal plant diseases and plant growth stimulants. The Iturin A compounds are cyclic heptapeptides with varying-length alkyl sidechains that impart surface activity and an affinity for fungal membranes [31]. In addition, Iturin A induces defense responses in plants [32]. The production of Iturin A by Bacillus cereus (B15) and its characterization using HPLC analysis provide valuable information on the potential use of this compound as a biocontrol agent against fungal plant diseases. Further studies are needed to determine the effectiveness of Iturin A produced by Bacillus cereus (B15) in controlling fungal plant diseases under field conditions.

5. Conclusions

In conclusion, this study highlights the potential of epiphytic bacteria as a biological control method against green mold post-harvest disease in oranges. The isolated strains Bacillus, Streptomyces, and Pseudomonas spp. demonstrated strong antifungal properties against P. digitatum, both in vitro and in vivo. Further studies on these bacterial strains could investigate the presence of inhibitory compounds. The detection of Iturin A in Bacillus cereus indicates the presence of inhibitory compounds that can be further explored for commercial applications. Further research on the potential use of epiphytic bacteria in citrus farming will assist the development of sustainable agricultural practices and promote safer food production.