Abstract
This work aimed to evaluate the efficiency of bioconfrol agents in controlling green mold (Penicillium digitatum) in 'pera' orange fruits. In vitro experiments were carried out with a fungus isolate grown on BDA medium and tested against different concentrations of commercial products based on biocontrol agents: Trichoderma harzianum (0, 5, 10, 15, 20 and 25 mL L-1), Bacillus subtilis (0, 2, 4, 6, 8 and 10 mL L-1) and B. licheniformis + B. subtilis + 7. longibrachiatum (0, 5, 10, 15, 20 and 25 g L-1) and the fungicide imazalil as standard (2 mL L-1). The experiment analyzed mycelial growth and spore germination. Based on the results, in vivo tests were carried out, evaluating the curative and preventive effect of applying bioconfrol agents as inoculating with 10 µL of conidial suspension (108 conidia mL-1). All biochemical agents displayed 100% control over mycelial growth and a linear effect to inhibit the germination of P. digitatum. The in vivo tests highlighted that all agents showed a linear effect, both in the curative and preventive effects, significantly reducing the development of green mold (AUDPC) in 'pera' orange fruits. The experiment concludes that the prophylactic application of 25 mL L-1 of 7. harzianum, 10 mL L-1 of B. subtilis, and 25 g L-1 of B. licheniformis + B. subtilis + T. longibrachiatum in orange fruits 'pera' control P. digitatum.
Keywords: citrus farming, citrus, biological control, orange
Introduction
Brazilian citrus farming has great relevance in the world economy, being the largest producer of citrus and the largest exporter of concentrated orange juice (Chagas et al., 2018; IBGE, 2020). As a result of the COVID-19 pandemic, eating habits changed, increasing the per capita consumption of fresh citrus fruits and orange juice in 2020, with a tendency to continue increasing consumption by around 40% per month (CITRUS BR, 2020).
The post-harvest quality is the primary factor for the fruits' commercialization. Attributes such as appearance, flavor, aroma, texture, and nutritional value directly reflect consumer acceptance (Chagas et al., 2018). The green mold caused by the fungus Penicillium digitatum (Pers.: Fr) Sacc. is among the most critical post-harvest diseases (Dukare et al., 2018; CarmonaHernandez et al., 2019).
The fungus P. digitatum is widely disseminated in all producing regions, being responsible for around 90% of total losses in citrus fruits (Benato et al., 2018; Pétriacq et al., 2018; Bazioli et al., 2019). Preventive and curative measures are required due to the aggressiveness of the fungus and its potential to cause losses, and to avoid or eliminate the incidence and severity of P. digitatum in fruits.
Its primary control method is still the application of systemic fungicides, such as imazalil (Costa et al., 2019). However, several studies have revealed the emergence of strains of P. digitatum resisfanf to the main fungicides, in addition to the severe implications that these products represent for the environment, animals, and humans (Ferraz et al., 2018; Pétriacq et al., 2018).
Among the most promising strategies is the use of bioconfrol involving beneficial microorganisms in the control of diseases in the pre- and post-harvesf of fruits (Dukare et al., 2018; Boffette et al., 2018; Carmona- Hernandez et al., 2019; Tian et al., 2020).
In citrus farming, the use of antagonistic microorganisms, such as those of the genus Trichoderma spp. and Bacillus spp., revealed positive results in the control of phytopathogens in citrus farming (Cunha & Kupper, 2018; Hussain, 2018; Cunha et al., 2018; Ahima et al., 2019; Tian et al., 2020). In general, research has emphasized the efficiency of T. harzianum and B. subtilis in the biocontrol of pre-harvest diseases, but little is known about the effectiveness of these agents in post-harvest rot, especially of B. licheniformis + B. subtilis + T. longibrachiatum in the biocontrol of P. digitatum in 'pera' orange fruits. Therefore, this work aimed to evaluate biocontrol agents' efficiency in controlling green mold (Penicillium digitatum) in 'pera' orange fruits.
Material and methods
Biocontrol agents and pathogen isolate
The experiment used three commercial products: Trichodermil® (2x109 viable Trichoderma harzianum spores mL-1), Serenade® (1x10'UFC g-1 of the Bacillus subtilis strain QST 713 bacteria), and Nem Out™ (3.75 x 108 UFC g-1 of the mixture of 6. licheniformis, B. subtilis, and T. longibrachiatum).
The biocontrol agents were incorporated into the melting BDA culture medium (50 °C) at concentrations of 0, 5, 10, 15, 20, and 25 ml L-1 for Trichoderma harzianum, 0, 2, 4, 6, 8, and 10 mL L-1 for Bacillus subtilis, and 0, 5, 10, 15, 20, and 25 g L-1 for 6. licheniformis + 6. subtilis + T. longibrachiatum. The fungicide imazalil (2 ml L-1) was the positive control.
P. digitatum was used on 'pera' orange from the commercial orchard. The fungus was cultivated on BDA medium (potato dextrose agar) and incubated in a BOD-type growth chamber at 25 °C, alternating 12-hour photoperiod, for seven days to keep the isolated strain alive.
In vitro tests: mycelial growth and spore germination
The pairing tests were performed in duplicate through direct comparison according to Campanile et al. (2007), calculating the antagonism index. The biocontrol agents were poured into Petri dishes (90 mm), and after two hours, in the center of each Petri dish, a disc (6 mm) of the pathogen colony that had been growing for seven days was placed, leaving the fungal structures in contact with the culture medium. Then, the plates were placed in a BOD-type growth chamber at 25 °C, with a 12-hour photoperiod, for seven days.
Mycelial growth was assessed daily by measuring the colony's mycelial radius (mm) using a digital caliper (two orthogonal measurements), calculating the average values per plate. The values obtained were used to calculate the antagonism index (IA): IA% = [(RM - rm ) x100] RM, where: IA= Antagonism index in percentage; RM= Mycelial radius without the presence of the antagonist (control); rm = Mycelial radius with the antagonist. The experimental design was completely randomized, with 17 treatments and 20 replications. Each experimental plot consisted of a Petri dish.
The potential of the biocontrol agents on spore germination was assessed by preparing spore suspensions of the pathogen obtained from pure colonies of the P. digitatum isolate. These suspensions were cultivated in BDA medium with distilled water + Tween 20, scraping the Petri dish with a Drigalski loop and filtering through gauze. The suspension was calibrated to 104 spores mL-1, with Neubauer chamber in an optical microscope. Aliquots of 40 µL of the spore suspension were pipetted into individual wells of Elisa test plates, where 40 µL_ of each treatment and their respective concentrations were also placed.
Spore germination was stopped with 20 µl_ of lactophenol after an incubation period of 26 hours, at 25 °C, and evaluated by counting 50 spores per cavity with subsequent calculation of the germination percentage. Spores that presented germ tube emission were considered germinated, regardless of their size. Data were expressed as a percentage of spore germination in relation to total spores.
In vivo tests: Obtaining plant material and spore suspension
The in vivo tests used 'pera' orange fruits from a commercial orchard at a stage of ripeness suitable for consumption. The fruits were selected and standardized and then washed with neutral detergent (0.2%), sanitized with sodium hypochlorite ( NaCIO ) 0.1% for 10 minutes, with subsequent rinsing in running water and then in distilled water, being allocated on the bench at room temperature until completely dry.
The suspension of pathogen spores was obtained as previously described in the in vitro tests but was calibrated to 108 spores mL-1 by a Neubauer chamber in an optical microscope.
Biocontrol agents' curative effect assessment
Fruits were inoculated with a suspension of P. digitatum spores (108 spores ml-1) by applying an aliquot of 10 µL in a 2 mm deep wound in the previously demarcated fruit epidermis to evaluate the curative effect. After four hours of inoculation, the fruits were immersed in the treatments for 2 minutes at the same concentrations as the in vitro test.
The fruits were immersed for 2 minutes in the treatments and then dried for four hours on a bench covered with Kraft®' paper to evaluate the preventive effect. After drying, a 100 µL syringe performed a 2 mm deep wound in the previously demarcated epidermis, which was later inoculated with ten µL of P. digitatum spore suspension (108 spores mL-1).
The fruits were stored at 25 °C (± 2 °C) and 80% (± 5%) RH, for seven days to evaluate the curative and preventive effects. During this period, the fruits from the control treatment remained wholly overtaken by green mold growth.
Five assessments of the diameter of the lesions on 'pera' orange fruits were carried out using a digital caliper to assess the severity. The data obtained were used to calculate the area under the disease progress curve (AUDPC) using the equation AUDPC = Z [(Y i+1 + YJ/2] [T i + 1 - Ti ] where Yi+1 = diameter of the lesion at time T¡+1 and Yi = diameter of the lesion at time Ti ( Campbell & Adden, 1990).
The experimental design was completely randomized, with five replications consisting of four fruits as an experimental unit and carried out in duplicate. The data obtained were submitted to homogeneity, normality, and variance analysis (F test), and the means were compared by the Tukey test at 1% and 5% probability. The regression analyses were carried out at 1% and 5% probability using the Sisvar program.
Results and Discussion
In vitro tests
All biocontrol agents tested induced a 100% inhibition of P. digitatum mycelial growth at all concentrations evaluated (Figure 1). The concentrations of T. harzianum, 6. subtilis, and 6. licheniformis + 6. subtilis + T. iongibrachiatum were efficient and equal to the control observed for the fungicide, with all treatments differing from the control (p0.01).
The spores' germination of P. digitatum displayed a linear effect (p<0.01) depending on the doses for all biocontrol agents (Figure 2), and there was a difference among the concentrations, the control, and imazalil treatments. The treatments with biocontrol agents significantly reduced the germination of P. digitatum spores. The fruits treated with 6. licheniformis + 6. subtilis + T. Iongibrachiatum (25 g L-1) displayed the most significant controls, followed by T. harzianum (25 ml L-1) and 6. subtilis (10 ml-1), showing germination inhibition of 94%, 88%, and 85% respectively, when compared to the control. The fungicide inhibited 100% spore germination.
In vivo tests: Curative and preventive control of P. digitatum in 'Pera' orange fruits
Regarding the incidence of P. digitatum, all fruits displayed disease symptoms except for the imazalil (fungicide) treatment. The symptoms started two days after inoculation. Regardless of the biocontrol agent tested, the disease developed. However, when evaluating the severity (AUDPC), the presence of biocontrol agents linearly reduced the progress of the disease compared to the control in both the curative and preventive forms of treatment application (Figure 3).
The curative effect of applying T. harzianum (Figure 3A) at a concentration of 5 mL L was not efficient in controlling green mold on 'Pera' orange, not differing from the control. However, from a concentration of 10 mL L there was a significant effect in inhibiting the P. digitatum development, showing a linear reduction in severity, reaching reduction percentages of 13.6%, 26.4%, 33.6%, and 53.6% at concentrations of 10, 15, 20, and 25 mL L-1, respectively when compared to the control. Concentrations between 10 and 25 mL L 1 differed from each other and differed significantly also from the control and imazalil treatments.
The preventive effect of T. harzianum application (Figure 3B) displays a response similar to that presented by the curative effect, with a linear reduction depending on doses. Even though, in this case, the control was efficient at all concentrations, which displayed significant differences between them and from both control and imazalil treatments. The effect on green mold control caused a reduction in severity by 16.3%, 26.8%, 45.6%, 56.9% and 69.8% at concentrations of 5, 10, 15, 20, and 25 mL L-1, respectively.
The comparison between the curative and preventive effects of T. harzianum concentrations (Figure 3) highlighted that the preventive effect presented better performance to control P. digitatum in 'Pera' orange fruits. For all concentrations of T. harzianum there was a difference between curative and preventive effects. In both treatments (curative and preventive), the 25 ml L concentration promoted the most significant reduction in the severity of P. digitatum. However, the reduction was even more significant in the preventive (69.8%) than in the curative effect (53.6%) when compared with their respective controls.
The curative effect applying 6. subtilis (Figure 4A) showed a response similar to that observed for treatment with T. harzianum, with a linear reduction depending on doses. The concentration of 2 mL L was inefficient in controlling green mold, not differing from the control. However, from a concentration of 4 mL L there was a significant effect on inhibiting the development of P. digitatum, showing a reduction in severity of 13.8%, 22.4%, 31.9%, and 47.8% at concentrations of 4, 6, 8, and 10 mL L , respectively, when compared to the control. Such concentrations differed from each other and both the control and imazalil treatments.
The preventive effect of treatment with different concentrations of 6. subtilis (Figure 4B) displayed a response similar to that presented by the curative effect, with a linear reduction depending on the doses. Still, in this case, there was control efficiency for all concentrations evaluated, presenting significant differences between them and differing from the control and imazalil treatments. The effect on the control of green mold on 'Pera' orange reduced the severity by 18.3%, 33.8%, 44.3%, 57.0%, and 71.7% at concentrations of 2,4, 6,8, and 10 ml L-1, respectively.
The comparison between the curative and preventive effects of 6. subtilis concentrations (Figure 4) highlighted that the preventive effect also presented the best performance for controlling P. digitatum in 'pera' orange fruits. For all concentrations of B. subtilis there was a difference between curative and preventive effects.
In both application effects (curative and preventive) (Figure4),the 10mLU B.subtilis concentration significantly reduced the P. digitatum severity. However, in the preventive effect, the reduction was more significant (71.7%) than in the curative effect (47.8%) when compared with their respective controls.
Both curative and preventive effects of applying 6. licheniformis + B. subtilis + T. longibrachiatum (Figures 5A and 5B) presented a response similar to that observed for the T. harzianum and B. subtilis treatments. Besides, the 5 g L -1 concentration was also inefficient in curing green mold on 'Pera' orange, not differing from the control. However, from a concentration of 10 g Luthere was a significant effect on inhibiting the P. digitatum development, showing a severity reduction of 13.0%, 22.2%, 31.3%, and 47.4% at concentrations of 10, 15, 20, and 25 g L , respectively, when compared to the control. Such concentrations differed from each other, the control, and imazalil treatments.
The preventive effect of treatment with 6. licheniformis + B. subtilis + T. longibrachiatum also had control efficiency for all concentrations evaluated, presenting significant differences between them and differing from the control and imazalil treatments. Again, there was a significant effect on the control of green mold on 'Pera' orange, with a reduction in severity of 17.4%, 31.5%, 52.3%, 61.8%, and 75.5% at concentrations of 5, 10, 15, 20 and 25 g L1, respectively.
The comparison between the curative and preventive effects of 6. licheniformis + B. subtilis + T. longibrachiatum (Figure 5) highlighted that the preventive effect presented the best performance for controlling P. digitatum . In both treatments (curative and preventive), the concentration of 25 g L-1 of 6. licheniformis + B. subtilis + T. longibrachiatum promoted the most significant reduction in the severity of P. digitatum. However, the decline was more significant in the preventive effect (75.5%) than in the curative effect (47.4%) compared to their respective controls.
Analyzing the curative and preventive effect of T. harzianum, B. subtilis, and 6. licheniformis + B. subtilis + T. longibrachiatum, verified that the preventive effect promoted the most significant green mold severity reduction on fruits in the tests carried out, and determined that the best concentration of each treatment was higher: 25 ml L-1 10 ml L-1, and 25 g L-1, respectively. The analyses also depicted a significant interaction (p<0.05) between the two sources of variation: treatment and application effect (Table 1).
The analysis of each effect separately revealed that the significant curative effect of the biocontrol agents. Applying T. harzianum on the fruits differed from other biocontrol agents, as it presented the lowest AUDPC. Treatments with B. licheniformis + B. subtilis + T. Iongibrachiatum, and B. subtilis , did not differ from each other and were less efficient in reducing AUDPC than T. harzianum. For the preventive effect, the treatments with B. licheniformis + B. subtilis + T. Iongibrachiatum and B. subtilis displayed the lowest AUDPC.
The comparison among the forms of treatment application stressed a significant difference between the curative and preventive application of treatments. The preventive effect of applying T. harzianum, B. subtilis, and B. licheniformis + B. subtilis + T. Iongibrachiatum reduced disease severity significantly compared to the curative effect. Prophylactic application of 25 ml L of T. harzianum, 10 mL L-1 of B. subtilis, or 25 g L-1 of B. licheniformis + B. subtilis + T. Iongibrachiatum reduced AUDPC by 29%, 46% and 51%, respectively, when compared to the curative application (Table 1).
Biocontrol agents showed different control responses depending on the application form, with the highest AU DPC in fruits treated curatively. The joint use of B. licheniformis + B. subtilis + T. Iongibrachiatum provided the best control of P. digitatum, followed by treatments with T. harzianum and B. subtilis , when applied preventively. Given these findings, it was possible to verify that the behavior of biocontrol agents on P. digitatum depends on the application form (curative or preventive) on the fruits.
The use of antagonistic microorganisms in citrus farming has emerged as a potential alternative to fungicides. Several studies show positive effects with the application of biocontrol agents in the suppression of phytopathogens, especially those related to the post-harvest stage, such as P. digitatum and P. italicum (Kupper et al., 2012; Ferraz et al, 2018; Hussain, 2018; Cunha et ak, 2018; Ahima et al., 2019; Tian et ak, 2020). To date, such research has emphasized the efficiency of T. harzianum and B. subtilis in the biocontrol of post-harvest diseases. However, little is known about their effectiveness post-harvest, especially B. licheniformis + B. subtilis + T. Iongibrachiatum in the biocontrol of P. digitatum in 'Pera' orange fruits.
According to the results obtained in the in vitro tests, biocontrol agents T. harzianum, B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum, completely inhibited P. digitatum, with all concentrations evaluated being as efficient as the fungicide imazalil. Studies indicate that this finding relates to the main mechanisms of action of the genera Trichoderma and Bacillus (Silva et al., 2019; Tian et al., 2020).
As seen for P. digitatum, species of Trichoderma spp., such as T. harzianum and T. longibrachiatum, offer potential control over phytopathogens, as they present different modes of action on the aggressor, such as competition for nutrients and space, antibiosis and mycoparasitism (Gajera et al., 2013; Di Francesco et al., 2016; Dukare et al., 2018; Hussain, 2018). Isolates of Trichoderma spp. were also effective against the fungus P. digitatum under in vitro conditions. In all results, the isolates proved their antagonistic capacity in double culture techniques and culture filtrate assays (Mishra et al., 2011; Hussain, 2018).
Antagonistic bacteria of the genus Bacillus, such as the species B. subtills and B. licheniformis, are efficient in competing for nutrients and space, synthesizing antibiotics and other compounds (antibiosis), acting through mycoparasitism, among other potential antagonistic mechanisms, such as the formation of biofilms, quorum sensing, and siderophores (Carmona-Hernandez et al., 2019). Therefore, these species effectively control P. digitatum and other pathogens.
Several studies have revealed positive results from the use of Bacillus spp. isolates in citrus farming, such as the studies by Chen et al. (2018) and Tian et al. (2020), who verified that the application of cell-free supernatant (CFS) obtained from B. subtilis isolate ET-1 and B. amyloliquefaciens Isolated DH-4 effectively inhibited P. digitatum in in-vitro and in-vivo tests, demonstrating that it can be used as part of the integrated management of green mold in citrus. Furthermore, in the study carried out by Tian et al. (2020), the authors detected that the intense antifungal activity of the CFS of B. subtilis isolated ET-1, against P. digitatum, occurred due to the synthesis of five types of antimicrobial substances: macrolactin, bacillaene, iturins, fengycin, and surfactin.
Mohammadi et al. (2017) studied the effect of ten antagonistic bacteria (4 strains of B. subtilis, 2 strains of B. pumilus, 2 strains of B. cereus, 1 strain of B. megaterium, and 1 strain of Agrobacterium radiobacterium) in vitro against P. digitatum. All bacteria inhibited mycelial growth and spore germination of the fungus (except A. radiobacter). These results emphasize the antagonistic potential of Bacillus spp. species as important biocontrol agents for P. digitatum. (Mohammadi et al., 2017).
The suppression of mycelial growth and germination of P. digitatum spores by antagonistic bacterial species, such as those of the genus Bacillus, acts mainly through antibiosis. These bacteria suppress the growth and development of the pathogen by secreting chemical substances and other antibiotic compounds, inhibiting cell wall synthesis, destroying and altering cell membrane structures, and damaging protein synthesis. That is why they are so efficient as biocontrol agents (DUKARE etai., 2018).
Furthermore, according to Di Francesco et al. (2016) and Dukare et al. (2018), the biological control mechanism of Trichoderma spp. and Bacillus spp. also correlates with the ability to produce volatile antifungal metabolites. Volatile organic compounds are active at low concentrations and belong to various chemical groups, such as alcohols, aldehydes, ketones, esters, lactones, terpenes, and sulfur compounds. Due to their volatility, these compounds can travel great distances in structurally heterogeneous environments, as well as in solid, liquid, or gaseous compounds, being an excellent advantage for the success of antagonistic microorganisms (Carmona-Hernandez et al., 2019).
Most fungal pathogens, such as P. digitatum, according to Wang et al. (2018), infect fruits through wounds, stomata, and lenticels, starting the process with the germination of spores and subsequent formation of the germ tube. Biocontrol agents T. harzianum, B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum also inhibited the germination of P. digitatum spores, showing a linear effect depending on the doses. However, the most outstanding germination control was observed for the highest treatment concentration with B. licheniformis + B. subtilis + T. longibrachiatum, indicating that combining species from the two genera in the same product presented greater efficiency of action.
The presence of two species of the genus Bacillus (B. licheniformis and B. subtilis) in the studied formulation possibly expanded the pathogen spectrum of action since the mode of action of this genus involves the synthesis of antibacterial and antifungal metabolites, such as surfactin, bacillomycin, and fengycin (DUKARE et al., 2018; TIAN et al., 2020). The isolated application of biocontrol agents, according to Dukare et al. (2018), is typically not sufficient to achieve a consistently high level (>95%) of disease control. Therefore, combining biocontrol agents is an advantageous approach, exploring the synergistic effects of the combination of microorganisms and, thus, improving the performance and effectiveness of the biocontrol method.
The in-vivo trials, both curative and preventive applications of T. harzianum, B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum in 'Pera' orange fruits, proved to be effective in controlling green mold in citrus, by significantly reducing AUDPC, especially at the highest concentrations of each biocontrol agent. These results indicate that biocontrol strategies involving these beneficial microorganisms represent a promising alternative to reduce or even replace fungicides to control post-harvest diseases.
The reality of many orchards shows that chemical control in pre- and post-harvest is still the most used method for controlling P. digitatum, as well as other phytopathogens (Erasmus et al., 2015; Ferraz et al., 2018; Costa et al., 2019; Fenta et al., 2019; Tian et al., 2020). However, the prolonged and uncontrolled use of fungicides has triggered a series of problems in agriculture in general. There are currently several cases of pathogen strains resistant to the modes of action of many active ingredients, in addition to the increasing detection of fruit residues, putting consumer health at risk (Fischer et al., 2013; Ferraz et al., 2018).
The continuous use of fungicides in citrus post-harvest has led to the development of many cases of resistance of strains of P. digitatum and P. italicum to synthetic fungicides, from the benzimidazole, sodium octaphenylphenate, and imidazole groups (Erasmus et al., 2015). Evaluating the sensitivity of 75 strains of P. digitatum to seven fungicides, Sánchez-Torres & Tuset (2011) found that among the strains assessed, 84% were resistant to thiabendazole and 77% to imazalil, which are the main fungicides used in post-harvest of citrus.
Worldwide, systemic fungicides, such as imazalil, are the most used post-harvest treatments to control green mold in citrus fruits ( Costa et al., 2019). In Brazil, control of P. digitatum in packing houses has been carried out for more than 25 years with imazalil and thiabendazole (Fischer et al., 2013). Furthermore, studies reveal that the repeated use of some active ingredients allows the emergence of resistant P. digitatum populations, besides the severe implications that these products represent for the environment, animals, and humans (FERRAZ et al ., 2018).
In many citrus-producing regions, the application of imazalil is still considered safe post-harvest. However, agricultural systems need to reformulate the practices adopted concerning the use of pesticides and the maximum residue limits (MRL) allowed in a product. Therefore, it is likely that the use of imazalil in post-harvest applications will begin to have restrictions due to the continued reduction of MRLs permitted in many countries (Pétriacq et al., 2018).
Therefore, research that explores the use of alternative products, as is the focus of this work, offers strategies that offer new dimensions and flexibility in the choice of disease management in the production of healthier foods and reduce pathogen resistance to products, traditional (Fischer et al., 2013; Dukare et al., 2018; Boffette et al., 2018; Tian et al., 2020).
As seen, citrus fruits are very susceptible to infection by P. digitatum, especially in the post-harvest phase, that is, during storage that precedes fruit consumption (Tian et al., 2020). According to Boffette et al. (2018), when the fungus is in ideal conditions, with a temperature in the range of 25 °C and relative air humidity above 80% (factors monitored in in-vivo tests), added to the availability of nutrients to stimulate spore germination, the initiation and success of the infection are favored (COSTA et al., 2019). According to Nicoli et al. (2009), under ideal temperature and humidity conditions, in less than two days, the lesion reaches approximately 50 mm in diameter, reaching the juice vesicles (pulp). In fact, in 'Pera' orange fruits, the first symptoms appeared 48 hours after inoculation with the pathogen.
Studying the infection process of P. digitatum, Costa et al. (2019) described that the fungus produces enzymes capable of dissolving the middle lamella of fruit tissues, which is why the first symptom observed is the appearance of soft rot at the site of infection (a watery spot with slight discoloration of the tissue). Subsequently, the presence of white mycelia and the production of olive-green spores appear in the initial lesion region. As the days go by, the disease progresses until it takes over the entire fruit (Cunha et al., 2018).
In in-vivo tests, the fungus took around 168 hours after inoculation to completely take over the fruits of the control treatment, that is, five days after the first symptoms. However, treatments with T. harzianum , B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum, especially at the highest doses, were efficient in containing the evolution of infection by P. digitatum, and it can be observed that the diameters of the lesions were smaller, with absence or reduction of sporulation.
Biocontrol agents displayed differences between curative and preventive effects, as fruits treated preventively had lower disease severity. In their study, Moretto et al. (2014) state that biocontrol agents' application after harvest may be too late to compete effectively against the pathogen since it may have established itself in the fruit while still in the field (latent infection). In cases like this, the antagonistic microorganism should present a curative action, controlling these preexisting infections and preventing subsequent infections by inhibiting the fungus sporulation. The highest AUDPCs in this study were in fruits treated curatively, demonstrating that the biocontrol agents displayed a higher antagonistic than preventive efficiency.
Biocontrol agents promoted a significant reduction in the disease severity when applied preventively, and the longer the fruit storage after treatments with T. harzianum , B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum, the lower the disease severity, indicating the effective action of these treatments in protecting the fruits from future infections. A similar study also found that the preventive application of biocontrol agents to fruits was more effective in containing P. digitatum infection. (Wang et al., 2018).
In commercial situations, the preventive effect of applying biocontrol agents is a promising alternative. Reinfection of the same fruit or healthy fruits can occur during handling and processing within the packaging (Moretto et al., 2014). A single spore of P . digitatum can infect a fruit and produce millions of spores after seven days under optimal environmental conditions (Benato et al., 2018).
The preventive effect of applying T. harzianum, B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum in 'Pera' orange fruits demonstrated that they showed an efficient capacity to grow and quickly colonize the fruit surface for a more extended period, regardless of environmental conditions. In addition to rapidly using available nutrients, they reduce their availability to pathogens (Dukare et al., 2018).
This effect is significant, as in citrus farming, the incidence of green mold on fruits is the main post-harvest rot, as alone, is responsible for around 90% of total losses in the citrus chain (Bazioli et al., 2019), reducing fruit quality, marketing period and shelf life (Fischer et al., 2008; Fischer et al., 2011). Faced with such aggression, biocontrol agents that act quickly to suppress P. digitatum are interesting.
Biocontrol agents employ direct and indirect inhibitors as mechanisms to suppress fungal growth by synthesizing antifungal compounds and specific metabolites that act on the pathogen, inhibiting its metabolism and development, besides damaging or killing pathogen propagules on fruits (Dukare et al., 2018). The synthesis of antibiotics is another primary strategy to control the possible deterioration of fruits during storage, as they reduce the growth of pathogens, even at very low concentrations (Wang et al., 2018; Wang et al., 2020).
Biocontrol agents have been successfully implemented in biological control strategies in the fruits' post-harvest, as they are faster in consuming the nutrients available on the surface of the fruits, colonizing with greater agility and thus reducing the chances of the pathogen spreading, install on site (Dukare et al., 2018).
Conclusion
All evaluated concentrations of T. harzianum, B. subtilis, and B. licheniformis + B. subtilis + T. longibrachiatum were 100% efficient in inhibiting the mycelial growth of P. digitatum.
The application of B. licheniformis + B. subtilis + T. longibrachiatum inhibited the germination of P. digitatum spores by 94%, followed by T. harzianum (88%) and B. subtilis (85%).
The preventive application of B. licheniformis + B. subtilis + T. longibrachiatum promoted the best control of P. digitatum in 'pera' orange fruits, followed by treatments with T. harzianum, and B. subtilis.
Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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REFERENCES
Ahima, J., Zhang, X., Yang, Q., Zhao, L, Tibiru, A.M., Zhang, H. 2019. Biocontrol activity of Rhodotorula mucilaginosa combined with salicylic acid against Penicillium digitatum infection in oranges. Biological Control 135: 23-32.
Bazioli, J.M., Belinato, J.R., Costa, J.H., Akiyam, A.D.Y., Pontes, J.G.M., Kupper, K.C., Augusto, F., Carvalho, J.E., Fill, T.P. 2019. Biological Control of Citrus Post-harvest Phytopathogens. Toxins 11:460.
Benato, E.A., Belletti, T.C., Terao, D., Franco, D.A.S. 2018. Essential oils and heat treatment in post-harvest control of green mold on oranges. Summa Phytopathol 44: 65-71.
Boffette, B.G., Lopes, M.G.F., Furlan, M.R., Souza, A.D. 2018. Control methods and quality of orange fruits Citrus sinensis (L.) osbeck cv pera in post-harvest. Magazine Electronics Thesis 30:1 -15.
Campanile, G., Ruscelli, A., Luisi, N. 2007. Antagonistic activity of endophytic fungi towards Diplodia corticola assessed by in vitro and in planta tests. European Journal of Plant Pathology 117: 237-246.
Campbell, C.L., Madden, L. 1990. Introduction to plant disease epidemiology. John Wiley & Sons, New York, USA. 532 p.
Carmona-Hernandez, S., Reyes-Pérez, J.J., Chiquit°Contreras, R.G., Rincon-Enriquez, G., Cerdan-Cabrera, C.R., Hernandez-Montiel, L.G. 2019. Biocontrol of Postharvest Fruit Fungal Diseases by Bacterial Antagonists: A Review. Agronomy 9:121.
Chagas, T.L.K., Soriani, R., Leite, C.A.M., Barbosa, C.A.C., Santos, E.D. 2018. Assessment of post-harvest conservation of 'pera' orange under ambient and refrigerated conditions. Revista Terra & Cultura 34: 1 -8.
Chen, K., Tian, Z., Luo, Y., Cheng, Y., Long, C. 2018. Antagonistic Activity and the Mechanism of Bacillus amyloliquefaciens DH-4 Against Citrus Green Mold. Phytopathology 108: 1253-1262.
CITRUS BR, National Association of Citrus Juice Exporters. Consumption of orange juice increases for breakfast during confinement. 2020. http://www.citrusbr.com/ < Accessed on May 14. 2020>.
Costa, J.H., Bazioli, J.M., Pontes, J.G.M., Fill, T.P. 2019. Penicillium digitatum infection mechanisms in citrus: What do we know so far?. Fungal Biology 123: 584-593.
Cunha, T., Ferraz, L.P., Wehr, P.P., Kupper, K.C. 2018. Antifungal activity and action mechanisms of yeasts isolates from citrus against Penicillium italicum . International journal of food microbiology 27 G: 20-27.
Di Francesco, A., Martini, C., Mari, M. 2016. Biological control of post-harvest diseases by microbial antagonists: how many mechanisms of action?. European Journal of Plant Pathology 145: 711 -717.
Dukare, A.S., Paul, S., Nambí, V.E., Gupta, R.K., Singh, R., Sharma, K., Vishwakarma, R.K. 201 Sexploitation of microbial antagonists for the control of post-harvest diseases of fruits: a review. Critical Reviews In Food Science and Nutrition , 59: 1498-1513.
Erasmus, A., Lennox, C.I., Korsten, L., Lesar, K., Fourie, P.H. 2015. Imazalil resistance in Penicillium digitatum and P. italicum causing citrus post-harvest green and blue mold : Impact and options. Post-harvest Biology and Technology 107: 66-76.
Fenta, L., Mekonnen, H., Gashaw, T. 2019.Biocontrol potential of trlchoderma and yeast against post-harvest fruit fungal diseases: A review. World News of Natural Sciences 27: 153-173.
Ferraz , L.P , Cunha, T., Kupper, K.C. 2018. Mechanisms of action of yeast isolates involved in the biocontrol of Penicillium digitatum , causal agent of green mold on citrus fruits. Citrus Research & Technology 39: e-1033.
Fischer, I.H., Almeida, A.M., Arruda, M.C., Bertani, R.M.A., Garcia, M.J.M., Amorim, L. 2011. Post-harvest damage to guavas in the Central-West Region of Sao Paulo. Bragantla 70: 570-576.
Fischer, I.H., Loureno, S.I., Amorim, L. 2008. Postharvest diseases in citrus and characterization of the environmental fungal population in the wholesale market in Sao Paulo. Tropical Plant Pathology 33: 219-226.
Fischer, I.H., Palharini, M.C.A., Spósito, M.B., Amorim, L. 2013. Post-harvest diseases in 'pera' orange produced in organic and conventional systems and Penicillium resistance digitatum to fungicides. Summa Phytopathol 39:28-34.
Gajera, H., Domadiya R., Patel S., Kapopara M., Golakiya B. 2013. Molecular mechanism of Trlchoderma as biocontrol agents against phytopathogen system - a review. Current Research in Microbiology and Biotechnology 1: 133-142.
Hussain, I. 2018. Biological Control of Fungal Plant Pathogen Penicillium digitatum Causing Green Rot of Citrus Fruit by Local Trichoderma Isolates from Bihar, India . International Journal of Current Microbiology and Applied Sciences 7:2970-2978.
IBGE. BRAZILIAN INSTITUTE OF GEOGRAPHY AND STATISTICS. Systematic Survey of Agricultural Production (LSPA): Planted area, harvested area and production, by harvest year and crop product. 2020. https://sidra.ibge.gov.br/ pesquisa/lspa/tabelas <Accessed on 13 May 2020>.
Kupper, K.C., Cervantes, All., Klein, M.N., Silva, A.C. 2012. Evaluation of antagonistic microorganisms, Saccharomyces cerevisiae and Bacillus subtilis for the control of Penicillium digitatum . Brazilian Journal of Fruticulture 35: 425-436.
Mishra, B.K., Mishra, R.K., Mishra, R.C., Tiwari, A.K., Yadav, R.S., Dikshit, A. 2011. Biocontrol efficacy of Trichoderma viride isolates against fungal plant pathogens causing disease in Vigna radiata L. Archives of Applied Science Research 3: 361-369.
Mohammadi, P., Tozlu, E., Kotan, R., Kotan, M.S. 2017. Potential of Some Bacteria for Biological Control of Post-harvest Citrus Green Mold Caused by Penicillium digitatum . Plant Protection Science 53:134-143.
Moretto, C., Cervantes, All., Batista Filho, A., Kupper, K.C. 2014. Integrated control of green mold to reduce chemical treatment in post-harvest citrus fruits. Scientia Horticulturae 165: 433-438.
Nicoli, A., Zambolim, L., Maro, L.A.C., Roma, R.C.C., Duarte, H.S.S. 2009. Comparison of the efficiency between alternative products and fungicides in controlling green mold in orange. Trópica Magazine - Agricultural and Biological Sciences 3: 12.
Pétriacq, P., López, A., Luna, E. 2018. Fruit Decay to Diseases: Can Induced Resistance and Priming Help? Plants 7:77-93.
Sánchez-Torres, P., Tusét, J.J. 2011. Molecular insights into fungicide resistance in sensitive and resistant Penicillum digitatum strains infecting citrus. Post-harvest Biology and Technology 59:159-165.
Silva, R.N., Monteiro, V.N., Steindorff, A.S., Gomes, E.V., Noronha, E.F., Ulhoa, C.J. 2019. Trichoderma/pathogen/ plant interaction in pre-harvest food security . Fungal Biology 123: 565-583.
Tian, Z., Chen, C., Chen, K., Liu, P., Fan, Q., Zhao, J., Long, C. 2020. Biocontrol and the mechanisms of Bacillus sp. wl 76 against post-harvest green mold in citrus. Postharvest Biology and Technology 159: 111 -022.
Wang, S., Zhou, Y., Luo, W., Deng, L., Yao, S., Zeng, K. 2020. Primary metabolites analysis of induced citrus fruit disease resistance upon treatment with oligochitosan , salicylic acid and Pichia membraneefaciens . Biological Control , 148: 104-289.
Wang, Z., Jiang, M., Chen, K., Wang, K., Du, M., Zalán, Z., Hegyi, F., Kan, J. 2018. Biocontrol of Penicillium digitatum on Postharvest Citrus Fruits by Pseudomonas fluorescens. Journal of Food Qualify ,2018:1-10.
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Abstract
This work aimed to evaluate the efficiency of bioconfrol agents in controlling green mold (Penicillium digitatum) in 'pera' orange fruits. In vitro experiments were carried out with a fungus isolate grown on BDA medium and tested against different concentrations of commercial products based on biocontrol agents: Trichoderma harzianum (0, 5, 10, 15, 20 and 25 mL L-1), Bacillus subtilis (0, 2, 4, 6, 8 and 10 mL L-1) and B. licheniformis + B. subtilis + 7. longibrachiatum (0, 5, 10, 15, 20 and 25 g L-1) and the fungicide imazalil as standard (2 mL L-1). The experiment analyzed mycelial growth and spore germination. Based on the results, in vivo tests were carried out, evaluating the curative and preventive effect of applying bioconfrol agents as inoculating with 10 µL of conidial suspension (108 conidia mL-1). All biochemical agents displayed 100% control over mycelial growth and a linear effect to inhibit the germination of P. digitatum. The in vivo tests highlighted that all agents showed a linear effect, both in the curative and preventive effects, significantly reducing the development of green mold (AUDPC) in 'pera' orange fruits. The experiment concludes that the prophylactic application of 25 mL L-1 of 7. harzianum, 10 mL L-1 of B. subtilis, and 25 g L-1 of B. licheniformis + B. subtilis + T. longibrachiatum in orange fruits 'pera' control P. digitatum.
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1 Universidade Estadual do Centro-Oeste, Guarapuava-PR, Brazil