RETRACTED:Chloramphenicol-Induced Inhibition of Phytophthora citrophthora in PDA Media: Implications for Fungicide Efficacy Studies

The use of chloramphenicol in Potato Dextrose Agar (PDA) media is widespread while making fungi cultivars to avoid the interference of external bacteria. It is used as well in the laboratory in vitro trials when a new potentially fungicide (or biofungicide) substance is tested against target fungi. But chloramphenicol must be treated with care in these kinds of trials. In this work, we monitorized the growth of Phytophthora citrophthora in PDA medium without chloramphenicol against PDA containing chloramphenicol at 0.025 and 0.05 g/L. The results showed a statistically significant reduction of P. citrophthora growth when PDA contained chloramphenicol at both rates. The highest rate 0.5 g/L avoided the growth of P. citrophthora 6 days. These findings demonstrate that while chloramphenicol is effective in preventing bacterial contamination in PDA media, it also significantly inhibits the growth of Phytophthora citrophthora. Both tested concentrations (0.025 g/L and 0.05 g/L) resulted in reduced fungal growth, with the higher concentration (0.05 g/L) completely inhibiting growth for six days postinoculation. Therefore, chloramphenicol is unsuitable for in vitro studies involving P. citrophthora, as it may compromise the accuracy of fungicide efficacy evaluations. Alternative strategies to manage bacterial contamination should be considered when conducting in vitro trials with this pathogen.

Introduction

Testing the efficacy of new fungicides and biofungicide products in vitro is essential to their development. This testing is usually done in the in vitro studies with fungicides, the most common culture medium is PDA (Potato Dextrose Agar), as it promotes fungal growth and facilitates the evaluation of antifungal activity. However, there are other culture media that are also used, depending on the type of fungus or the specific objective of the study. Some examples include:

• MEA (Malt Extract Agar): Promotes the growth of a wide range of fungi and is useful for studies requiring a more nutrient-rich medium than PDA.
• SDA (Sabouraud Dextrose Agar): Primarily used for isolating dermatophytic fungi and yeasts, particularly from clinical samples.
• V8 Agar: Commonly used in studies involving oomycete fungi such as Phytophthora and Pythium.
• CMA (Corn Meal Agar): Utilised for isolating and studying fungi that require simpler growth media.

Regarding chloramphenicol, it is a commonly used antibiotic to prevent bacterial growth in fungal culture media [1]. However, other antibiotics are also employed for the same purpose, such as:

• Streptomycin: Highly effective against Gram-negative bacteria and frequently used in fungal culture media.

Where the fungal growth can be measured through time in controlled conditions. These trials are of great importance also to identify the rates at which the tested products are effective against the target fungi before taking them to regulatory studies in the field. Before conducting regulatory studies for various fungicides and biofungicides, preliminary laboratory tests are essential to screen doses and identify effective products that inhibit fungal growth in vitro in Potato Dextrose Agar (PDA) media.

In in vitro studies with fungicides, the most common culture medium is PDA (Potato Dextrose Agar), as it promotes fungal growth and facilitates the evaluation of antifungal activity. Additionally, there are other culture media that are also used, depending on the type of fungus or the specific objective of the study.

A common challenge during these studies is bacterial contamination in the products, which can hinder the normal growth of the target fungi. To prevent this, antibiotics like chloramphenicol, known for its broad-spectrum activity against both Gram-positive and Gram-negative bacteria, are often added to the media. According to [2], chloramphenicol is used at a concentration of 0.025 - 0.05 g/L to prevent bacterial contamination.

Regarding chloramphenicol, there are other antibiotics also used for the same purpose, such as:

• Streptomycin: Highly effective against Gram-negative bacteria and frequently used in fungal culture media.
• Gentamicin: Similar to streptomycin, it is used in some studies to inhibit bacteria.
• Tetracycline: Broad-spectrum against Gram-positive and Gram-negative bacteria, sometimes used in combination with other antibiotics.

However, they were not the object of this study.

Phytophthora citrophthora is a notorious pathogen affecting citrus crops worldwide, causing significant losses due to diseases like foot rot, root rot, and gummosis. These infections lead to severe damage in the roots, trunk, and fruits, ultimately reducing both yield and fruit quality. The pathogen thrives in moist conditions and can persist in soils, making it a persistent challenge for citrus growers [3].

Phytophthora citrophthora causes significant economic losses in the citrus industry. Globally, citrus production is an essential agricultural sector, particularly in countries like Spain, the U.S., Brazil, China, and India. The impact of Phytophthora diseases can reduce crop yields by as much as 20% - 30% in heavily infested regions, depending on the severity of the outbreaks.

In specific cases, losses can exceed 50% when appropriate control measures are not in place. Infected trees can experience diminished vigor, smaller fruit sizes, and increased fruit drop, directly impacting the profitability of citrus farms.

Effective fungicide treatments, combined with other management practices, are crucial to maintaining healthy orchards and ensuring sustainable citrus production across the globe [4].

Materials and methods

Materials

The materials and equipment used are as follows:

• Potato Dextrose Agar (PDA): This culture medium was used for fungal growth and to evaluate the efficacy of the fungicides.
• Petri dishes: Standard 9 cm diameter dishes were used for culturing the fungi.
• Sterile cabinet: A sterile environment was maintained throughout the procedure to prevent contamination.
• Mother plates with inoculum: From the CECT Phytophthora citrophthora
• Distilled water: Used for preparing solutions and diluting products.
• chloramphenicol: Used as an antibacterial agent to prevent contamination in the culture media at 0.025 g/L and 0.05 g/L.
• Incubator: Set at 26 °C to provide optimal growth conditions for the fungi.

Methodology

1. Preparation of media and petri dishes:

• PDA media was prepared according to standard protocols. In experiments requiring contamination control, chloramphenicol was added to the PDA at a concentration of 0.025 g/L and 0.05 g/L to prevent bacterial growth, this rates were chosen according to [2].
• The media was poured into sterile 90 mm Petri dishes under aseptic conditions and allowed to solidify.
• 5 replicates per treatment were used in this experiment.

2. Inoculation procedure:

• Inside the sterile cabinet, small sections (approximately 2 cm x 5 cm) were cut from the mother plates containing the inoculum of P. citrophthora using sterilized tools.
• These sections, of active fungal inoculum, were carefully transferred onto the PDA media in the Petri dishes.
• For each test, inoculum blocks were placed on the PDA media containing the different rates of chloramphenicol product being evaluated.

3. Control plates:

• Control plates, also known as untreated controls, consisted of PDA media with distilled water and without chloramphenicol. These plates were inoculated with P. citrophthora as the other treatments.
• Controls are critical in these experiments to assess the natural growth of the fungi and to detect any inhibitory effects that might not be related to the fungicidal treatments being tested in other studies.

4. Incubation:

• All inoculated Petri dishes, including both treated with chloramphenicol at 2 different rates and control plates without bactericidal chloramphenicol, were incubated at 26 °C in a controlled environment. This temperature was chosen as it supports optimal growth for P. citrophthora
• The dishes were monitored for fungal growth at regular intervals, specifically at 4, 5, 6, and 10 days after inoculation (DAI).

5. Observation and data collection:

• Fungal growth was measured at each time point by observing and recording the radio of the fungal colonies in each Petri dish. At 4,5,6,10 days after inoculation.
• Special attention was given to the control plates to ensure that the presence or absence of chloramphenicol did not inadvertently influence fungal growth, particularly in the case of Phytophthora citrophthora.
• Data were statistically analysed using ARM 2024.1 software.

Results

The data collected from the different doses of chloramphenicol in PDA media and control plates were analyzed to determine the inhibition of fungal growth.

The results of the study (Tables 1,2) revealed a significant inhibitory effect of chloramphenicol on the growth of P. citrophthora in PDA media. Plates that were prepared with PDA medium supplemented with chloramphenicol (0.025 - 0.05 g/L) and inoculated with P. citrophthora showed an inhibition of fungal growth across all replicates. In contrast, plates containing only PDA medium without chloramphenicol, inoculated with the same mother culture of P. citrophthora, exhibited normal fungal growth (Figure 1).

Figure 1: Growth observations. Top: Graph representing the growth of Phytophthora citropthora at 10 days. Bottom: Control chloramphenicol 0 g/L, chloramphenicol 0.025 g/L, chloramphenicol 0.05 g/L.

Quantitative measurements:

• Colony diameters were measured at each time point (4, 5, 6, and 10 DAI) to assess the extent of fungal growth.

Statistical tests were done using the statistical software ARM 2024.1. Statistical analysis (ANOVA) confirmed that the differences between treatments were statistically significant (p < 0.05; Student-Newman-Keuls).

• In the chloramphenicol 0.05 g/L treated plates, the diameter remained at 0 mm throughout the 6 days of the experiment, indicating no growth, at 10 days after inoculation the growth was 0.28 cm diameter growth.
• In the untreated control plates, the average colony diameter increased progressively over time, with maximum growth observed at 10 DAI with a mean of 8.40 cm diameter. In contrast, the chloramphenicol 0.025 g/L treated plates had 2.36 cm diameter growth.

Discussion

in vitro studies evaluating fungicidal products against Phytophthora citrophthora should be conducted without the addition of chloramphenicol to the culture medium, as its presence can inhibit fungal growth and render the results of efficacy tests invalid. To address the potential issue of bacterial contamination in such studies, increasing the number of replicates is recommended as a precautionary measure. This ensures that any contaminated samples can be identified and excluded from the analysis, allowing for accurate evaluation of the fungicidal treatments.

The use of chloramphenicol at a concentration of 0.05 g/L has demonstrated a strong inhibitory effect on the growth of Phytophthora citrophthora in vitro. This pathogen is a major cause of disease in citrus crops worldwide, making it essential to carefully consider the composition of culture media in studies aimed at identifying effective treatments. While chloramphenicol is useful for preventing bacterial contamination in other fungal studies, its inhibitory effect on P. citrophthora highlights the need for alternative contamination control methods when working with this particular fungus.

However, this study highlights an unintended consequence of using chloramphenicol in Potato Dextrose Agar (PDA) when working with Phytophthora citrophthora. Our findings reveal that chloramphenicol inhibits the growth of P. citrophthora, rendering any fungicide efficacy studies invalid under these conditions [5]. While successful inhibition studies have been conducted with other fungi such as Mycosphaerella fijiensis and Fusarium oxysporum and using chloramphenicol, its use in P. citrophthora studies requires caution. The results emphasize the need to avoid altering the media with chloramphenicol in such studies and to mitigate potential contamination by increasing the number of replicates. This study underscores the critical importance of media composition in the accurate assessment of fungicide efficacy against P. citrophthora, a pathogen responsible for significant damage to citrus crops.

Conclusion

These findings demonstrate that chloramphenicol, while effective in preventing bacterial contamination in fungal culture media, exhibits a significant inhibitory effect on the growth of Phytophthora citrophthora. At both tested concentrations (0.025 g/L and 0.05 g/L), fungal growth was markedly reduced, with the higher concentration (0.05 g/L) completely suppressing growth for six days post-inoculation.

The results indicate that chloramphenicol is unsuitable for in vitro studies involving Phytophthora citrophthora, as its inhibitory effect could compromise the accuracy and reliability of fungicidal or biofungicidal efficacy trials. In contrast, its use in studies with other fungal pathogens, such as Mycosphaerella fijiensis and Fusarium oxysporum, has not shown similar inhibitory effects, suggesting that the impact of chloramphenicol is species-specific.

To address bacterial contamination without compromising fungal growth, it is recommended to:

• Increase the number of replicates to identify and exclude contaminated samples.
• Explore alternative antibacterial agents that do not interfere with P. citrophthora growth.

This study underscores the critical importance of selecting appropriate culture media compositions in in vitro studies, particularly when evaluating fungicidal efficacy against pathogens with unique sensitivities, such as Phytophthora citrophthora. Future research should focus on identifying alternative contamination control strategies that do not inhibit the growth of this economically significant pathogen.

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