Eco-Friendly Management of Penicillium Fruit Rot of Citrus

entry into fruit via wounds caused by insects, branches, or improper human handling during harvesting and transportation (Kellerman et al., 2016) [7] . The cycle of infection and sporulation can occur multiple times throughout the season, particularly in packing facilities. While the fruit rot rate generally ranges between 10-30%, it can escalate to as much as 50% under adverse conditions, resulting in substantial economic losses. Penicillium species also been reported to produce toxic secondary metabolites such as patulin and citrinin which are responsible to have negative effects on human health (Yu et al., 2020) [19] . Control of post-harvest diseases in citrus relies heavily on synthetic fungicides like imazalil, pyrimethanil, fludioxonil, and thiabendazole. However, their use raises concerns, including human health risks, environmental contamination, reduced fungicide efficacy due to resistant fungal strains, and restricted access to organic markets demanding lower pesticide levels (Palou, 2018) [13] . Consequently, safer, ecofriendly alternatives, such as plant extracts, medicinal plant extracts, citrus extracts, and microbial antagonists, are gaining attention for disease management. This study aims to assess the incidence of Penicillium rot in citrus, identify the pathogen, and explore eco-friendly management methods.

Materials and Methods

Collection, isolation and purification of the pathogen

Citrus fruits with typical rotting symptoms were collected from Dudia Talav market, Navsari. Diseased samples were surface sterilized with 0.1% sodium hypochlorite for 1 minute, rinsed with distilled water, and plated on potato dextrose agar (PDA). After incubation at 28 ± 2°C for 7 days, fungal growth was sub-cultured using the hyphal tip method for purification. The pure culture was maintained on PDA slants for further analysis.

Pathogenicity Test

To confirm the pathogenicity of the fungus, two inoculation methods were employed. In the Pin Prick Method, healthy citrus fruits were surface sterilized with 1% sodium hypochlorite and washed with distilled water. Sterilized pins were used to make small injuries on the fruit surface, and a spore suspension (10^6 spores/mL) was applied to the wounds. In the Deep Wound Method, fruits were disinfected with 1% sodium hypochlorite, rinsed, and dried. A sterile blade created wounds around the equatorial region, and the pathogen was inoculated into these wounds.

Cultural and morphological characterization

Cultural characterization of Penicillium digitatum was conducted by placing a 5mm mycelial disc from a sevenday-old culture onto sterilized PDA in Petri plates, followed by incubation at 28 ± 2°C for seven days. Observations on mycelial growth, colony color, and diameter were recorded. For morphological characterization, a small amount of pure culture was transferred to a glass slide from four positions on the culture plate. Measurements of conidia length and width, and conidiophore size and shape were taken using a light microscope at 40X magnification, with ten observations per characteristic, and mean values were calculated.

Eco-friendly management of penicillum fruit rot of citrus 2.4.1 Evaluation of antagonists against penicillium in vitro

In vitro evaluation of biocontrol agents was performed using the dual culture technique. The in vitro evaluation of various antagonists against Penicillium digitatum involved the following treatments: Trichoderma viride (T1), Trichoderma harzianum (T2), Bacillus subtilis (T3), and Pseudomonas fluorescens (T4), with a control group (T5). The experiment was conducted using a Completely Randomized Design (CRD) and included four replications for each treatment. A 5mm mycelial disc of the biocontrol agent and the pathogen was placed on the periphery of the Petri plate with a 60mm distance between them. For bacterial biocontrol agents, the plate was streaked, and the pathogen disc was placed at the corner. In the control, only the pathogen was placed in the centre. Plates were incubated at 27 ± 1 °C for seven days. Mycelial growth and percent growth inhibition (PGI) of the pathogen were recorded. PGI was calculated using the formula, Vincent, 1947 [17] .

PGI=(DC-DT)/DC×100

where PGI = Per cent growth inhibition, DC is the average colony diameter of the control (mm), and DT is the average colony diameter of the treated plate (mm).

Effect of plant extracts against fruit rot pathogen in vitro and in vivo

The efficacy of plant extracts against the fruit rot pathogen Penicillium was tested both in vitro using poisoned food technique and in vivo. In vitro, plant parts (100g) were washed, crushed with water, and filtered to prepare 1:1 extract, which were mixed with sterilized PDA medium at concentrations of 5, 10, and 15ml. Petri plates were inoculated with a 5mm mycelial disc of the pathogen, and mycelial growth and percent inhibition were recorded after seven days of incubation. In vivo, effective extracts were applied to disinfected, wounded citrus fruits. The wounded fruits were dipped in botanical solution and the control fruit was treated with same volume of sterile distilled water. After two hrs., of incubation at room temperature, each wound was inoculated with 25µl of spore suspension (10 6 spores/mL) of Penicillium, the fruits were stored for ten days, and disease severity was assessed. The botanical extract used are listed in Table 1 with CRD design and 3 replications.

Effect of edible chemicals on penicillium growth in vitro and in vivo

The impact of edible chemicals on Penicillium growth was evaluated both in vitro and in vivo. For the in vitro assessment, edible chemicals were mixed with PDA containing streptocycline at 250, 500, and 750ppm, and 5mm mycelial discs of the pathogen were placed on the plates. After seven days of incubation at 25 ± 1 °C, the mycelial growth and percent inhibition were measured. In vivo, the six most effective edible chemicals from the in vitro tests were applied to wounded citrus fruits, and disease severity was recorded on the eighth day, following the same method as used for plant extracts. The list of different chemicals used are listed in Table 2 with CRD design and 3 replications.

Results and Discussion

Survey for Penicillium Fruit Rot Incidence at Markets of Navsari

The weekly survey was carried out from first week of January to fourth week of April, 2021. The survey revealed the presence of Penicillium fruit rot pathogen (Penicillium digitatum) at Dudia Talav, Viraval and Ganadevi markets of Navsari. The data is presented in Table 3.1. Verma (2009) [16] observed that major fruit spoilage of Kinnow mandarins during the months of November to April was dominated by incidence of P. digitatum (5.18-8.85%) and P. italicum (2.59-4.82%) compared to remaining post-harvest fungal rot pathogens.

Isolation, purification and Pathogenicity test of fungal pathogen

The fungal pathogen responsible for citrus fruit rot was isolated from diseased samples collected at Dudia Talav market, Navsari, Gujarat. The tissue isolation technique was used to obtain a pure culture of Penicillium digitatum from naturally infected fruits. This pure culture was maintained on fresh PDA slants and stored under refrigeration for further investigation. The pathogenicity test of P. digitatum was carried out on healthy citrus fruits under in vitro conditions following both pin prick and deep wound methods. The results indicated that the symptoms appeared on the fruits after six days of inoculation which were identical to the symptoms appeared under natural infection. Pathogen re-isolated from such inoculated fruits was identical to the original pathogen thereby proving Koch's postulates.

Cultural and morphological characteristics

The cultural characteristics of P. digitatum were studied on PDA medium incubated at 27 °C. Colonies appeared olive green, fast-growing, irregularly shaped, and velvety to powdery in texture, with a diameter of 68mm and visible conidiophores and conidia (Photo 1). These findings align with earlier studies. El-Gali (2014) [3] observed P. digitatum forming radial colonies with white airy mycelia and olivegreen substrate mycelium on PDA, displaying a clear zone at the plate's bottom. Similarly, Khan et al. (2015) [8] reported bluish-green, fast-growing colonies with a powdery texture.

The morphological characterization of P. digitatum revealed hyaline, septate, branched mycelium with terverticillate conidiophores arising from subsurface or aerial hyphae. Cylindrical phialides (10.5-15.0 × 3.5-5.0 µm) produced smooth, green, ellipsoidal to cylindrical conidia (6.0-12.5 × 3.5-6.0 µm) (Photo 2). These findings align with standard descriptions by Khokhar et al. (2013) [9] , and Rasool et al.

(2014) [14] . [18] , and Ferreira et al. (2020) [5] , which highlighted the role of antagonists in inhibiting fungal growth via rapid nutrient uptake and antifungal compound production.

Eco-friendly management of

Effect of plant extracts against Penicillium digitatum in vitro

Six plant extracts, including turmeric, neem, tulsi, garlic, aloe vera, and eucalyptus, were tested at concentrations of 5%, 10%, and 15% to assess their in vitro efficacy against Penicillium digitatum using the standard poisoned food technique (Nene and Thapliyal, 1979) [11] . Observations on average colony diameter and percentage inhibition of linear growth are summarized in Table 3.2 and illustrated in Photo 4. At all tested concentrations (5%, 10%, and 15%), turmeric consistently showed the highest efficacy against Penicillium digitatum, with the lowest mycelial growth (2.59-3.06 mm) and the greatest growth inhibition (90.18-93.11%). Garlic ranked second, with growth inhibition ranging from 83.92% to 89.63%. In contrast, extracts of tulsi, neem, aloe vera, and eucalyptus demonstrated comparatively lower efficacy, with growth inhibition below 32% even at higher concentrations. A positive correlation was observed between plant extract concentration and fungal growth inhibition across all treatments. These findings highlight the superior antifungal potential of turmeric and garlic, as supported by the findings of Obagwu and Korsten (2003) [12] and Jhalegar et al. (2014) [6] , who similarly identified the effectiveness of garlic and aloe vera extracts against P. digitatum and related pathogens.

Effect of plant extracts against the pathogen in vivo

Phyto-extracts demonstrating efficacy in vitro were further assessed in vivo on citrus fruits using the fruit dip method at varying concentrations. Disease severity data was recorded to calculate the percent disease index (PDI) and decay reduction index (DRI) for each treatment (Table 3.3, Photo 5). All treatments effectively controlled the pathogen, achieving complete inhibition of Penicillium fruit rot compared to the untreated control after inoculation at 25 ± 1°C. Similar findings were reported by Daniel et al. (2015) [2] , who evaluated garlic extracts and clove oil for their ability to reduce decay caused by Botrytis cinerea, Penicillium expansum, and Neofabraea alba on apple cultivars, demonstrating reduced lesion diameters relative to controls.

Table 5: In vivo evaluation of plant extracts against P. digitatum

1 Turmeric 5 0.71 * (0.00)

Effect of edible chemicals on penicillium growth in vitro

Four edible chemicals-sodium bicarbonate, potassium metabisulphite, sodium chloride, and calcium chloride-were tested at 250, 500, and 750 ppm against Penicillium digitatum using the poisoned food technique depicted in Table 3.4. At 250 ppm, potassium metabisulphite showed the highest growth inhibition (93.78%), followed by calcium chloride (91.03%) and sodium bicarbonate (91.00%), while sodium chloride was less effective (58.70%). At 500 ppm, sodium bicarbonate (94.30%) and potassium metabisulphite (94.22%) were most effective, with sodium chloride at 73.00%. At 750 ppm, all except sodium chloride showed complete growth inhibition. The growth inhibition increased with higher concentrations.

These results are consistent with previous studies, such as Fallanaj et al. (2013) [4] , who found sodium bicarbonate effective against P. digitatum, and El-Gali (20140) [3] , who reported reduced growth with higher calcium chloride concentrations.

Effect of edible chemicals on penicillium growth in vivo

Edible chemicals effective in vitro were tested in vivo on citrus fruits using the fruit dip method. Disease severity data were used to calculate the percent disease index (PDI) and decay reduction index (DRI). All treatments significantly controlled the pathogen compared to the control, showing complete mycelial growth inhibition at 25±1°C depicted in Table 3.5.

Summary and conclusion

Citrus, the third most important fruit crop in India, faces severe post-harvest losses due to Penicillium digitatum and P. italicum, which cause fruit rot during storage and transit. A survey conducted in Navsari markets a disease incidence ranging from 5% to 26.7%, with the highest in Dudia Talav market in February. The pathogen P. digitatum was isolated, and its pathogenicity confirmed through Koch's postulates. Cultural studies showed olive-green, fastgrowing colonies, while morphological analysis identified hyaline, septate mycelium and green, ellipsoidal conidia. For management, P. fluorescens exhibited the highest mycelial growth inhibition (93.13%) among biocontrol agents, followed by T. harzianum (86.18%). Turmeric and garlic extracts were highly effective in controlling the pathogen both in vitro and in vivo. Among edible chemicals, sodium bicarbonate, potassium metabisulphite, and calcium chloride achieved complete inhibition at 750 ppm, while sodium chloride was less effective. These findings suggest that turmeric, garlic, and selected edible chemicals offer sustainable solutions for managing citrus fruit rot effectively.

Acknowledgments:

Authors are very thankful to the Department of Plant Pathology, Navsari Agricultural University, Navsari, Gujarat, India, for assisting and providing funds and facilities to carry out the research.

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