Post-harvest fungal diseases of Vazhakulam pineapple: Isolation, identification, and management strategies

Introduction

Pineapple (Ananas comosus) is one of the most valuable and widely cultivated fruits globally, with India ranking among its major producers. This tropical fruit is highly appreciated for its exceptional juiciness, vibrant flavor, and notable health-promoting properties. As a non-climacteric fruit with rich nutritional content, pineapple is consumed fresh or processed into various products, including canned slices, juices, jams, jellies, fiber, and even wine (Lalhruaitluangi et al., 2024) [11] . Fruits are broadly classified as climacteric or non-climacteric based on their ripening behavior. Climacteric fruits exhibit a marked rise in respiration and ethylene production during ripening and can continue to ripen after harvest. In contrast, pineapple is a nonclimacteric fruit, showing no post-harvest respiratory peak and little improvement in flavor or sweetness once removed from the plant. As a result, pineapples must be harvested at optimum maturity and handled with great care during post-harvest operations to preserve their quality, as ripening cannot compensate for poor harvesting, storage, or transport conditions. (Coates et al., 1997) [4] . Pineapple availability in most markets fluctuates with the seasons, and its naturally high perishability makes it difficult to store and transport for long periods. As a result, substantial post-harvest losses occur, which significantly reduce the volume that actually reaches consumers and processors. Consequently, these losses have been identified as a major factor contributing to the overall decline in pineapple production. Such losses result around 24 percent reduction with gross margin of pineapple farmers. The reasons may be attributed to cultivable area, transportation, storability and number of bruised fruits (Mirza et al., 2016) [18] . [4] . The process of infection follows the development of fungal penetrating structure called appressorium. The colonization process by fungi involves their ability to establish themselves within the host. This is initiated when the fungi following adhesion and release of enzymes depolymerize certain cell wall polymers such as prospection, the cementing substance of the produce (Onuorah et al., 2013) [20] . The pineapple primary cell wall contains roughly 10% proteins and 90% polysaccharides, mainly comprising cellulose, hemicellulose, and pectin. Many fungal pathogens secrete a wide range of cell wall-degrading enzymes that enable them to penetrate and utilize these structural components as nutrients. Their abundant production of extracellular pectinases and hemi-cellulases is a key factor contributing to pineapple spoilage, ultimately shortening post-harvest life and causing quality deterioration and fruit rot.

The long-standing challenge of post-harvest decay in fruits and vegetables remains unresolved, as evidenced by consumers who continue to discard spoiled produce despite selecting high quality fresh items. Traditional reliance on post-harvest fungicide applications has diminished due to increasing pathogen resistance, limited availability of alternative chemicals, public concerns over pesticide safety, and resulting regulatory constraints (Janisiewicz and Lise., 2002) [8] . Effective post-harvest fungal management is vital for maintaining fruit quality, ensuring food safety, and minimizing economic losses, and can be achieved through diverse biological strategies that enhance the commodity's natural defence systems while employing antagonistic microorganisms and natural antimicrobial agents (Mari et al., 1998) [16] .

Materials and methods

Samples collection

Pineapple fruits (both diseased and healthy) were collected from Vazhakulam, one of Asia's largest pineapple markets supplying fruit across India. The samples were placed in separate sterile polythene bags and transported to the Plant Pathology Laboratory, Pineapple Research Station, Vazhakulam, for further analysis

Isolation and identification of fungus:

The collected fruits were aseptically cut into small segments (approximately 3 mm in diameter) using a sterile blade. The segments were surface sterilized in 1% sodium hypochlorite for 1 min, followed by rinsing with sterile water to remove residual sterilant. The sterilized tissue pieces were then placed on sterile Potato Dextrose Agar (PDA) plates supplemented with antibiotics to suppress bacterial growth. The plates were incubated in an inverted position at room temperature for 5 days. Emerging fungal colonies were sub-cultured to obtain pure cultures (Guo et al., 2023) [7] . [20,6,19,17,20] .

Pathogenicity test & Re-isolation

Pathogenicity tests were conducted to verify Koch's postulates. Healthy pineapple fruits were washed with tap water, rinsed with sterile distilled water, and surface sterilized with 75% ethanol. The fruits were wounded and inoculated by injecting the fungal suspension using a sterile syringe. Non-inoculated fruit served as control.

All fruits were incubated at room temperature and observed regularly for symptom development (Fig. 1). After symptom appearance, the pathogen was re-isolated and compared with the original culture to confirm Koch's postulates (Guo et al., 2023) [7] .

Evaluation of Antifungal Activity of Biocontrol Agents and Natural Oil

Microbial biocontrol agents-Bacillus subtilis (Sting), Ampelomyces quisqualis (Biodewcon), Trichoderma viride (Biocure F), and Pseudomonas fluorescens (Biocure B)-at 1.5% concentration, along with 100% tea tree oil as a botanical treatment, were evaluated for their antifungal efficacy using the agar well diffusion method (Magaldi et al., 2004) [13] with slight modifications. The plates were incubated at room temperature, and antifungal activity was assessed by measuring the zone of inhibition around each well. All experiments were conducted in triplicate.

Results

Symptomatology and Identification of fungus

Examination of the symptomatic pineapple fruits revealed distinct post-harvest fungal diseases including those caused by molds (figure 2, figure 3, figure 4) and yeast (figure 5).

Samples showing dark, water-soaked lesions beginning at the stalk end, progressing internally with firm black necrosis and characteristic tar-like mycelial masses were diagnosed as Ceratocystis paradoxa, confirming black rot. Fruits exhibiting irregular brown to black surface lesions, tissue softening, and rapid decay were associated with Curvularia spp., and cultural and microscopic characteristics verified Curvularia fruit rot. In contrast, fruits that appeared externally healthy but displayed internal browning of individual fruitlets, tissue breakdown around the core, and blue-green sporulation were identified as Penicillium spp., consistent with fruitlet core rot. Candida spp. produced cream-colored, smooth, moist, circular, convex colonies with distinct margins. (Table 1)

Activity of biocontrol agents & natural oil against isolated fungus

The in vitro inhibition assay revealed limited antifungal activity of the tested biological control agents. Bacillus subtilis (Sting), Ampelomyces quisqualis (Biodewcon), Trichoderma viride (Biocure F), and Pseudomonas fluorescens (Biocure B) did not produce measurable zones of inhibition against Ceratocystis paradoxa, Curvularia spp., Penicillium spp., or Candida spp. under the experimental conditions. (figure 6).

In contrast, tea tree oil exhibited notable antifungal activity (table 2), with the highest inhibition observed against Penicillium spp. followed by Ceratocystis paradoxa, Curvularia spp. and Candida spp. The antifungal efficacy of tea tree oil is likely associated with its volatile bioactive constituents that disrupt fungal cell membrane integrity.

Discussion

Post-harvest diseases of pineapple are caused not only by filamentous fungi but also by yeasts and yeast-like fungi, which contribute to fruit decay, off-flavors, and reduced marketability during storage and distribution (Snowdon, 2010) [22] . The isolation of Ceratocystis paradoxa, Curvularia spp., Penicillium spp. and Candida spp. from market samples highlights the complex fungal and yeastassociated microbiota involved in post-harvest deterioration of pineapple. The present findings align well with previously documented post-harvest pathogens associated with pineapple. Penicillium spp., identified in the current samples as the causal agent of fruitlet core rot, has also been reported from decaying pineapple fruits by Onuorah et al., 2013 [20] , confirming its relevance as a major spoilage fungus in the supply chain. Similarly, the isolation of Curvularia spp. corresponds with the work of Ferreira, who demonstrated its role in inducing post-harvest fruit rot in pineapple. In addition, the detection of Ceratocystis paradoxa causing characteristic black rot supports earlier observations by Oniah et al., 2018 [19] emphasizing its importance as a destructive pathogen particularly under poor handling and storage conditions. Yeast-associated pineapple fruit rot has been reported by Korres et al. (2010) [10] , who identified yeasts such as Candida spp., Saccharomyces spp., and Kloeckera spp. in collapsed pineapple fruits and demonstrated their role in fruit decay through pathogenicity tests.

Previous studies report that during harvesting, storage, packaging, and transportation, fruits are prone to physical damage, which increases post-harvest losses and the likelihood of fungal contamination. This risk is further amplified by inadequate fruit management in markets. Unsanitary marketing environments characterized by poor vendor hygiene, the use of microbially unsafe containers, improper handling practices, and unfavorable environmental conditions create situations that promote contamination and spoilage (Mailafia et al., 2017) [14] . The effectiveness of essential oils observed in the present study is consistent with earlier reports. Post-harvest application of essential oils has been shown to improve quality and extend the shelf life of Indian pineapple (Mandal & Vanlalawmpuia, 2020) [15] and to effectively control fusariosis (Vilaplana et al., 2018) [24] . Similarly, tea tree oil has been reported to reduce post-harvest decay in strawberries (Shao et al., 2013) [21] . The antifungal potential tea tree oil has been further demonstrated against Botrytis cinerea in strawberries, where concentrations of 125 and 1000 μL L⁻¹ resulted in 10% and 48% reductions in the mycelial growth index, respectively, indicating a concentration-dependent suppression of pathogen development (Lorenzetti et al., 2011) [12] . Comparable inhibitory effects were also reported by Souza et al in 2015, who observed a reduction in the mycelial growth rate of Stemphylium spp. following treatment with tea tree oil. These findings, together with the results presented in Table 2, highlight the potential of tea tree oil as a natural alternative for managing post-harvest fungal diseases of pineapple, warranting further in vivo evaluation. Botanical antifungal agents have gained increasing attention in post-harvest disease management due to growing concerns over fungicide residues, pathogen resistance, and environmental safety. Essential oils, including tea tree oil, are particularly attractive as they are of natural origin and generally well accepted by consumers seeking residue-free and eco-friendly alternatives (Bakkali et al., 2008) [2] . Tea tree oil demonstrated strong in vitro antifungal activity against the isolated post-harvest pathogens, corroborating earlier reports of its broad-spectrum efficacy against fungi and yeasts through membrane disruption and interference with cellular metabolism (Carson et al., 2006) [3] . Consumer acceptance plays a critical role in the adoption of botanical treatments in post-harvest systems. While essential oils are perceived as safe and natural, their strong aroma and volatility may influence sensory quality, necessitating careful dose optimization and formulation strategies. In addition to post-harvest application, pre-harvest use of botanicals has been reported to reduce field inoculum levels, thereby lowering post-harvest disease incidence and delaying symptom development during storage (Tripathi et al., 2008) [23] .

Conclusion

Post-harvest deterioration of pineapple is caused by a diverse group of fungal pathogens that significantly compromise fruit quality during marketing and storage. The present investigation confirmed the occurrence of major post-harvest diseases in the Vazhakulam market, including black rot caused by Ceratocystis paradoxa, fruit rot associated with Curvularia spp. and Candida spp. and fruitlet core rot caused by Penicillium spp., highlighting the persistent risk of microbial spoilage during post-harvest handling and distribution. Among the evaluated treatments, tea tree oil exhibited notable in vitro antifungal activity against Penicillium spp., Ceratocystis paradoxa, Curvularia spp. and Candida spp., indicating its potential as a botanical alternative for post-harvest disease management, while the tested biocontrol agents were ineffective under the experimental conditions. The growing consumer demand for natural and residue-free treatments further supports the exploration of botanical fungicides such as tea tree oil. However, successful commercial application will require optimized formulations, assessment of sensory impacts, and validation of both pre-harvest and post-harvest application strategies, along with improved market hygiene practices including sanitation of handling equipment, minimization of mechanical injury, and proper storage conditions to effectively reduce inoculum spread and disease incidence.

Acknowledgement

AcknowledgementsWe are grateful to Kerala Agricultural University, Thrissur for providing research facilities and financial support.

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