Background

Citrus fruits especially sweet orange is a good source of vitamin C, folic acid and fibres. In fact, sweet oranges are important exportable cash crops that serve as major source of foreign exchange to Nigeria prior to discovery of crude oil in 1951. In fact, it ranks as the most widely planted tree and earns substantial foreign exchange having produced 4.1 million tonnes in the year 2021 (FAOSTAT, 2023). The fruits are either eaten fresh or used for making canned orange juice. In Nigeria, the recommended varieties of sweet orange fruits include Hamlin, Valencia, Amber sweet, Agege Parson Brown, Umudike, Bende, Etir and Meran (Olaniyan et al., 2000). Nevertheless, the major cause of loss of orange fruits after harvest is pathological deteriorations. Thus, the potential foreign exchange earnings through the export of citrus fruits (sweet oranges) are under serious threats due to post-harvest infections, which could result in up to 40% yield loss. In fact, 40 to 50 percent of horticultural crops including fruits and vegetables are lost before they reach consumers while post-harvest losses in tropical fruits vary widely from 10 to 80 percent in both developed and developing countries.

Damage so produced by diseases and pests is probably the major cause of loss in orange fruits. It was reported that Pencillium species, especially P. digitatum and P. italicum with Geotrichum citriaurantii are the major mould species causing post-harvest decay of sweet oranges (Eckert and Ogawa, 1985; Ohr and Eckert, 1985). However, Lasiodiplodia sp IMI50324 was reported as a major post-harvest pathogen of orange fruits in Akure, Nigeria, accounting for over 75 % disease incidence of the total rots in their survey study (Oladele and Aborisade, 2015). The fungus was identified by ITS Rdna sequence analysis using the FASTA algorithm with the fungus data base from European Molecular Biology Laboratory (EMBL) and the sequence showed 100% identity to numerous ITS sequences described from different Lasiodiplodia species or their Botryosphaeria teleomorphs. Even best matches with the fungus included sequences of Lasiodiplodia species reported in peer reviewed literature (Orlandelli et al., 2012).

Similarly, Lasiodiplodia was also reported as a frequent pathogen among sweet oranges in Brazil (Slippers and Wingfield, 2007), which is consistent with (Dantas et al., 2003) results for ‘Pera’ variety where the Lasiodiplodia rot was 53% of the total fruit rots while Wright and Harmon (2009) reported Lasiodiplodia rot as an important post-harvest disease in warm and humid growing regions such as Florida and Caribean. Twenty-four species of Lasiodiplodia are known to have been distinguished based on their DNA phylogeny together with their conidial morphology and morphology and size of their paraphyses (Burgess et al., 2006). Generally, all the known species of Lasiodiplodia are associated with various symptoms such as dieback, root rot, fruits rot and leaf spots among many others (Punithalingham, 1980). So, Lasiodiplodia sp IMI50324 is a common soil pathogen associated with woody hosts in the tropics and which causes brown rot in orange fruit (Oladele and Aborisade, 2015). Initial infection shows as light brown discolouration on any area of the fruit surface. As the decay develops, the lesion becomes more brown, firm and slippery (Ismail and Zhang, 2004).

Currently, these pathogens are primarily controlled by application of fungicides either as dips, sprays, fumigants, treated wraps and box liners or in waxes and coatings. Citrus growers and sellers have routinely applied synthetic fungicides (chemicals) on their fruits for the management of post-harvest diseases. Numerous studies have been conducted to evaluate the effectiveness of various fungicides against common post-harvest pathogens affecting oranges, such as Penicillium digitatum and Penicillium italicum (Rosenberger et al., 2018), with none on Lasiodiplodia. Those studies provide important information for selecting appropriate fungicides and application protocols for combating post-harvest diseases in citrus fruits. 

Nonetheless, synthetic fungicides could result in health hazards during application process as well as residual accumulation in the fruits. Synthetic preservatives have raised concerns regarding their potential health risks and environmental impact, underscoring the need for innovative and eco-friendly solutions (Gupta et al., 2014). Besides, export markets are increasingly more sensitive to the use of chemicals for disease control coupled with the fact that most chemicals are expensive and inaccessible to local farmers who are the major bulk producers of this fruit in Nigeria. The escalating demand for safe and sustainable food preservation methods has prompted researchers to explore alternative solutions beyond conventional synthetic preservatives. Hence, the use of non-chemical ecofriendly means of control such as botanicals have emerged as viable alternatives.

Besides, the rising awareness of health and environmental concerns associated with synthetic additives has led to a renewed interest in natural antimicrobial compounds derived from plant sources, thus making the utilization of plant-derived compounds as potential alternatives to synthetic additives to gain prominence, Therefore, the research investigated the antifungal efficacy of aqueous extract of Tridax procumbens against brown rot pathogen of orange fruits during ambient storage and its potential as a suitable alternative to synthetic preservatives. Phytochemical and bio active compositions of the aqueous extract was also investigated.

1 Materials and Methods

1.1 Source of fruits

Mature, green healthy orange fruits were harvested from a commercial orchard in September 2023 from a citrus farm in Igbatoro, Akure North, Nigeria. Fruits of uniform size and colour were selected. Before treatment, the fruits were washed with clean water, disinfected for 10 min in 10 % sodium hypochlorite and allowed to air-dry at room temperature.

1.2 Preparation of spore suspension

A ten-day old agar slant culture of Lasiodiplodia sp (IMI Number: 503248) on malt extract agar (MEA) was used to prepare spore suspension. Sterile water was poured into the slant and shaken vigorously to dislodge the spores from the vegetative hyphae. The wash water was collected in a sterilized beaker. One milliliter of the suspension was spread on an area of 1 cm² and allowed to dry on a clean microscope slide before counting spores using the formula of Breed Direct Counting Technique (Ogundana, 1989) under the high dry ×40 objective microscope (Olympus).

1.3 Pathogenicity test

Spore suspension of Lasiodiplodia was used to inoculate fresh fruits 1mm deep at the equator and incubated at 28±2℃ and 75% relative humidity inside sterilized desiccators. The disease symptoms were noted and re-isolation from infected fruit tissue was performed on fresh sterile MEA plate and its cultural characteristics were compared with the original isolate.

1.4 Preparation of extract of Tridax procumbens

Detached Tridax procumbens leaves were air dried for 14 days. The dried leaves were pulverized with a blender to a smooth powdery form. About 28 g of the pulverized leaves were dissolved in 1 L of water, thoroughly shaken together and later filtered with a muslin cloth to obtain the crude /stock solution. Varying concentrations (5%, 10%, 15%and 100%) of the aqueous extract were then prepared from the stock / crude extract with appropriate volumes of water.

1.5 Inoculation and Treatment of orange fruits with Tridax procumbens extract

The orange fruits were artificially infected with spores (6.50×10⁴ cfu/ml) of Lasiodiplodia and later dipped separately into each concentration (5%, 10%, 15% and 100%) of the prepared extract for 5 min while benlated (fungicide) and untreated orange fruits served as positive and negative control respectively. Each set up consisted of 3 fruits. After treatment, the fruits were then placed inside a sterilized Petri dish and transferred into desiccators, and stored at 28± 2℃ and 75% relative humidity and assessed daily for disease severity。

1.6 Assessment of rot severity

Assessment of disease severity was done using the scale of (Arekemase and Oyeyiola, 2007) but with slight modification where 1=disease free, 2=slight rot /decay up to 10% of the fruit, 3=moderate rot /decay up to 25% of the fruit, and 4=severe rots/decay ≥35% of the fruit surface. Rot/decay was recognized by light brown discolouration on the fruit or by appearance of mycelium on the fruit surface.

1.7 Phytochemical screening of the aqueous extract of Tridax procumbens

The phytochemical screening of the aqueous extract was done according to the method described by (Trease and Evans, 2004). The phytochemicals screened for were tannin, saponin, phlobatinnin, flavoniod, alkaloid and cardiac glycosides.

1.8 Gas chromatography-mass spectrophotometry (GC-MS) of Tridax procumbens extract

The Tridax extract was subjected to chromatographic analysis using a Varian 3800/4000 gas chromatograph mass spectrometer equipped with an Agilent Technologies and a BP5 (30 m×0.25 mm×0.25 microns) capillary column. Nitrogen was used as a gas carrier. 1.0 µL volumes of the extract were injected using a splitless mode at an injector temperature of 270 ℃. The oven temperature was ramped from 80 to 200 ℃ (1 min hold) at a rate of 5 ℃/min. The oven temperature was held at 280 ℃ for 6 min following each analysis. The total run time for each sample was approximately 45 min. The GC-MS interface temperature was set to 280 ℃. The peaks of the organic compounds in the samples were identified in Wiley’s NIST 08 Mass Spectral Library and expressed in terms of its balance and retention time.

1.9 Statistical analysis

The data obtained for the disease severity were subjected to analysis of variance and where significant, the means were compared at 5% level of probability using Tukey’s Test (SPSS version 20).

2 Results

2.1 Effects of different concentrations of Tridax procumbens extracts on disease severity in orange fruits pre- inoculated with Lasiodiplodia sp and stored at 28±2 ºC and 75 % relative humidity

All treated and control fruits recorded mean disease severity values of 1.00±0.00 by day 5 of storage (Figure 1), indicating that they were all disease free. As storage duration progressed till day 10, except the control fruits that had shown slight rottenness as evident by their mean disease severity values of 2.00±0.52, all treated fruits were still disease free (healthy), though with mean disease severity values that were not significantly different (p≥0.05) (Figure 2). For instance, fruits treated with both 15 and 100 % extracts of T. procumbens still maintained 1.00±0.00 as their disease severity values while fruits treated with 5, 10 % extracts of T. procumbens and fungicide had their severity values increase non-significantly (p ≥ 0.05) to 1.67±0.44, 1.33±0.38 and 1.33±0.38 respectively (Figure 2).

Figure 1   Effect of different concentrations of Tridax procumbens on disease severity in orange fruits pre-inoculated with Lasiodiplodia sp and stored for 5 days

Figure 2 Effect of different concentrations of Tridax procumbens on disease severity in orange fruits pre-inoculated with Lasiodiplodia sp and stored for 10 days

Meanwhile by day 15 in storage, only fruits treated with 15 and 100% T. procumbens extracts maintained their diseased free status (1.00±0.00) when compared with control and other treated fruits. In fact, the severity values of control fruits had increased to 3.00±0.33 (moderate rottenness) while all the other treated fruits recorded same severity values (2.00±0.25) and had started showing slight rottenness (Figure 3). By day 20 in storage, only fruits treated with 15 and 100% T. procumbens extracts still maintained their diseased free status (1.00±0.00) when compared with control and other treated fruits (Figure 4). Unfortunately, the control fruits had become completely rotten (4.00±0.52) but fruits treated with 5 and 10% extracts of T. procumbens and fungicide still showed slight rottenness as evident by their respective severity values of 2.67±0.44, 2.50±0.50 and 3.00±0.33 (Figure 4).

Figure 3 Effect of different concentrations of Tridax procumbens on disease severity in orange fruits pre-inoculated with Lasiodiplodia sp and stored for 15 days

Figure 4 Effect of different concentrations of Tridax procumbens on disease severity in orange fruits pre-inoculated with Lasiodiplodia sp and stored for 20 days

Again by day 25 in storage, except fruits treated with 15 and 100% T. procumbens extracts that were still diseased free and control fruits that had become completely rotten, the severity values of all the other treated fruits with fungicide, 5 and 10% T. procumbens extracts had increased significantly to 3.67±0.67, 3.33±0.37 and 3.00±0.33 respectively, indicating moderate rottenness (Figure 5). The same trend of results was observed for all the fruits (control and treated) as the storage durations became further extended to day 30 (Figure 6).

Figure 5 Effect of different concentrations of Tridax procumbens on disease severity in orange fruits pre-inoculated with Lasiodiplodia sp and stored for 25 days

Figure 6 Effect of different concentrations of Tridax procumbens on disease severity in orange fruits pre-inoculated with Lasiodiplodia sp and stored for 30 days

2.1 Phytochemical compositions of the aqueous extract of Tridax procumbens

Tannins, alkaloids, cardiac glycosides, saponins and flavonoids were all detected in the Tridax extracts (Table 1).

Table 1 Phytochemical constituents in Tridax procumbens

2.2 Bioactive compounds detected in aqueous Tridax procumbens extracts

Table 2 shows the bioactive compounds with their peak area (%) detected in Tridax extract included 2,3-Butanediol (10.05), Hexanoic acid (11.77), Benzaldehyde,4-ethyl-(12.92), 4H-Pyran-4-one, 2,3 dihdro-3,5-dihydroxyl-6-methy-(6.89), Ethanone,1-(2-hydroxy-5-methylphenyl) (2.58), Methyl 4-(hydroxymethyl) benzoate (8.04), p-Menth-3-en-9-ol (3.73), Eugenol (1.44), Acetamide, N-tetrahydrofurfuryl-2-methoxy-(4.30), n-Hexadecanoic acid (12.64), 9,12-Octadecadienoic acid methy ester (1.03), Oleic acid (10.33), Octadecanoic acid (6.32) while the chromatograph of the aqueous extract of Tridax procumbens using GC-MS is shown in Figure 7.

Table 2 Bioactive compounds detected in aqueous Tridax procumbens

Figure 7 Chromatogram of aqueous extract of Tridax procumbens

3 Discussion

This study explored the efficacy of aqueous leaf extracts of Tridax procumbens in controlling brown rot of orange fruits during ambient storage. In fact, the study showed that all the orange fruits treated with all the varying concentrations (5, 10, 15 and 100%) of the Tridax extracts were disease free (healthy), showing no sign of any infection by day 10 in storage, whereas the control fruits had started showing slight rottenness. Even when the storage durations became extended till day 30 and the control fruits had become completely rotten, fruits treated with 15 and 100% T. procumbens extracts were still disease free, without any sign of infection. Even performed better than the fungicide used (mancozeb) because all mancozeb treated fruits were moderately rotten by day 30. Though Rosenberger et al. (2018) demonstrated the effectiveness of various fungicides in controlling post-harvest rots of stone fruits, this work has further confirmed the use of botanicals in extending the shelf life of fruits better than fungicide because the aqueous leaf extracts of Tridax procumbens particularly 15 and 100% extracts, best protects the orange fruits for 30 days with no sign of brown rot/infection. This observation is also consistent with the works of Efunwoye et al. (2024) who observed that all the Padma tomato fruits treated with Tridax procumbens in their investigation exhibited significantly lower disease incidences (p ≤ 0.05) than the control fruits throughout the storage period.

This showed the efficacy of botanicals in controlling fruit pathogens as equally buttressed by Zakawa et al. (2018) who investigated the use of neem leaf extracts in the control of post-harvest fungal pathogens on mango fruits in Yola, Adamawa state, Nigeria emphasizing the potential of the extract as a biofungicide and Udomlak et al. (2008) who demonstrated the antifungal efficacy of clove and cinnamon oil in preventing post-harvest decay in grape fruits, showing the synergistic potential of both oils in combating post-harvest fungi of grape fruits in vitro. Equally, Oladele (2019) investigated the antifungal activity of garlic extracts against three post-harvest pathogens that affect fruits, demonstrating garlic's potential as a natural preservative. In recent years, researchers have investigated the antimicrobial and antioxidant properties of Tridax procumbens extracts in fruit preservation. No wonder, Narendhirakannan and Subramanian (2005) reported that Tridax procumbens has been traditionally employed in various medicines for its pharmacological properties and antimicrobial activities in food preservation. Similarly, it has been reported that T. procumbens has played a major role in food preservation due to its rich phytochemical compositions with potential antimicrobial properties (Gupta et al., 2019). Notably, tannins, alkaloids, cardiac glycosides, saponins and flavonoids were all detected as phytochemical constituents in the Tridax extracts in this work. This is in consonance with the findings of Jisha et al. (2016) and Palou et al. (2001) who reported a number of active chemical constituents such as alkaloids, flavonoids, carotenoids, β-sitosterol, fumeric acid, myristic, palmitic, arachidic, linoleic acid and tannin from botanicals and all these phytochemical composition, makes the plant a natural bio pesticide with potential antimicrobial efficacy against a broad spectrum of microorganisms (Nair et al. 2015; Mishra et al., 2018).

Each of these phytochemical constituents as enunciated by others authors, is connected with significant antimicrobial properties. For instance, alkaloids and flavonoids when present exhibit antimicrobial properties. These compounds inhibit the growth of spoilage-causing microorganisms, extending the shelf life of fruits (Das, 2014). These compounds possess antioxidant properties, which play a crucial role in fruit preservation by inhibiting oxidative processes and prevent the degradation of fruits by scavenging free radicals that contribute to spoilage (Nair et al., 2015). This is further buttressed by the reports of Vasudevan et al. (2017) that alkaloids and flavonoids act as natural antimicrobial agents, inhibiting the growth of bacteria and fungi that contribute to fruit spoilage. Also, tannins act as natural preservatives by forming complexes with proteins and enzymes, thereby inhibiting microbial growth and enzymatic browning (Okigbo et al., 2010).

Even the GC - MS analysis of the Tridax extract revealed a lot of bioactive compounds and these natural compounds exert their effects through various mechanisms, making them effective against different types of microorganisms. For instance, n-Hexadecanoic acid identified in the extract is reported to have pesticide activities (Rajasekaran and Cary, 2014). Also, 9-octadecenoic acid methyl ester possesses anti-microbial properties while stearic acid were also found to exhibit antibacterial and antifungal activities (Gehan et al., 2009). Remarkably, the presence of bioactive compounds in Tridax procumbens may contribute to natural pest resistance, presenting opportunities for sustainable pest management in agriculture. The method typically involves the application of plant extracts onto the fruit surface or as part of coatings, creating a protective barrier against fungal colonization. Besides, their anti-microbial properties inhibit the growth and development of post-harvest pathogens, thereby preserving the quality of the orange fruits.

4 Conclusions

The study demonstrates antifungal efficacy of the aqueous extracts of Tridax procumbens against Lasiodiplodia sp (IMI: 50324), the brown rot pathogen of amber sweet orange fruits. The findings reveal the effectiveness of the aqueous extract at all tested concentrations against the brown rot pathogen by keeping the infected orange fruits healthy (disease free) for the first 10 days of storage under ambient temperature when compared with the untreated fruits. Further investigations even show that 15 and 100% concentrations of the aqueous extract still maintain the healthy status (wholesomeness) of the orange fruits for 30 days without any sign of rottenness as against the untreated and fungicide treated fruits that had already become unwholesome and diseased. Thus, we adjudge 15 and 100% of the Tridax aqueous extracts the most effective among the tested concentrations. They show potential for further development as safe, ecofriendly and effective natural substitutes against fruit pathogens.

Author’s Contributions

Oladele O.O. carried out the conceptualization, methodology, investigation, supervision, formal analysis and writing of original manuscript while Oyedokun F.D. and Adebomi D.M. were involved in the methodology, investigation, resources and data analysis. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Life Technologies, UK for PCR purification and sequencing reactions of the fungi isolate.

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