INTRODUCTION
Introduction
Mango, often referred to as the "king of fruits," is a vital and highly valued fruit in Vietnam, known for its significant economic benefits The selling price ranges from 25,000 to 30,000 VND per kilogram, with off-season prices reaching up to 50,000 VND per kilogram Mango cultivation yields impressive revenues of approximately 350-450 million VND per hectare and can be intercropped with low-canopy trees to enhance farm income This high-value crop not only generates profits but also creates jobs, helping to alleviate poverty and contributing to Vietnam's development Additionally, mango cultivation has expanded to various regions worldwide, including Africa, the Americas, and the Caribbean, while its consumption continues to rise in developed countries.
Mango contains a variety of beneficial chemical compounds, including phenolic compounds, polyphenols, phenolic acids, hydrocarbons, fatty acids, amino acids, and triterpenes These compounds have been shown to exhibit numerous biological activities, such as anticancer, anti-inflammatory, antidiabetic, antioxidant, antibacterial, antifungal, anthelmintic, gastroprotective, hepatoprotective, immunomodulatory, antiplasmodial, and antihyperlipidemic effects (Ediriweera et al., 2017).
Mango production faces significant challenges due to losses occurring at various stages, including field, storage, transit, and handling processes from growers to consumers (Chukwuka et al., 2010; Barth et al., 2013) The storage phase is particularly critical, as mangoes are prone to spoilage Factors such as high concentrations of sugars, minerals, vitamins, amino acids, and low pH levels create an environment conducive to the growth of saprophytic and parasitic fungi (Bhale, 2011) Notably, the most common pathogenic fungi found in mangoes include Colletotrichum gloeosporioids, Botryodiplodia theobromae, and Dothiorella dominicana.
D Mangiferae, Phomopsis mangiferae and Aspergillus niger (Sangchote, 1987) Species belonging to Rhizopus, Aspergillus, Colletotrichum, Botrydiplodia, Phomopsis and Diplo- dia have been reported to cause rotting of fruit during transit, storage and marketing inju- ries (Dasgupta and Bhat, 1946; Kanitkar and Uppal, 1939; Thakur and Chenulu, 1970; Laxinarayan and Reddy, 1975; Thakur, 1972) Fungal spoilage created unpleasant odours and flavor, reduced quality and caused foodborne disease (Rawat, 2015)
The decline in mango productivity is adversely impacting the economy This study aims to isolate and analyze the morphological and biochemical characteristics of fungal strains affecting post-harvest mangoes in various markets in Gia Lam district The findings will help identify effective control measures for these isolated fungal strains in mango fruits.
Objective
Isolation, determination the morphological and biochemical characteristics of pathogenic fungi associated with spoilage of mango fruits in the different markets of Gia Lam district.
Requirement
Isolate and select of fungi associated with spoilage of mango fruit in the different markets of Gia Lam district area
Re-infect the fungi into the healthy mangoes
Study biological characteristics of fungal strains causing spoilt on mango fruits: morphology colony, mycelium and spore of fungus
Evaluate of the ability of extracellular enzyme production of isolated fungal strains Examine some factors affecting on the growth of those fungal strains.
LITERATURE REVIEW
General introduction of mango
The mango (Mangifera indica L.) is a tropical stone fruit from the Anacardiaceae family, primarily cultivated for its delicious edible fruit Native to South Asia, particularly eastern India, Burma, and the Andaman Islands, mangoes have been cherished and cultivated since ancient times Recognized as the national fruit of India and the national tree of Bangladesh, Mangifera indica L has gained global distribution, making it one of the most widely cultivated fruits in tropical regions.
Mangoes, originally from South Asia, particularly eastern India, Burma, and the Andaman Islands, have been cultivated in India for 4,000 to 6,000 years The first recorded introduction of mangoes to the outside world was by Hsuan-tsang, a Chinese Buddhist monk, who highlighted their significance Subsequently, mangoes were transported on voyages to Malaya and eastern Asia, expanding their reach and popularity.
Mangoes were first carried to China by Buddhist monks in the 7th Century and spread to East Africa by the 10th Century AD, reaching the Philippines in the early 15th Century The rapid dissemination of mangoes from South and Southeast Asia began in the late 15th Century, with introductions to West Africa and Brazil in the early 16th Century After establishing in Brazil, mangoes were introduced to the West Indies, first planted in Barbados around 1742, and later in the Dominican Republic, Jamaica in 1782, Hawaii in 1809, and Mexico and America during the 19th Century The popularity of mangoes surged globally, with many popular varieties today originating from Florida, USA The widespread geographic popularity of mangoes was largely achieved in the second half of the 19th century, reaching locations as distant as Florida, Hawaii, Fiji, Queensland, and Natal.
The taxonomic classification of the common mango is:
Kingdom – Plantae Class – Magnoliopsida Phylum – Magnoliophyta Order – Sapindales Family – Anacardiaceae Genus – Mangifera Species – Indica b Distribution of mango in the world
In 2013, the global mango production reached around 43 million metric tons This figure increased significantly to around 55 million metric tons in 2018 ( Amber Pariona, 2018; Shahbandeh, 2020)
From 2000 to 2018, India emerged as the world's leading mango producer, contributing over 18 million tons, which accounted for nearly 50% of the global mango supply by 2018 The primary mango-producing states in India include Andhra Pradesh, Bihar, Gujarat, Karnataka, Maharashtra, and Orissa, although many other states also engage in mango cultivation As of 2018, approximately 2,309,000 acres were dedicated to mango farming across the country.
China and Thailand were the next largest producers with 4.77 and 3.4 million tons of mango, respectively (Amber Pariona, 2018)
Figure 2.2: World's top 25 largest mango producers in 2018 (tonnes)
The wholesale price of mangoes is influenced by factors such as size and variety, with the Free on Board (FOB) price for imported mangoes in the US ranging from about $4.60 to $5.74 per 4kg box in 2018, according to the United States Department of Agriculture (NMB crop reports, 2019) Additionally, the distribution of mangoes in Vietnam plays a significant role in the market dynamics.
Vietnam is the 13th largest mango producer globally, with over 87,000 hectares dedicated to mango cultivation and an annual yield of approximately 969,000 tons This tropical fruit is primarily grown in the Mekong Delta, which accounts for 48% of the country's total mango area In central Vietnam, Binh Thuan, Ninh Thuan, Khanh Hoa, and Binh Dinh provinces are key production areas, with Khanh Hoa leading at 8,000 hectares and a yield of 60 quintals per hectare Additionally, Son La province is noted for having the largest mango cultivation in northern Vietnam, covering 4,300 hectares.
In Vietnam, the majority of mangoes are consumed domestically, with only 4% of total production being exported The primary border trade export market is China Officially, the main export destinations for Vietnamese mangoes are South Korea, which accounts for 43% of total exports with 1,181 tons, followed by Japan at 34% with 934 tons, and Singapore at 7% with 186 tons (vntrade, 2016).
Table 2.1: Acreage and yield of mango in different regions in Vietnam in 2017
Figure 2.3: Map of mango in Vietnam
A report from the Agency of Foreign Trade under the Ministry of Industry and Trade reveals that mango imports surged by 99.9% to $2.79 million compared to 2019, based on U.S official figures The average import price increased by 6.7% to $2,064.8 per tonne for fresh and frozen fruit.
The United States ranks as the 12th largest mango import market for Vietnam, representing 0.3% of the total mango imports According to the Agency of Foreign Trade, the U.S presents a significant opportunity for Vietnamese companies, particularly in the fresh fruit sector However, it is crucial for exporters to adhere to strict standards regarding farming practices, packaging, and origin tracing Notably, Vietnam's inaugural mango export to the U.S took place in April 2019.
The tropical mango tree is a tall, erect tree with a broad, rounded canopy that can reach heights of 30 to 54 meters and widths of 30 to 38 meters, primarily found in the rainforests of South and Southeast Asia Its root system extends up to 20 feet deep, allowing it to thrive in various soil conditions Known for their longevity, some mango trees can bear fruit for up to 300 years The evergreen leaves are simple and alternate, measuring 15–35 cm long and 6–16 cm wide, transitioning from orange-pink to dark green as they mature Mature trees can produce hundreds to thousands of small, fragrant flowers, which are typically white with five petals and appear in terminal panicles With over 500 varieties of mangoes, most experience spoilage in summer, while some can yield a double crop The flowering to ripening period lasts approximately four to five months.
Ripe mangoes exhibit a wide range of characteristics based on their cultivar, including variations in size, shape, color, sweetness, and overall fruit quality These fruits can display colors such as yellow, orange, red, or green, and come in shapes like round, oval, or kidney-shaped Depending on the cultivar, mangoes can measure between 5 to 25 cm in length and weigh anywhere from 140 grams to 2 kilograms Each mango contains a single flat, oblong pit that may be fibrous or hairy and is difficult to separate from the pulp The skin of the mango is smooth, waxy, and fragrant, with colors that transition from green to yellow, yellow-orange, yellow-red, or blushed as they ripen, ultimately showcasing shades of red, purple, pink, or yellow when fully ripe.
Ripe mangoes emit a unique sweet and resinous aroma Each mango contains a single seed, which is encased in a thin lining measuring 1–2mm thick and is 4–7cm long The seeds of mangoes are recalcitrant, meaning they do not endure freezing or drying However, mango trees can be easily grown from seeds, with the highest germination success achieved when seeds are sourced from mature fruits.
Fruit development occurs in four distinct stages: the juvenile stage, lasting up to 21 days post-fruit set, is characterized by rapid cellular growth; the maximum growth stage, from 21 to 49 days, involves cell enlargement and maturation; the maturation and ripening stage, spanning 49 to 77 days, sees an increase in respiration and ethylene production, marking the climacteric phase of ripening; finally, the senescence stage begins on the 77th day, where the fruit becomes susceptible to microbial attack and decay.
2006) Mango fruits normally reach maturity about 4–5 months after flowering
Figure 2.6: Mangoes with different colours
Figure 2.7: Unripe mango and ripe mango
Vietnam is home to 10 popular types of mangoes, including Hoa Loc mango, Thailand mango, Mangifera mekongensis (xoài thanh ca), Australian mango, Mangifera indica (xoài tượng), blockchain mango (xoài cát chu), xoài keo, xoài tứ quý, xoài giống đài loan đỏ, and xoài bào tử.
Fruits are essential for human nutrition, providing energy, growth factors, carbohydrates, dietary fibers, and antioxidants crucial for health The quality of fruit is determined by its physicochemical and nutritional characteristics, which improve during ripening Mango, in particular, is a rich source of sugars, carbohydrates, fats, vitamins, minerals, dietary fibers, antioxidants, tannins, polyphenols, pigments, and flavor compounds, offering a high energy value of 250 kJ (60 kcal) per 100g Both fresh and processed mango are significant components of the human diet, making it a commercially valuable food crop.
Common diseases of mango fruits in the world and in Vietnam
Mango crops are significantly impacted by anthracnose disease, caused by the fungus Colletotrichum gloeosporioides, which belongs to the order Melanconiales This disease poses a serious threat, leading to economic losses ranging from 30% to 60%, and in some cases, up to 100% under wet or humid conditions The ideal temperature for the germination and infection of conidia is between 25°C and 30°C, particularly when free moisture is present Major losses typically occur from the flowering stage to fruit set and again post-harvest Leaf infections manifest as small, dark spots that can merge into larger necrotic areas, while flower panicles show small brown or black spots that can lead to flower death The fungus rapidly invades small fruits upon infection, and on nearly mature or ripe fruits, black spots can merge to cover extensive areas.
Source: (Pacific Pests and Pathogens, 2017)
Figure 2.8: Anthracnose on mango fruits
At 25°C, colonies of Colletotrichum gloeosporioides exhibit an average growth rate of 6.5–8.7 mm per day on PDA The mycelia start as white and gradually turn pale brown, accompanied by orange conidial masses Aerial mycelia appear white to gray, while the conidia are hyaline, cylindrical to fusiform, aseptate, and smooth, measuring 5-7.5 × 15-17.5 μm The appressoria are brown, ovoid, and occasionally clavate, with dimensions of 4.7-5.8 × 7.9–8.4 μm.
Figure 2.9: Colletotrichum gloeosporioides a Colony on PDA; b Conidia; c Appressoria Scale bars = 10 μm.
In mangoes from drier regions, stem end rot poses a more significant post-harvest threat than anthracnose This disease, caused by fungi such as Botryosphaeria spp., Lasiodiplodia theobromae, and Phomopsis spp., manifests as a soft, watery rot originating from the stem end as the fruit ripens post-harvest Severe losses due to stem end rot are particularly pronounced during long-term storage Symptoms include a dark rot that starts at the stem end, characterized by a dark brown ring that spreads towards the opposite end, along with dark streaking in the water-conducting tissues These fungi are naturally present on mango tree branches and can infect the fruit before harvest, with additional risks of infection from soil, bark, or twig litter when fruit is placed on the ground for desapping.
Figure 2.10: Stem end rot of mango caused by Lasiodiplodia theobromae
Transit rot, caused by the fungus Rhizopus stolonifer, often emerges post-harvest, leading to significant fruit losses in high-humidity environments Characterized by pale, watery lesions and white fungal growth, the disease also produces black spores It spreads easily between fruits and through contaminated materials like wood wool, particularly affecting those in contact with the soil or infested fruit The severity of transit rot increases in warm, wet conditions, resulting in substantial deterioration during transit or storage.
Figure 2.12: Morphological characterization of Rhizopus stolonifer
Figure 2.13: Transit rot of mango caused by Rhizopus stolonifer
Aspergillus rot is a significant postharvest disease affecting mangoes, leading to substantial fruit losses during storage This disease manifests as light yellow lesions around the stem end, which progressively enlarge, resulting in a soft rot condition with sunken centers covered in brownish-black spores Reports indicate that Aspergillus rot can cause losses of 25-35% in regions like Allahabad and Lucknow, India The economic impact is particularly severe for home garden growers in Jaffna, highlighting the urgent need for effective control measures against this pathogen.
Isolated colonies of the causal organism on potato dextrose agar (PDA) began as white in color After 36 hours of inoculation, black conidia production was observed, leading to the development of black colonies with a notable diameter.
Three days post-inoculation, the culture plates measured 8 cm and exhibited an off-white coloration on the reverse side, displaying a fractured appearance The presence of hyaline, septate mycelia alongside black conidia and spore-bearing structures is indicative of Aspergillus niger (Bennett, 2010).
‘T’ shaped foot cells that produce a single conidiophore were observed
Figure 2.14: Spoilage mango caused by A.niger
Figure 2.15: Morphological characterization of Aspergillus niger.
Control of spoilage and ripening in mango fruits
Post-harvest decay significantly hinders the successful export of mangoes To mitigate ripening and physiological deterioration, refrigeration during transit and storage is crucial, as it effectively reduces post-harvest decay in various commodities Research indicates that decay-causing microorganisms proliferate more slowly and result in less decay at temperatures between 32° F and 50° F compared to higher temperatures However, it is important to note that even optimal storage temperatures do not eliminate these organisms.
To enhance efficiency and economic value, it is often essential to use chemical treatments to mitigate postharvest decay before or after harvest However, any chemical intended for this purpose must be registered with the Plant Pest Control Division of the Agricultural Research Service at the United States Department of Agriculture before it can be transported across state lines.
Various post-harvest chemical treatments have been evaluated for their effectiveness in controlling mango decay, showing varying levels of success (Lonsdale, 1992, 1993; Lonsdale et al., 1991; Pelser, 1985; Pelser and Lesar, 1989, 1990).
For fruit intended for the local market in South Africa, a 2 min dip in a heated
To effectively control anthracnose on mangoes caused by C Gloeosporioides, it is recommended to dip export fruits in a prochloraz emulsion (90 ml / 100 L) for 20 seconds at 25°C, followed by a 5-minute hot water treatment at 50°C Additionally, a 5-minute hot dip in a benomyl suspension (100 g / 100 L) can also help manage anthracnose, as well as stem-end and soft brown rot, particularly in South Africa.
In the European Union (EU), the Maximum Residue Limit (MRL) for benomyl is currently set at the limit of detection (LOD), prohibiting its use as a post-harvest chemical The EU has utilized prochloraz for controlling anthracnose on mangoes, particularly in combination with other treatments, while establishing the MRL for carbendazim at 0.1 ppm Thiabendazole is registered in various countries as a post-harvest chemical for anthracnose, with the EU's MRL set at 5 ppm; however, it is not registered for use on mangoes in South Africa (Nel et al., 2003).
Fludioxonil is an effective fungicide used to manage stem-end and soft brown rots in mangoes, with a maximum residue limit (MRL) ranging from 0.01 ppm to 5 ppm in Japan, the EU, South Africa, and other regions.
In Vietnam, products derived from prochloraz, benomyl, carbendazim, thia- bendazole and fludioxonil are used widely with MRL ranging from 0.01 to 5 ppm
Figure 2.16: Products derived from prochloraz
In the past, inorganic fertilizers were commonly employed to combat fungal phytopathogen infestations, which negatively impact crop yield and quality However, there has been a recent shift towards prioritizing biological control methods over chemical sprays, focusing on natural antagonists and their products to manage diseases effectively.
Recent research is focusing on the biocontrol potential of local microbial inoculants as effective and appealing alternatives to chemical fungicides Studies have shown that natural substances, including animal-derived antimicrobials like lactoperoxidase, lysozyme, and chitosan, as well as plant-based antimicrobials such as essential oils, aldehydes, esters, herbs, and spices, can significantly reduce pathogenic and spoilage microorganisms in fruits Additionally, microbial-origin antimicrobials like nisin have also demonstrated effectiveness in this area.
Research has shown that adding organic acids to fresh-cut fruits and fruit juices effectively inhibits spoilage and pathogenic microorganisms Furthermore, combining organic acids with preservation methods like mild heat, high-intensity pulsed electric fields, dehydration, freezing-thawing, and low temperatures significantly enhances their antimicrobial effects For instance, a study by Sifat Rahi (2017) found that a 2% concentration of citric acid completely prevented the growth of fungal strains responsible for post-harvest spoilage in mangoes.
Chitosan, a natural polysaccharide derived primarily from marine crustacean shells, serves as an effective edible coating for fruits, significantly reducing harmful microorganisms and extending shelf life (Lavall et al., 2007; Arbia et al., 2013).
Chitosan, a derivative of chitin, is a hetero-polysaccharide made up of 2-amino-deoxy-β-D-glucopyranose and 2-acetamido-deoxy-β-D-glucopyranose Its key properties arise from three functional groups (primary-OH, secondary-OH, and -NH2) and its solubility in acidic conditions When applied to fruits, chitosan interacts with microbial cell walls, leading to effective microbial control while preserving the taste, odor, and palatability of fresh-cut fruits and vegetables.
Chien et al (2007) demonstrated that an edible chitosan coating applied to sliced mango at concentrations of 0.5%, 1%, and 2% (w/v) significantly inhibits microbial growth during storage at 6 °C The study revealed a reduction in the growth of naturally occurring microorganisms, with counts increasing from 3.82 to 5.53 log CFU/g for the chitosan-coated mango, compared to an increase from 3.82 to 6.41 log CFU/g in the control group However, increasing the chitosan concentration beyond 2% did not provide additional benefits in delaying microbial growth Therefore, a chitosan concentration of 2-3% is recommended to effectively inhibit microbial growth on fresh-cut fruits while maintaining their sensory qualities.
In Vietnam, chitosan is utilized for fruit preservation due to its effectiveness in inhibiting the growth of microorganisms on fresh-cut fruits while maintaining their sensory qualities This biodegradable substance is derived from abundant and inexpensive shrimp shells, which are available year-round, making chitosan supply convenient Furthermore, using shrimp shell waste from seafood processing for food preservation significantly helps mitigate environmental pollution caused by this waste.
Method of preserving fruits by chitosan/chitin
Shrimp and crab shells undergo a thorough cleaning process, starting with a wash and soaking in a 5% NaOH solution at 80-90°C for 6 hours, repeated three times with a 5% HCl solution for 2 hours, followed by a 1% HCl solution until no air bubbles are observed The shells are then soaked again in a 5% NaOH solution for 6 hours to ensure complete deproteinization After neutralization and washing, the shells are treated with a 5% H2O2 solution at 50°C for 2 hours, resulting in white chitin The final step involves deamethylation, where the chitin is cleaned, dried, and soaked in a 50% NaOH solution at 90°C for 8 hours to produce chitosan.
To prepare a 2% chitosan stock solution, dissolve 2g of chitosan in a 1% acetic acid solution Chitosan can be utilized in various ways to preserve fruits, including spraying the stock solution onto the fruit surface, soaking the fruits in a moderately diluted chitosan solution tailored to the specific type of fruit, or applying a chitosan biopolymer film.
Source: (Tạp chí công nghệ và tiêu dùng, 2016)
Figure 2.17: Bio-polymer film for preserving fruits.
MATERIAL AND METHODS
Materials
Post-harvest spoiled mangoes and healthy looking mango fruits are purchased in the different markets of Gia Lam
Chemicals used for SDA, PDA, ISP2, Czapek, MEA, enzyme activity medium
The tools: petri dishes, a sterile blade, test tubes, measuring cylinders, glass sam- pling bottles, alcohol burner, scissors, inoculating loop, glass spreader, microscope slide cover glass, eppendorf tubes, pipettes
The essential laboratory equipment includes precision balance scales, analytical balance scales, microscopes, water distillers, pH meters, centrifuge systems, refrigerated centrifuges, incubator chambers, laminar airflow cabinets, refrigerators, freezers, drying ovens, microwave ovens, and autoclaves.
Location: This study was conducted in the laboratory of the Department of Micro- bial Biotechnology-Faculty of Biotechnology-Vietnam National University of Agriculture
Time: Study was conducted from 8/2020 to 2/2021
Sabouraud Dextrose Agar (SDA): distilled water 1000ml; dextrose 40g; peptone 10g; agar 15g; pH = 6
Potato Dextrose Agar (PDA): distilled water 1000ml; potato infusion 200g; dex- trose 20g; agar 20g; pH = 5.6
Czapek Dox Agar (CDA): distilled water 1000ml; sucrose 30g; sodium nitrate 2g; dipotassium phosphate 1g; magnesium sulphate 0.5g; potassium chloride 0.5g; ferrous sulphate 0.01g; agar 15g; pH = 7.3
ISP2: distilled water 1000ml; yeast extract 4g; malt extract 10g; dextrose 4g; agar 20g; pH = 7.2
Malt extract agar (MEA): distilled water 1000ml; malt extract 20g, dextrose 20g; peptone 6g; agar 15g; pH = 5.5
Enzyme activity medium: 2% agar and 0.5-1% substrate (starch solution, CMC, gelatin, chitin or pectin) in phosphate buffer 0.1M, pH = 7
The mediums were autoclaved at 121 degree celsius, 1.4 atm for 15 minutes.
Research content
Isolate and select of fungi associated with spoilage of mango fruit
Re-infect the fungi into the healthy mangoes
Study biological characteristics of fungal strains causing spoilt on mango fruits: morphology colony, mycelium and spore of fungus
This study evaluates the extracellular enzyme production capabilities of isolated fungal strains while examining the impact of various factors on fungal growth, including the culture medium, temperature, and pH levels.
Methods
3.3.1 Method of isolation and purification of fungal pathogens from rotten mango fruit
Post-harvest spoiled mangoes were sourced from three markets in the Gia Lam district The diseased fruits underwent a thorough cleaning process, which included washing with tap water, sterilizing the surface with 75% alcohol for 3-5 seconds, and rinsing twice with sterilized distilled water before drying Infected tissue samples were excised and cultured on Potato Dextrose Agar (PDA) medium using a sterile knife, followed by incubation at 30°C for 5-7 days The resulting fungi were re-inoculated on PDA medium multiple times to achieve pure cultures, which were then stored at 4°C for preservation.
Healthy mango fruits were sterilized with 70% alcohol and incised to create artificial wounds Agar pellets containing fungal mycelia were inoculated into these wounds, which were then sealed with muslin cloth Control samples were prepared with artificial wounds inoculated with PDA pieces Both inoculated and control fruits were placed in individual clean polythene bags and incubated at 30°C Daily observations were made for disease symptoms, allowing for the comparison of isolated samples to identify fungal strains capable of causing disease through artificial infection These selected fungal strains will be utilized in further experiments to study their extracellular enzymes, biological characteristics, and classification based on morphological and microscopic traits.
3.3.3 Method of studying biological characteristics
The study of pure cultures of fungal isolates focused on their morphological characteristics, as detailed by Tafinta et al (2013) Fungal strains were cultivated on potato dextrose agar (PDA), with glass laminae positioned at a 30-45° angle Mycelial growth was examined under a light microscope at 40x and 100x magnification every 12 hours to identify spores, hyphae, and other specialized structures The morphological and cultural traits of the fungal isolates from rotten fruits were then compared to established descriptions from previous research (Ellis, 1971; Samson and Varga, 2007).
3.3.4 Optimal growth conditions of fungi
To determine the optimal growth conditions for three fungal strains, we inoculated them at 30°C across various media, including Potato Dextrose Agar (PDA), Sabouraud Dextrose Agar (SDA), International Streptomyces Project 2 (ISP2), Malt Extract Agar (MEA), and Czapeck Dox Agar (CDA) Daily observations were conducted to identify the most suitable medium for fungal growth.
The study investigated the impact of temperature and pH on the growth of a fungal strain by incubating it in Potato Dextrose Agar (PDA) at various temperatures (20°C, 30°C, 37°C, 40°C, and 50°C) and pH levels (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) Daily observations of the fungal strains were conducted to identify the optimal conditions for maximum mycelial growth, with the diameter of the fungal colony being measured as the key outcome.
3.3.5 Method of testing enzymatic activity
3.3.5.1 Experimental materials for detecting extracellular enzyme
Table 3.1: Experimental materials for detecting extracellular enzyme
Black amino 10B 0.1% in methanol, acetic acid, distilled water (3:1:6)
Colloidal chitin was synthesized by mixing 1 g of chitin powder with 85% H3PO4 and incubating the mixture at 4°C for 24 hours After preparation, the colloidal chitin samples were thoroughly washed with distilled water multiple times to achieve a pH of 7.0 (Alam & Mathur, 2014).
3.3.5.2 Detection of enzyme activity on plates
Fungal strains were cultivated for 48 hours in potato dextrose agar (PDA) at 30°C with shaking at 180 rpm Following centrifugation at 6000 rpm for 15 minutes, 50 µl of enzyme solution from each strain was added to the wells of the plates The plates were then refrigerated at 4°C for 2 hours before being incubated at 30°C for 24 hours After incubation, Lugol solution was applied to the plates to observe the clear zones around the wells, indicating substrate hydrolysis Each assay was performed in triplicate, and the mean values were calculated Enzyme activity was determined using the formula \( K = D - d \), where \( K \) represents enzyme activity, \( D \) is the diameter of the clear zone, and \( d \) is the diameter of the well (7 mm).
3.3.6 Molecular identification of fungal species
3.3.6.1 DNA extraction and PCR amplification
Fungal isolates were cultured on potato dextrose broth at 28±2°C for approximate- ly 3 days The fungal isolates were used for DNA extraction by using CTAB methods
PCR amplification was carried out using ITS1 and ITS4 primers Two primers have respective sequences:
The final volume for the PCR amplification was 30àl including 1.5àl primer ITS1, 1.5àl primer ITS4, 2àl DNA template, 10àl H2O, and 15àl Mastermix 2X
The reaction was conducted using specific amplification processes, starting with an initial denaturation at 95°C for 5 minutes This was followed by 35 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 40 seconds, and extension at 72°C for 30 seconds The final extension was performed at 72°C for 5 minutes, followed by rapid cooling to 4°C prior to analysis.
The results of PCR reaction are visualized by using gel electrophoresis The size of DNA fragments in the PCR sample was compared to DNA ladder
The PCR products were sequenced in Singapore, and the resulting sequences were analyzed by comparing them with related sequences using a BLAST search in GenBank (NCBI) (Liu et al., 2000; Landeweert et al., 2003; Javadi et al., 2012).
RESULTS AND DISCUSSION
Isolation of fungal pathogens causing spoil in post-harvest mango fruits
The isolation experiment utilized spoiled mango fruits collected from three markets in Gia Lam district Infected samples were cultured on PDA plates and incubated at 30°C The results revealed three unknown fungal strains, identified based on morphological features such as form, margin, elevation, surface, color, mycelium, and spores Strains M1 and M3 were associated with post-harvest spoilage from Cuu Viet, Ecopark, and Long Bien markets, while strain M2 was exclusively found in the Ecopark market Detailed morphological characterization of the three isolated strains is provided in Table 4.2.
Table 4 1: The presence of fungi strains in three chosen markets
Fungi strains Cuu Viet market Long Bien market Ecopark market
Figure 4.1: Isolated fungal strain from the postharvest spoiled mangoes
Table 4.2: Morphological characterization of three isolated fungal strains
Surface Rough with black co- nidial production
Rough with wavy growth Rough
Front colour White to light brown- ish White to pale yellow White to brown
Back colour White with brown center
White with yellow center White to brown
Artificial infection
This study investigates the impact of spoilage diseases on the ripening stages of mango fruits through a re-infection experiment Fungal strains were isolated and inoculated into artificial wounds on both healthy ripe and unripe mangoes The findings are detailed in Table 4.3.
The data indicates that all three isolated strains led to spoilage in both ripe and unripe mangoes, with an increase in disease severity corresponding to the ripening of the fruit Symptoms of spoilage appeared within just 2 days in ripe mangoes and spread more rapidly than in green mangoes across all strains In the unripe stage, symptoms were also observable.
3 rd day and spreaded strongly in the following days
Table 4.3: Effect of different ripening stages on mango fruit rotting
Table 4.4: Symptoms of post-harvest fungal diseases in mango fruits
The initial symptoms appeared as small, light yellow lesions that gradually enlarged, leading to a depressed mesocarp and a soft rot condition Over time, the center of these lesions became sunken and was covered with brownish-black spores.
The initial symptoms appear as dark-brown to black rot on the fruit, which rapidly progresses into decay, resulting in brown, soft tissues that emit a foul odor Additionally, the lesions are soft and can quickly enlarge, indicating a severe deterioration of the fruit.
This fungus did not sporulate on infected fruit
Table 4.5: Prevalence of post-harvest diseases in mango fruits
This study revealed that all fungal isolates were pathogenic during both the ripe and unripe stages of mango fruits, with rot symptoms consistent with those previously observed in isolated mangoes.
Ripe mangoes exhibit faster symptoms and spread of fungal infection compared to green mangoes, indicating that green fruits do not provide the necessary nutrients for the fungus The antifungal activity of enzymes is more potent in green mangoes, as they contain higher levels of pectin, a crucial gelling sugar As mangoes ripen, the percentage of pectin decreases, resulting in a lower molecular weight that accelerates the rotting process.
Figure 4.2: Isolated samples of M1, M2 and M3 strain
Figure 4.3: Re-infection of isolated fungal strain (M1, M2, M3) in mangoes fruit and
Characterization of isolated fungal strains
Figure 4.4: Microscopic observation of the isolated fungal strain-M1
(A): Septae hyphae of M1 strain; (B, C): Conidiophores and conidia of M1 strain
Hyphae are long, colorless, and branched with septae The conidial heads are large, globose, and dark brown, becoming radiate and splitting into loose columns as they age Conidiophore stipes are smooth-walled and hyaline, darkening towards the vesicle The conidial heads are biseriate, with phialides on brown, often septate metulae Conidia are globose to subglobose, dark brown to black, and have a rough-walled texture.
Microscopically, the isolated M2 exhibits branched-septate hyphae that are colorless The sclerotia are sub-ovoid and light yellow, maturing to a pale brown color No fungal spores were detected The conidiophores generate both alpha-conidia and beta-conidia, with alpha-conidia being hyaline, aseptate, and ellipsoidal to cylindrical in shape, rounded at both ends In contrast, beta-conidia are hyaline, filiform, hamate, and aseptate.
Figure 4.5: Microscopic observation of the isolated fungal strain-M2
(A): Septae hyphae of fungal strain M2; (B) sclerotium; (C): Conidiophores; (D) α and β conidia; (E) α conidia (F): irregular hyphae, like chlamydospore
Microscopically, M3 isolated showed septate, branched hyphae Not produce spores
Figure 4.6: Microscopic observation of the isolated fungal strain-M3
Table 4.3: Time to form hyphae, sclerotium, conidiophores, conidia and chlamydo- spore Hyphae Sclerotium Conidiophores Conidia Chlamydospore
M2 3 days 4 days 5 days 6 days 9 days
Effect of different media on the growth of isolated fungal strains
This study investigates the impact of various media on mycelium growth, utilizing five different solid growth media: Potato Dextrose Agar (PDA), Czapek Dox Agar (CDA), Sabouraud Dextrose Agar (SDA), ISP2, and Malt Extract Agar (MEA) at an incubation temperature of 30°C The findings reveal that SDA and PDA significantly enhance the growth of the fungal strain M1, while strains M2 and M3 exhibit robust mycelial growth on PDA and MEA In contrast, all three strains demonstrate weak growth on CDA, as illustrated in figures 4.7, 4.8, and 4.9.
Figure 4.7: Fungal strain-M1 in different media on the 5 th day
Figure 4.8: Fungal strain-M2 in different media on the 5 th day
Effect of different pH on the growth of isolated fungal strains
The growth of fungi is significantly influenced by pH levels In a study of fungal strains M1, M2, and M3 cultivated in PDA media, mycelial growth was assessed across various pH values ranging from 4.0 to 10.0 The findings revealed that strain M1 exhibited optimal growth at pH 5.0, while strain M2 thrived at pH levels of 7.0, 8.0, and 10.0 Additionally, strain M3 demonstrated suitable growth at pH values of 4.0, 6.0, and 10.0, as illustrated in figures 4.10, 4.11, and 4.12.
Figure 4.10: Fungal strain-M1 in different pH on the 5 th day pH=4 pH=5 pH=6 pH=7 pH=8 pH=9 pH
Figure 4.11: Fungal strain-M2 in different pH on the 5 th day pH=4 pH=5 pH=6 pH=7 pH=8 pH=9 pH pH=4 pH=5 pH=6 pH=7 pH=8 pH=9 pH
Effect of different temperatures on the growth of isolated fungal strains
This study investigates the impact of temperature on mycelial growth across different fungal strains on PDA media, specifically at temperatures of 20°C, 30°C, 37°C, 40°C, and 50°C The strain M1 exhibited optimal growth between 30°C and 40°C, while still maintaining vegetative growth at extreme temperatures ranging from 20°C to 40°C Strain M2 achieved its highest growth at 30°C and can thrive between 20°C and 37°C Similarly, M3 demonstrated maximum mycelial growth within the 30°C to 37°C range and is capable of growing at temperatures from 20°C to 37°C Notably, all three strains failed to grow at 50°C, as illustrated in figures 4.13, 4.14, and 4.15.
Figure 4.13: Fungal strain-M1 in different temperatures on the 5 th day
Figure 4.14: Fungal strain-M2 in different temperatures on the 5 th day
Figure 4.15: Fungal strain-M3 in different temperatures on the 2 rd day
Testing extracellular enzyme activity
4.7.1 Producing chitinase activity of isolated fungal strains
Chitosan effectively controls various pre and post-harvest diseases in horticultural commodities, particularly by inhibiting fungal pathogens Research indicates that chitosan application directly impacts the morphology of treated microorganisms, showcasing its fungistatic or fungicidal properties Notably, increasing chitosan concentrations (ranging from 0.75 to 6.0 mg/ml) significantly reduce the radial growth of fungi such as Alternaria alternaria, Botrytis cinerea, Collectotrichum gloeosporioides, and Rhizopus stolonifer (EI Ghaouth et al., 1992c).
This study investigated the chitinase activity of three isolated strains The selected strains were cultured in liquid PDA medium for 48 hours at 30°C with shaking at 180 rpm Following centrifugation at 6000 rpm for 15 minutes, 50 µl of enzyme solution from each strain was added to wells containing chitin The plates were refrigerated at 4°C for 2 hours, then incubated at 30°C for 24 hours, and subsequently stained with Lugol's solution.
If the fungus has enzymatic activity, a ring (halo) of substrate resolution will be formed around the wells
Among the three isolated strains, only M3 exhibited no chitinase activity (Figure 4.17) The interaction between the amino groups of chitosan and the cell wall of M3 inhibits its activity Consequently, chitosan has been utilized to manage post-harvest diseases in mango fruits caused by the M3 fungal strain, as illustrated in Figure 4.16.
Chitinases have been identified in the cell walls of various fungal species, including Aspergillus, Penicillium, Trichoderma, Paecilomyces, Sporotrichum, Beaueria, and Mucor, with some causing fruit spoilage In this study, M1 and M2 exhibited chitinase activity, which may explain the continued rotting of fruits even after the application of chitosan technology due to infection by these fungi To mitigate this issue, producers can increase the concentration of chitosan to between 2% and 3% without affecting the sensory attributes of mangoes, as illustrated in figure 4.16.
Figure 4.16: Chitinase activity of the isolated strains based on their clear zones around the wells after a day’s incubation 4.7.2 Producing cellulase activity of isolated fungal strains
Cellulase production has been observed in various bacteria and fungi, highlighting its significance in microbial biology Fruit cells are fortified with cellulose, which serves as a barrier against fungal pathogens To penetrate these cells, fungi must effectively degrade cellulose Notable fungal pathogens such as Fusarium oxysporum, Aspergillus oryzae, and Aspergillus niger are recognized for their efficient cellulase production, enabling them to invade fruit tissues.
This study evaluated cellulase activity in three isolated strains The selected strains were cultured in liquid PDA medium for 48 hours at 30°C with shaking at 180 rpm Following centrifugation at 6000 rpm for 15 minutes, 50 µl of enzyme solution from each strain was added to wells containing carboxymethyl cellulase (CMC).
The result show that only M1 has cellulase activity among three isolated strains (figure 4.17) The size of the halo around the well represents the activity of the enzyme cellulase
The M1 strain's ability to produce cellulase enzyme enables it to break down cell walls, facilitating its intrusion into cells and subsequent spread In contrast, fungal strains M2 and M3, which do not produce cellulase, may utilize alternative mechanisms for cell invasion This highlights M1 as a significant agent in fruit spoilage, as illustrated in figure 4.17.
Figure 4.17: Cellulase activity of the isolated strains based on their clear zones around the wells after a day’s incubation
4.7.3 Producing pectinase activity of isolated fungal strains
This study evaluated the pectinase activity of three isolated strains The selected strains were cultured in liquid PDA medium for 48 hours at 30°C with shaking at 180 rpm Following centrifugation at 6000 rpm for 15 minutes, 50 µl of enzyme solution from each strain was added to wells containing pectin The plates were refrigerated at 4°C for 2 hours, then incubated at 30°C for 24 hours, and subsequently stained with Lugol's solution.
If the fungus has enzymatic activity, a ring (halo) of substrate resolution will be formed around the wells
The result show that all three isolated strains have pectinase activity (figure 4.17) The size of the halo around the well represents the activity of the enzyme pectinase
Three isolated strains demonstrate the ability to produce pectinase enzyme, which effectively breaks down the gelling sugar pectin found in mango pulp, thereby accelerating the ripening process of mango fruits.
Figure 4.18: Pectinase activity of the isolated strains based on their clear zones
Phylogenetic analysis
4.8.1 DNA extraction of isolated fungal strains
Figure 4.19: Isolated DNA of isolated fungal strains (M1, M2 and M3) 4.8.2 PCR amplification of three isolated strains
Figure 4.20: Banding pattern in the 2 fungi samples from spoilt fruits (M1, M2)
M: 100bp size DNA ladder, Lane 3: Positive control of M1 (600bp), Lane 4: Positive control of M2 (550bp) isolated from mango
Figure 4.21: Banding pattern in the fungi sample from spoilt mango fruits (M3)
M: 100bp size DNA ladder, Lane S: Positive control of M3 (550bp) isolated from mango
The 16s rRNA sequence of the M1 strain was analyzed using the BLAST tool against the Genebank nucleotide database The phylogenetic tree was constructed with MEGA-X software, revealing that the M1 strain exhibited a high homology of 98% with Aspergillus niger, leading to its identification.
Figure 4.22: Phylogenetic tree of M1 strain 4.8.4 Phylogenetic analysis of M2 strain
The study focuses on various Aspergillus isolates, including Aspergillus niger isolate Asp-7205, Aspergillus sp isolate FPZSP374, and Aspergillus tubingensis strain AM2 Additionally, it examines multiple Aspergillus niger isolates such as RIZ9-2, RIZ9-1, RIZ5-1, and RIZ3-6, along with Aspergillus tubingensis strain USMI03.
The 16s rRNA sequence of the M2 strain was analyzed using the BLAST tool against the Genebank nucleotide database The phylogenetic tree was constructed with MEGA-X software, revealing that the M2 strain exhibited a 94% homology with Phomopsis sp.
Figure 4.23: Phylogenetic tree of of M2 strain
CGCCGAGGTCTTTGAGGCGCGTCCGCAGTGAGGACGGTGCCCAATTCCAA- GCAGAGCTTGAGGGTTGTAATGACGCTCGAACAGGCATGCCCCCCGGAA- TACCAAGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTG- CAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAAC- CAAGAGATCCGTTGTTGAAAGTTTTAGTTTATTAACTTGTTTATCAGAC-
The 16s rRNA sequence of the M3 strain was analyzed using the BLAST tool against the Genebank nucleotide database The phylogenetic tree was constructed with MEGA-X software, revealing that the M3 strain exhibited a 100% homology with Lasiodiplodia theobromae, leading to its identification as Lasiodiplodia theobromae PaP-2.
Figure 4.24: Phylogenetic tree of M3 strain
Lasiodiplodia hormozganensis strain CMM39 Lasiodiplodia brasiliensis CMM 4015
Lasiodiplodia sp CMM4011 Lasiodiplodia brasiliense strain CMM 0354 18 Lasiodiplodia theobromae isolate 18-00584 Lasiodiplodia theobromae isolate PBBG186 M3
Lasiodiplodia theobromae strain PaP-2 Lasiodiplodia theobromae isolate PBBG179
CHAPTER V: CONCLUSION AND SUGGESTION 5.1 Conclusion
- Three fungal strains were isolated from spoiled mango fruits
The mycelium of the M1 strain starts as white but transitions to black after a few days when it produces conidial spores This strain features smooth-colored conidiophores and conidia, with large, globose conidial heads that darken to brown and become radiate, eventually splitting into several loose columns as they age The conidiophore stipes are smooth-walled and hyaline, darkening towards the vesicle, while the conidia are globose to subglobose, dark brown to black, and rough-walled.
The mycelium of M2 starts as white and transitions to a pale yellow on potato-dextrose agar (PDA), exhibiting circular wavy growth patterns It features branched, septate hyphae that are colorless The sclerotia are sub-ovoid and light yellow, maturing to a pale brown Additionally, conidiophores generate both alpha-conidia and beta-conidia.
- The mycelium of M3 is initially white and turns to a brown color after a few days M3 isolated showed septate, branched hyphae Not produce spores
The experiment investigating the influence of culture medium, temperature, and pH on fungal strain growth revealed that strain M1 thrives on SDA and PDA at a pH of 5.0 and temperatures between 30-37°C Strain M2 exhibits optimal mycelial growth on PDA and MEA at pH levels of 7.0, 8.0, and 10.0, with a preferred temperature of 30°C Meanwhile, strain M3 shows favorable growth on PDA and MEA at pH levels of 4.0, 6.0, and 10.0, also within the temperature range of 30-37°C.
- In terms of extracellular enzyme activity, M1, M2 and M3 can produce pectinase; M1 and M2 have the ability to produce chitinase whereas only M1 can produce cellulase among three isolated strains
- Through molecular identification, the isolated fungal strains were identified as A
Niger (M1), Phomopsis sp (M2) and L Theobromae (M3) with approximately 98%, 94% and 100% identity, respectively
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