Isolation, identification, and enzyme screening of fungi infested various vegetables in the local markets of Visakhapatnam, Andhra Pradesh

This study aimed to examine the fungi present in fruits and vegetables obtained from the local vegetable markets, Visakhapatnam and assess their enzymatic activity. seven fungal pathogens responsible for vegetable spoilage were isolated using sterilization and spread plate techniques on potato dextrose agar (PDA) medium. The isolated fungi were identified based on macroscopic and microscopic characteristics. The identified fungal strains included Aspergillus, Curvularia, Alternaria, Cladosporium, Fusarium, sclerotinia, Mucor, Penicillium , and Rhizopus . Only three selected strains were tested for extracellular amylase, pectinase and cellulase activity, which were showing 15mm,20mm,30mm clear zone capability. These findings provide essential information about the degradative properties of filamentous fungi and their secretion of extracellular enzymes. The study also highlights the presence of toxigenic fungi in local vegetables, emphasizing the importance of removing spoiled fruits to prevent the accumulation of toxins associated with fungal growth.


Introduction
In the Indian states of Andhra Pradesh and Telangana, there is a type of farmers' market called Rythu bazaar.It is managed by the Andhra Pradesh and Telangana governments for small-scale farmers with modest farmlands.In the month of January 1999, Chief Minister N. Chandrababu Naidu opened the inaugural market [1] This encouraged small and medium-sized farmers to sell their products directly to end-users at a profit and helped them maintain a modest level of financial stability.Customers in towns and cities can purchase these goods directly from farmers for a lower price.This allowed farmers to sell their products directly to consumers in towns and cities without the use of intermediaries, which helped them economically to some extent.Additionally, consumers can get high-quality agricultural products at reasonable prices.
Vegetables play a vital role in human nutrition, providing essential vitamins, minerals, and dietary Fiber.However, the presence of plant pathogenic fungi in vegetables can pose significant risks to both crop productivity and human health.These fungi are known to cause devastating diseases in plants (table-1), resulting in economic losses for farmers and affecting food security.Moreover, emerging research suggests that plant pathogenic fungi can also impact human health directly through infections and indirectly through the production of mycotoxins.Understanding the prevalence and implications of plant pathogenic fungi in vegetables is crucial for developing strategies to ensure both crop safety and human well-being.Vegetables, including leafy greens, root crops, and vegetables, are susceptible to infections by various plant pathogenic fungi.These fungi can infect different parts of the vegetables, such as the leaves, stems, roots, and flowers, leading to visible symptoms such as wilting, rotting, and discoloration.The diseases caused by these pathogens can result in significant yield reduction, economic losses, and compromised nutritional value of the vegetables.
Apart from the direct impact on vegetable crops, the presence of plant pathogenic fungi can also pose risks to human health.These fungi can produce mycotoxins, toxic compounds that can contaminate vegetables during cultivation, storage, and processing.Consumption of mycotoxin-contaminated vegetables can have severe health consequences, including carcinogenic, mutagenic, immunosuppressive, and hepatotoxic effects [2][3].It is essential to understand the occurrence and levels of mycotoxins in vegetables to mitigate health risks associated with their consumption.
Fungi is a diversified eukaryotic organism.They can be found in natural habitats such as water, soil, and decaying organic matter.Some fungi establish symbiotic relationships with animals, plants, and others act as parasites, causing diseases or even death.Fungi are characterized by their rigid cell walls containing chitin, mannan, and other polysaccharides.They reproduce both sexually and asexually [4].
Fungi have the ability to produce both intracellular and extracellular enzymes.While small molecules like monosaccharides, disaccharides, fatty acids, and amino acids can be directly utilized by fungi, larger complex compounds such as cellulose, starch, and pectin, require the secretion of extracellular enzymes for breakdown.[5] The production of extracellular enzymes by fungi plays a crucial role in facilitating the degradation process.This ability allows fungi to efficiently obtain nutrients from complex substrates and participate in nutrient cycling within ecosystems.Understanding the enzymatic capabilities of fungi can have significant implications in various fields, including biotechnology, agriculture, and environmental sciences.In this study, we explore the diverse enzymatic activities exhibited by fungi and their relevance to different applications and processes [6].

Collection of samples
Vegetables available at the bazaar were identified, and a comprehensive list of the different vegetable varieties was compiled, and Fungal-infected vegetables were targeted for observation and analysis.The identified vegetables were carefully examined for any visible signs of fungal infection, including discoloration, spots, and Molds growth.Samples of both the infected and non-infected vegetables were collected to facilitate a comparative analysis, and Multiple samples were collected to ensure representative results.Each sample was appropriately labelled with essential information, such as the date, location, and type of vegetable, to maintain accurate records.The collected samples were stored in sterile containers or bags.The Storage conditions involved maintaining a cool and dry environment to prevent contamination and minimize further fungal growth or decay.The collected samples were subjected to laboratory analysis.In the laboratory, the samples were examined to identify the specific type(s) of fungi present and determine the extent of the infection.

Isolation of fungal pathogen
The diseased leaves were washed thoroughly under running tap water to remove soil particles and the infected portions of the leaves were cut into 1.0 to 1.5 cm.fragments.The pieces were surface sterilized by 70% ethyl alcohol for 1-2 minutes and then rinsed in sterile distilled water for three to four times.Finally, the leaf bits were washed with 0.01% mercuric chloride for 1 or 2 minutes followed by washing with sterile autoclaved double distilled water 2 or 3 times.These fragments were transferred onto Potato dextrose agar (PDA) plates supplemented with 1.0% streptomycin sulphate (antibiotic solution) under completely sterile conditions in an inoculation chamber.

Identification of fungal pathogens
Identification of the fungal isolates was made with help of the relevant literature [7][8] [9][10] [11].The diagnostic characteristics of each isolate were compared with the previous work of the various authors.Fungal morphology was studied macroscopically by observing colony features (colony diameter, color, texture, and pigmentation) and microscopically by staining with Lacto phenol cotton blue (mounting fluid).A small pinpoint mycelium of each isolate was examined under a compound microscope for the observation of the conidia, conidiophores, and arrangement of spores.

Enzyme screening
The screening was performed by plate assay method.Isolated fungal strains were primarily tested for enzyme activity by culturing on CZA medium supplemented with 1% Carboxymethyl cellulose (CMC), 1% pectin, and 1% starch as a carbon source.Tetracycline was added to the media to control the bacterial contamination (pH 7.0).The plates were incubated at 30°C for 3 days.After incubation, the plates are flooded with following reagents (table no.2).The presence of clear zones around the fungal colonies indicates the production of respected enzymes.

Results
Rhythu Bazaar is a farmer's vegetable market located at Visakhapatnam, Andhra Pradesh.This markets hub for local farmers to sell their fresh produce directly to consumers, cutting out the middlemen and allowing farmers to earn a fair price for their crops.The term "Rythu Bazaar" translates to "Farmer's Market" in the local language.

Figure 1 Field survey on Rhythu Bazaar
In the survey conducted at Rythu Bazaar, A total variety of vegetables was recorded and categorized in the following table (Table 3), including both taxonomical and vernacular information.Additionally, during the survey, fungal diseased vegetables were identified and are represented in Figure 1, as outlined in Table 4.
To determine the plant pathogenic species responsible for the fungal infections, the isolated fungal species were identified.The results, presented in Table 3, show the following fungal species: Alternaria, Curvularia, Rhizopus, Penicillium, Aspergillus flavus, Aspergillus niger, and Mucor.These fungal species were associated with specific host plants.The table above summarizes the results of the enzyme screening for amylase, cellulase, and pectinase activities in three different (figure 7) fungal species: Rhizopus, Colletotrichum, and Sclerotinia.The screening was conducted by testing for the presence or absence of enzyme activity using standard assays.The results show that Rhizopus and Colletotrichum species have all three types of enzyme activities, these findings suggest that Rhizopus and Colletotrichum species may be useful sources of enzymes for industrial processes that require the breakdown of complex carbohydrates, such as starch, cellulose, and pectin.However, further studies are needed to determine the specific properties and potential applications of these enzymes.

Discussions
In total, 13 fungal endophytes were isolated from 13 different vegetables verities and five different tissue types like leaf, stem, root, flower and fruits.Our findings align with the results reported by [12] in their study on Soil Mycoflora in different soil samples from crop fields in Chintalapudi Mandal, West Godavari District, Andhra Pradesh, India.They also isolated genera such as Aspergillus, Mucor, Curvularia, Fusarium, Penicillium, and Rhizopus, which are similar to the genera we isolated during our investigation.
In another study conducted by [13] in agricultural fields of Tekkali Mandal, Srikakulam District, Andhra Pradesh, they identified 18 species belonging to 6 genera in Soil Mycoflora.They found that Aspergillus and Penicillium were dominant in their study.In our current study, we isolated a total of 27 fungal species belonging to 19 genera, and we also observed that Aspergillus and Fusarium were the dominant genera in all the agricultural fields we examined.
[14] examined the fungal diversity in rhizospheric soils from various fields in Nanjangud taluk of Mysore district, Karnataka.They identified 10 fungal species representing 7 genera, including Aspergillus, Alternaria, Mucor, Curvularia, Fusarium, Penicillium, and Rhizopus.Similarly, in our study, we isolated 27 species belonging to 19 genera, and the dominant genera we observed were Aspergillus, Fusarium, and Alternaria.
To summarize, our findings are consistent with previous studies, highlighting the presence of common fungal genera such as Aspergillus, Fusarium, Alternaria, and Penicillium in agricultural.
Among the isolated fugal isolates, Colletotrichum and Rhizopus species had the greatest production cellulase, amylase and pectinase enzymes these results corroborated [15], All isolates evaluated in this study showed cellulolytic activity as a yellow halo around the colony in plates including stained with Congo red and secured with NaCl.C. chrysophilum showed a significantly larger mean degradation halo of 8 mm in cellulolytic activity assay when compared to C. noveboracense and C. fioriniae (mean halo zone 6 and 6.5 mm, respectively).

Conclusion
Firstly, the presence of fungal infestations in vegetables sold in local markets indicates a potential health hazard to consumers.The fungi isolated from the vegetables belonged to different genera, including Aspergillus, Alternaria, Colletotrichum, Penicillium, Rhizopus, and Mucor, which are known to produce mycotoxins that can cause food poisoning and other health problems.
Secondly, the study showed that the incidence of fungal infestation was higher in leafy vegetables, spinach and coriander, than in root vegetables, carrot and potato.This suggests that leafy vegetables may be more susceptible to fungal infections, possibly due to their higher moisture content and surface area.
Thirdly, the enzyme screening of the isolated fungi revealed that they produced a wide range of extracellular enzymes, including amylase, protease and cellulase.This indicates the potential of these fungi for industrial applications, such as bioremediation and bioprocessing.
These findings suggest that Rhizopus and Colletotrichum species may be useful sources of enzymes for industrial processes that require the breakdown of complex carbohydrates, such as starch, cellulose, and pectin.However, further studies are needed to determine the specific properties and potential applications of these enzymes.
Overall, the study highlights the need for improved hygiene and quality control measures in local markets to prevent the spread of fungal infections in vegetables.It also underscores the importance of further research on the enzymatic properties of fungi and their potential applications in biotechnology.

Figure 2 Figure 3 Figure 4
Figure 2 Different types of fungal infected leafy vegetables

Figure 4
Figure 4 Isolated fungal pathogens grown on PDA medium plates, A. Colletotrichum spp.B. Sclerotinia spp.And C.Rhizopus spp.

Figure 5 Figure 6
Figure 5 Graphical representation of enzyme screening with isolated plant pathogenic fungi

Figure 7
Figure 7 Enzyme activity of isolated fungal spp.With amylase, cellulase and Pectinase clear zones.

Table 1
Different types Fungal diseases in vegetable crops

Table 2
Different types enzyme screening reagents and subtracts

Table 4
List of Identified fungal pathogens from infected vegetables

Table 4
Enzyme screening assay with isolated plant pathogens