# Metagenomic insights into the fungal diversity of flood-affected areas in Kuttanad, Kerala

Jisha J

Department of Bioinformatics, Union Christian College, Aluva, Kerala

Director, Rajiv Gandhi Centre for Biotechnology, Thiruvanathapuram, Kerala

Dr. Shijulal Nelson Sathi

Interdisciplinary Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvanathapuram, Kerala

## Abstract

Flood is the most common natural disaster occcuring world-wide which can impact both individuals and communities and have social, economic and environmental consequences. The increased rate of microbial loads that seen in flood-damaged areas has resulted in high incidences of infectious diseases including respiratory disorders such as allergy and asthma. Reports also reveal that fungal infections contribute to major percentage of observed microbial infections in flooded areas. The impact of climate change on fungal species and its habitat is not well studied. The 2018 flood crisis that occurred in Kerala has largely affected its economy and ecological habitat. The Kuttanad region of Kerala was one of the severely affected areas and thousands of hectares of land were submerged under water. This project aims to understand the fungal diversity in the soil sediments collected from flood affected Ramankary region of Kuttanad, Kerala. The fungal diversity will be elucidated by analyzing the shotgun metagenomic data from Divakaran et.al., 2019. The bioinformatics technique will be employed to identify the taxonomic classification in that data. It is found that the sample flood water taken from Ramankary, Kuttanad contains mainly seven kinds of fungal communities, including Leotiomycetes and Rhodotorula, most of them can cause serious plant and animal diseases. These results will give an overall idea about the common fungal communities that is present in the flood-affected Kuttanad region and its increase in the percentage of concentration when compared to a normal environment. For a better understanding of the fungal communities that occur as a result of the flood, large scale sampling of environmental sediments from different areas of flood-affected Kuttanad is needed.

Keywords: flood, metagenomics, microbial community, fungal diversity

## Abbreviations

Abbreviations
 BLAST Basic Local Alignment Search Tool PEAR Paired End Read Merger QC Quality Control MEGAN Meta Genomic Analyser LR Long Read LCA Lowest Common Ancestor DNA Deoxyribo Nucleic Acid NCBI National Centre for Biotechnology Information SRA Sequence Read Archeive SRR SRA Run_accession

## INTRODUCTION

Flood is considered as one of the most devastating natural disasters which can destroy both life and infrastructure. This disaster can occur due to various reasons including rain, storm, snow or through an overflow of dams or other water bodies. On 16th August 2018, Kerala, a South Indian state was severely affected by floods because of the heavy monsoon rainfall. The Kerala monsoon in 2018 was 42% higher than the Monsoon average and thus Kerala received a cumulative rainfall of 2346.3 mm [1]. This flood is reported as the worst flood in Kerala in nearly a century. The Indian Government declared that flood as the "calamity of severe nature". It is the worst flood in Kerala after that took place in 1924 [2]. One-sixth of the total population i.e., nearly 23 million people are said to be affected in this flood and related events. Over 500 people died and nearly 150 are reported missing [1]. Due to this heavy rainfall, 35 out of 54 dams within the state were opened. Heavy rain happened in Wayanad and Idukki district has resulted in landslides which isolated the hilly areas. The flood destroyed more than 10,000 houses completely and more than 1 million houses partially. Almost 83,000 kilometers of road and 60,000 hectares of the agriculture field got affected. The flood resulted in damage worth 2.9 billion Indian rupees [1,3].

Climatic conditions can affect epidemic infections [7]. The adverse health events due to flood water is a threat to human health. The outbreak of infectious diseases after the natural disaster is a common issue. There are various health impacts associated with flood including the risk of vector-borne and water-borne diseases. People in the flood-affected areas have more chances of getting affected by various diseases such as cholera, malaria, dengue fever, typhoid, yellow fever, skin infections, leptospirosis, etc. [4] This may be because of the low vaccine coverage, poor sanitation facility, limited access to the health care facility, inadequate hygienic infrastructure and massive population displacement [5]. The stagnant water can act as a home for various microbial pathogens [4]. This can be because the severe flood runoff combines water from various sources like sea, river, well with sewage from domestic waste, hospital, waste treatment, industries, etc., and thus these water altogether get exposed in the natural environment [1]. So there is an increased chance of transmission of infectious diseases [5].

As Kerala is one of the most monsoon rainfall regions in India, it is more vulnerable to infectious diseases associated with monsoon rain [7]. In 2018, the region that was most affected by the heavy monsoon rainfall was the Kuttanad regions of Kerala. These regions mainly consist of paddy fields and therefore it is known as the 'rice bowl of Kerala'. As it is almost 2.5 meters below sea level, the region is mostly water-logged [18]. This low-lying nature is the main reason for the massive destruction in Kuttanad due to heavy rainfall every year [7]. As a result of the devastating flood, there is a chance of an epidemic outbreak in Kuttanad, Kerala including waterborne diseases typhoid, fever, cholera, leptospirosis, and hepatitis viral influenza and vector-borne diseases like malaria, dengue and yellow fever [6,7]. As health centers in this region are also submerged under water, the condition became worse [6]. Many of those diseases are spreading like a wildfire.

The outbreak of fungal infections after a disaster is now a matter of concern. During a natural calamity, the pathogenic fungi can be displaced from their habitat which can lead to their increased growth and occurrence in the areas where they are not commonly found. The respiratory diseases and wound contaminations due to fungal communities present in floodwater is a serious issue as there is an increased chance of fungal contact from the contaminated water. It is reported that people in Kuttanad are severely affected by skin diseases every year after the Monsoon season [7]. Most of these infections are happening due to the inhalation of fungal spores or cutaneous inoculation of fungi from the disaster environment [5,19]. So, fungal infections after a natural disaster is a matter of clinical and public health issue. It is a matter of research by the health care providers, public health professionals and community members [5].

For a better understanding of the infections associated with pathogenic fungi, it is very important to have a detailed investigation regarding the fungal communities in the floodwater. This project aims at understanding the fungal diversity present in the flooded environment of Kuttanad. Flooded soil sediment is collected from the Ramankary region of Kuttanad. For a better understanding of the fungal community that occurs due to the flood by comparing it with a normal environment sample, the soil sample was collected from the Ponmudi region of Thiruvananthapuram. This region was not affected by the 2018 flood. Using shotgun metagenomics approach, the fungal communities are already sequenced. These sequences has bioinformatically analysed to obtain the taxonomic classification of fungal communities in it. It is found that the flood-affected Kuttanad region has a higher abundance of different kinds of plant pathogenic fungi. The presence of pathogenic fungi might be or might not be due to flood. This dilemma can only be solved by the large sampling and analysis of sediments from flood-affected Kuttanad regions. As Kuttanad is an agricultural area, these results will pave first pebbles to take necessary steps to prevent plant infections that might occur as an after-effect of flood and thus can control the fungal disease outbreaks related to a disastrous flood happened in Kuttanad regions of Kerala.

## METHODOLOGY

The samples were collected from two different locations, from flood-affected Ramankary region of Kuttanad and Ponmudi region, Thiruvananthapuram which was not affected by the flood. The latter is used as the control sample in this project for getting a better understanding about the fungal communities in flood affected and normal environment.

Figure 1 represents the collection sites of the two environment sample used in this project.

Map showing the sites from where the sample is collected. The first sample is collected from Ramankary region of Kuttanad and the second sample is collected from Ponmudi region, Thiruvanathapuram.

The fungal metagenomic dataset of the Ramankary region is reported in NCBI-Sequence Read Archive (SRA) by Divakaran et.al, 2019 during their investigation about the bacterial profiles present in the flood-affected Kuttanad region (Accession no: SRR 9620087). This data was downloaded from SRA for further analysis of the fungal communities in it. To get a better understanding of the flood associated fungal profile, an environment sample was collected from Ponmudi, Thiruvananthapuram, Kerala which was not affected by the flood season of August 2018. From that sample, metagenomic DNA was extracted. Libraries were prepared from these DNA using shotgun metagenomics [17]. The quality of the sequences was analyzed using FASTQC. The sequences that have Q value less than 20 and have adapter sequences were removed using the FASTX toolkit. All the commands that were used in this bioinformatics analysis were in the UBUNTU operating system

## Merging Forward and Reverse Strands

The library obtained had forward and reverse strands separately. It should be merged before starting the analysis. Using PEAR Merger which is a paired end read merger, the forward and reverse strands of the sample is merged using the command:

## Creating a Local Database

A biological database is a structured collection of biological data. A local database should be created for doing nucleotide-BLAST to identify the fungal species in the sample. The UNITE database was used here which is a web-based database that is used for the molecular identification of fungi [9, 21]. From the UNITE database, a FASTA zip file containing fungal taxon was downloaded. Using this file, a local database was created using the command:

## Filtering the Results

The output file were filtered to get the relevant results using the command:

$awk '{ if ($1>0) print }' resultfile.out>outputfile.txt

This output file is used for further steps in fungal taxonomic analysis.

## Taxonomic Classification using MEGAN

MEGAN is a computer software that is used for the analysis of the metagenomic dataset. It is mainly used for the analysis of genome sequences from the environmental sample by analyzing biodiversity in them [8]. The output data obtained after filtering the BLAST results were analyzed using MEGAN-LR 6.18.0. The default parameters that were used in MEGAN are: Minimum score = 50 , Top percent= 10, min Support = 1, min-complexity filter = 0. The weighted LCA algorithm was used as the parameter for the analysis with a minimum percentage of coverage as 80. From the results, the fungal species that were present in the sample were identified [20].

## RESULTS AND DISCUSSION

Though there was 21,24,021 and 32,05,802 sequences in the Ramankary and Ponmudi samples respectively, only 9 different kinds of fungal species had identified from the sample.

Fungal diversity present in the sample collected from Ramankary, Kuttanad . The sample is abundant with Leotiomycetes (28.9%), Lignincola laevis (19.16%), Tomentalla (15%), Bannoa ogasawarensis (13.7%), Rhodotorula (9.72%), Knoxdaviesia cecropiae (8.8%) and Cystoagaricus hirtosquamulosus (4.72%).

Figure 2 represents a pie cart containing the fungal diversity that is present in the sample collected from Ramankary region of Kuttanad, Kerala. The percentage of each of the fungal communities that were present in the Ramankary sample is shown in Table 1.

Percentage of concentration of fungal communities  in the sample from Ramankary region
 Fungal species Percentage of concentration Leotiomycetes 28.9% Lignincola laevis 19.16% Tomentalla 15% Bannoa ogasawarensis 13.7% Rhodotorula 9.72% Knoxdaviesia cecropiae 8.8% Cystoagaricus hirtosquamulosus 4.72%

The fungal species that were found in the Ramankary sample were Leotiomycetes (28.9%), Lignincola laevis (19.16%), Tomentalla (15%), Bannoa ogasawarensis (13.7%), Rhodotorula (9.72%), Knoxdaviesia cecropiae (8.8%) and Cystoagaricus hirtosquamulosus (4.72%). It is found that Leotiomycetes and Lignincola laevis are the most abundant fungal species in the environment sample from Ramankary.

Fungal diversity present in the sample collected from Ponmudi, Thiruvananthapuram. The sample is abundant with Bannoa ogaswarensis (65.5%), Tomentella (25%), Erysiphaceae (3.4%), Lignincola laevis (3%),Beauveria velata (1.7%) and Knoxdaviesia cecropiae (1.4%).

Figure 3 represents a pie chart containing the fungal diversity that is present in the Ponmudi region of Thiruvananthapuram, Kerala which was not affected by 2018 flood. The percentage of each of the fungal communities that were present in the Ponmudi sample is shown in Table 2.

Percentage of concentration of fungal communities in the sample from Ponmudi region
 Fungal Species Percentage of concentration Bannoa ogaswarensis 65.5% Tomentella 25% Erysiphaceae 3.4% Lignincola laevis 3% Beauverta velata 1.7% Knoxdaviesia cecropiae 1.4%

The sample that was taken from Ponmudi, Thiruvananthapuram has 6 different kinds of fungi that was present in the sample were Bannoa ogaswarensis (65.5%), Tomentella (25%), Erysiphaceae (3.4%), Lignincola laevis (3%), Beauveria velata (1.7%) and Knoxdaviesia cecropiae (1.4%). As the pie chart shows, the Ponmudi environment sample is more abundant with Bannoa ogaswarensis species.

Both the samples have a common presence of Knoxdaviesia cecropiae, Lignincola laevis, Tomentella and Bannoa ogasawarensis but their percentage of concentration seems to be higher in the flood-affected area when compared to the control sample. Knoxdaviesia ceceropiae is an insect pathogen [16]. Lignincola laevis is a kind of marine fungi that is found to have cytotoxic activity against both plant and animal cells. They are the most common fungi in Mangrove trees and are also known as Mangrove fungi [11,12]. Bauveria velata is a fungal species that is capable of infecting both plants and animals [14]. Bannoa ogaswarensis is a plant pathogen that is capable of infecting the leaves [13].

Besides the abundance of above fungal communities, the flood-affected Ramankary region of Kuttanad had an abundance of Leotiomycetes species which is a plant pathogen that forms a kind of white powdery dust in flowering plants. This dust can usually be seen in vegetative and reproductive parts of the plant that is capable of absorbing nutrients from the plants [10]. The presence of Rhodotorula species in the flood-affected areas is a matter of concern. Though it is not in abundance in the Ramankary sample, it can act as a causative agent of mycoses especially in vulnerable patients like those who have HIV infections or Leukemia [15]. The other fungi, Cystoagaricus hirtosquamulosus is a mushroom that is non-pathogenic.

In the previous researches that were done in the flood-affected areas of Ramankary, Kuttanad, it was found that those areas were heavily contaminated and had a high concentration of pathogenic bacterias including Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella typhimurium, Klebsiella pneumoniae, Vibrio cholerae etc. [1]. Compared to that the fungal communities in that region is not severe in pathogenecity except for the traces of Rhodotorula species which is highly pathogenic in nature. The level of concentration of fungal colonies seems to be more in the flooded areas when compared to the control sample. This might be because the favourable condition arisen for fungal growth due to a disastrous flood happened in Kerala. As most of the fungal communities identified were plant pathogens, there is a chance of a fungal epidemic outbreak in the regions like Kuttanad which is famous for its agricultural productivity. This can only be confirmed after larger sampling of the environmental sediment from different locations of Kuttanad that was affected by the flood as it will give a more vivid idea about the fungal communities in the flood-affected regions. The drug resistance study of the identified fungal species can also be done so that nature and pathogenicity of fungi can be studied as it is very necessary to be known before taking any steps to prevent the epidemic outbreaks or infections.

## CONCLUSION

The project was aimed to identify the fungal community that is present in the flood-affected Ramankary region of Kuttanad, Kerala. The flood that happened in August 2018 in Kerala has severely affected the Ramankary region of Kuttanad. The environmental sample was collected from both Ramankary region and Ponmudi region, Thiruvananthapuram for analysing the fungal taxonomy present. Using Shotgun Metagenomics, the fungal community was sequenced. The bioinformatics approach is implemented to identify the fungal species present in both the sample. It is found that the flood-affected region has a presence of plant pathogenic fungi like Leotiomycetes, Lignincola laevis, Tomentalla, Bannoa ogasawarensis, etc. Though many of these fungal communities were common, the flood-affected areas are found to have the presence of Rhodotorula fungal species which is highly pathogenic both to plants and animals. The percentage of concentration of fungal communities in the flood-affected region seems to be more when compared to the control sample. The presence of these fungal communities is might be because of the favourable environmental conditions for fungal growth that have occurred after flood. These fungal species can produce mycoses to both plant and animal species. As Kuttanad is an agricultural area, the high concentration of the above fungal communities might affect the agricultural productivity of that region. For the more detailed investigation, the large scale analysis of environment sample from different flood-affected regions of Kuttanad and drug resistance study of the identified fungal species is needed, as it will give a clear picture about the nature and abundance of fungi in the flood-affected regions for taking necessary actions for preventing fungal epidemic outbreaks if any.

## REFERENCES

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## ACKNOWLEDGMENTS

I am very thankful to the Indian Academy of Sciences (Bangalore), Indian National Science Academy (New Delhi), National Academy of Sciences (Allahabad) for providing me the Summer Research Fellowship Programme-2019 and allowing me to work under such experienced professionals. I am highly obliged to my project guide, Prof.M.Radhakrishna Pillai, Director at Rajiv Gandhi Centre for Biotechnology, Thiruvanathapuram, Kerala for giving me the privilege to work in such a prestigious institution. I am also very thankful to Dr. Shijulal Nelson Sathi, Scientist at Rajiv Gandhi Centre for Biotechnology, Thiruvanathapuram, Kerala for allowing me to work in his lab under his supervision.

I would take a moment to thank Mr. Shyam Mohan, Head of the Department of Bioscience, Union Christian College, Aluva, Kerala who had insisted me to apply for this fellowship. I am highly grateful to all the faculty members of my college for recommending me to apply for this fellowship. I have gained a lot of knowledge and experience from this summer project which will be highly useful for my future studies. I am highly thankful to Almighty God for always blessing me. I am obliged to my parents who are always by my side to help me, my friends who are my constant supporters, my fellow trainees for encouraging me for the completion of this project.

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