SURVEY AND INVENTORY OF SOIL MICROBIAL BIOTA IN THE POST FLOOD SCENARIO (MANIMA RIVER)

A Report Submitted to State Biodiversity Board Govt of Kerala

Dr. Shiburaj Sugathan (Principal Investigator) Division of Microbiology

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SURVEY AND INVENTORY OF SOIL MICROBIAL BIOTA IN THE POST FLOOD SCENARIO ( RIVER)

Final Report to Kerala State Biodiversity Board (KSBB), Government of Kerala

Submitted by Division of Microbiology Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala-695562.

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Title: Survey and inventory of soil microbial biota in the post flood scenario ()

Thrust area: Soil biota

Study Areas: Manimalayar

Investigating Team Principal Dr. Shiburaj S. Investigator: Senior Scientist & Head, Division of Microbiology Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala-695562. Email: [email protected] Tel.: 0472 2869226 Mob: 9495826669

Co-Principal Investigator 1. Dr. Rachanamol RS Post Doctoral Fellow Division of Microbiology Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala-695562

Co- Investigators: 2. Dr. Biju H Technical Officer Gr. III Division of Microbiology Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala-695562

3. Dr. Sabeena A Technical Officer Division of Microbiology Jawaharlal Nehru Tropical Botanic Garden and Research Institute Palode, Thiruvananthapuram, Kerala-695562

Budget Sanctioned (No. 3371/48/2018AGBB dated 22.10.2018) Item Amount (Rs) Man power 66,000.00 Consumables 3,00,000.00 Travel 1,50,000.00 Contingencies 70,000.00 Other items (Sequencing Charges) 1,00,000.00 Institutional overhead (5 %) 34,300.00 Total 7,20,300.00 Fund Released 5,76,240.00 Fund Utilised 7,11,000.00

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Introduction

Recent heavy rains have created large scale stagnant flooded soil and seriously affected soil conditions including biotic and abiotic factors. These factors are primary concerns for crop producers. A disturbance such as flooding affects both above- and belowground ecosystem processes. Floods affect soil structure and fertility, reducing nutrient availability and initiating primary succession processes in the case of strong disturbances. Soil microorganisms are vital for the functioning and long-term sustainability of ecosystems (Pankhurst et al., 1996). They play key roles in organic matter decomposition and nutrient cycling and thereby for plant growth. Soil microorganisms are sensitive to disturbance, and shifts in soil microbial community structure are expected when anaerobic conditions develop from flooding. Other soil conditions are also affected. The rapid depletion of soil oxygen causes a shift in nitrogen cycle soil micro-organisms that lead to the denitrification processes. This loss of soil oxygen is the greatest challenge to established natural and crop plants. Flood also results in soil oxygen depletion, fostering anaerobic conditions and microorganisms able to survive these conditions. Such changes in soil abiotic conditions can alter soil microbial community composition.

Flooding frequently results in higher levels of plant diseases that reduce stands and yields. Cool, wet soils are most conducive to disease development. Soilborne plant pathogens respond differently to soil flooding, and there can be both positive and negative effects of flooding in a soil environment. As a rule, populations of soil microorganisms like fungi, bacteria, and actinomycetes generally decline in flooded soils. The actinomycetes in soil are one of the major players in soil ecosystem, controlling most of the soil born plant pathogens by their antagonistic properties. Further, disease in host plants may be influenced by soil waterlogging depending on (i) the growth and ability of a pathogen to reach host tissues (if it is a motile propagule such as a zoospore); (ii) whether host resistance is altered; and (iii) if the activities of antagonistic microorganisms are changed. Direct digestion of fungal structures by microorganisms also has been proposed as one mechanism of inhibiting fungi through flooding (Mitchell and Alexander 1962). There is always the danger of spreading soilborne pathogens via a flooding program, bringing risk to the practice. However, these conditions are not only optimal for some soil pathogens, but also delay emergence and plant development. These delays keep seedlings from out-growing damage by soil-borne diseases that attack seeds and seedlings. In addition to seedling diseases like Pythium, other diseases may also infect the plant during flooding, but their symptoms may not be evident until much later.

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Study Area

Though all the 14 districts of Kerala had a share of the havoc caused by disaster, the districts of received the major chunk of damage. Our study area includes the Manimala River basins (from high range to low lands).

Manimala River is about 92km long often mistaken as a tributary of Pamba. But clearly it’s evidence that a branch of Pamba flows to Manimala from and joins Manimala at Kallunkal later branches out from Manimala at and flows through ,, , , Nedumudy and finally to and empties into lake. Manimala is a separate independent river for all geographical purposes.

It has it’s origin on the Hills (2500 feet above sea level) on the Western Ghats ,in of Kerala ,. The river passes through the districts of , Pathanamthitta and . The villages and small towns like , Mundakkayam, Erumeli, Manimala, Kulathurmuzhy, Mallappally, Niranam, Kuttor, Nedumpuram, Neerettupuram, , Kidangara, , and Mankompu lie on the banks of Manimala river.

Approved Objectives The objectives of this project are to:

1. The primary objective of the proposed study is to determine the effect of flooding on soil microbial communities. 2. To provide information on the negative effects of recent flood events on soil biota particularly on microorganisms. 3. To provide a comparative information on increase or decrease soil born phytopathogen populations and plant growth promoting organisms towards predicting their effects on crops.

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SOIL MICROBIAL BIOTA ON RIVER BED Total biome on river bed is protruded as a result of geological and climatic variables like Salinity, pH, Mineral concentration, Water availability, Wind, Temperature etc. So, variations in temperature and humidity affect the local soils, which in turn alter the microbial flora of that particular river basin. Natural calamities like Tsunami, Flood, Hurricanes are major problems that affect the natural biota as well as the spontaneous process of our ecosystem. So, the microbial diversity of soil collected along the banks of flood affected river basin formulates the actual picture of microbial ecosystem and fertility of soil after and before the flood.

Fig 1: Map of Manimala River

MATERIALS USED

All media, bases and media supplements used throughout the study were purchased from Hi Media (Mumbai). The fine chemicals were purchased from Sigma (USA), Qualigens (Mumbai) and SRL (Mumbai). All the glass wares (Test tubes and petriplates) used was manufactured from Borosil Co Ltd Mumbai.

COLLECTION OF SAMPLES AND ANALYSIS Soil samples were collected from various flood affected regions in Alappuzha and Pathanamthitta districts taken as study material. We have collected samples from Flooded area, Border area and Control. Samples were brought to lab aseptically for further analysis.

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Sample A – Flooded Area Sample B – Midway from source (buffer zone) Sample C – Adjacent area unaffected by flood (control)

Collection spots We conducted different field trips. Total of 87 samples were collected. No Visited Locations 1 Adamattom, 2 3 Chennappady 4 Manimala Town 5 Manimalakavu Devi Temple 6 Puthenpally 7 Kulathor Devi Temple 8 St Marys Old Church, Vaipar 9 East Bridge 10 Chakkulathukavu 11 Thalavadi 12 Muttar, Kumaranchira 13 Kidangara 14 Veliyanad 15 Ramankari 16 Vezhapra 17 Thekkekkara, Mankombu 18 Thekkekkara Boat Jetty 19 Chambakkulam, Powerhouse Road 20 Thirumoolapuram, 21 Vembalam, 22 Kallumankavu, ARS Junction, Thiruvalla 23 Iramallikkara, Thiruvalla 24 Valanjavattom, , 25 Pulikeezhu, Thiruvalla, Pathanamthitta 26 Pattode, Thiruvalla 27 Manakkachira, Thiruvalla, Pathanamthitta 28 Vallalamkulam Bridge 29 Elanthore, Thiruvalla, Pathanamthitta

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ADAMATTOM – MALLAPPALLY

Near CHAKKULATHUKAVU – KIDANGARA

Level of water during flood

THIRUVALLA AREA

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ISOLATION OF SOIL MICROBES The Serial dilution method was followed to isolate microorganisms from collected soil samples. 10g soil samples from all the three site samples labelled as A, B, C were weighed and added to 90ml sterile distilled water and vortexed. The soil sample was then serially diluted and 1ml diluted soil sample from four dilutions, 10-3 to 10-6 were pour plated on NA (Nutrient agar ) media for bacterial isolation, GA (Glucose –asparagine ) media for Actinomycetes and SDA (Sabouraud’s dextrose Agar) media for fungi. After 24h bacterial colonies from nutrient agar plates were enumerated. After 5 days hours of incubation fungal strains and after 7 days Actinomycetes from the selective media were also enumerated. The total cfu/gram soil was calculated for each group of organisms. The colonies appeared with difference in morphology were sub-cultured and stored for further analysis.

Fig.3: Samples collected Fig. 4: Media Used

Fig.5: Fungal colonies appeared on SDA Plates

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Fig.6: Bacterial colonies on NA Fig.7: Actinomycetes colonies on GAA

RESULTS

Soil bacteria, fungi and Actinomycetes were isolated on nutrient agar, Glucose –Asparagine media and SDA (Sabouraud’s Dextrose Agar) plates respectively following dilution plate method were counted and tabulated. CFU (Colony forming units) per gram soil was calculated. The isolates were made into pure cultures and stored. The flooded area, the presence of coliform bacteria, anaerobic bacteria etc are more comparing to control samples. The fungal populations show the presence of plant pathogenic species. As per plat assays the Buffer zone is having more Actinomycetes, bacterial and fungal load. The bacterial load in flooded area is more than that of control but lower than that of buffer zone. Fungal load is comparatively lower than that of Control and buffer zones, while Actinomycetes load is almost equal to that of buffer zone.

Samples of Vazhoor to Mallappally: The enumeration of bacterial load from nine spots, Adamattom to Mallappally East Bridge at Pathanamthitta showed a lower bacterial load near the flood affected areas which are inhabited by human populations. In these areas we found a massive dumps of plastics and waste. Mostly the buffer zone (Area B) where plantations was found to be favourable niche for microbes. The control area, where flood was not affected also had a lesser microbial diversity (Chart1, 2 & 3).

Samples of Chakkulathukavu to Kidangara : The ten areas we have located for the soil collection from Chakkulathukavu to Kidangara were mostly very close to Manimalayar and are mainly surrounded by cultivation fields. Different soil samples collected from these areas very near to overflown river had a higher proliferation of diverse microbial community. Also the

9 major crops in this regions were also noticed. From these areas the microbial load was high especially at highly flooded areas (A) at Veliyanad, Kidangara and Muttar. In this areas the count of Actinomyctes were also observed to be high.

Samples of Thiruvalla regions in Pathanamthitta: A diverse bacterial population was enumerated from the ten areas we chosen from Thiruvalla. The bacterial load was high at those plantation areas very close to Manimalayar. Here we like to spot two areas Vallalakulam Bridge and Iramallikara Bridge, Area (A) very close to riverbed with cultivation lands reported to be a favourable habitat of microbes (both bacteria and actinomycetes) than buffer zone (B) and control area (C) which was not affected by flood.

Chart-1: Total CFU of Bacterial isolates in different sampling spots (Comparison of Flooded Area, Buffer Zone and non-flooded Area).

Soil Bacterial load 1400 Flooded Area Buffer Zone Control 1200

1000

800

600

Cfu/gm(x10³) 400

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Sampling areas

Chart-2: Total CFU of Actinomycetes isolates in different sampling spots (Comparison of Flooded Area, Buffer Zone and non-flooded Area).

Soil Actinomycetes Load 3000

2500 Flooded Area Buffer Zone Control 2000

1500

1000 Cfu/gm(x10³)

500

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Sampling areas

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Chart-3: Total CFU of Fungal isolates in different sampling spots (C0mparison of Flooded Area, Buffer Zone and non-flooded Area).

Soil Fungal Load

1600

1400 Flooded Area Buffer Zone Control 1200

1000

800

600 Cfu/gm Cfu/gm (x10³)

400

200

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Sampling areas

Comparative Table of Colony forming Units (cfu) of Bacteria, Fungi and actinomycetes in different Sampling places based on dilution plating on selective media

AdamattomVazhoor (9.511072/9.511072) Major Plants: Coconut, Arecanut, Teak Microbial Count (Cfu/g) Flooded Area (A) Buffer Zone (B) Control (C) Bacteria 23x103 51x103 43x103 Fungi 34x103 14.2x104 47.7x102 Actinomycetes 49.5x102 63.7x102 55.3 x102 Cheruvally S.C.T.M. UP School (9.504184/ 76.751472) Major Plants: Coconut Bacteria 12.1x103 27.4 x103 99.3x102 Fungi 16.3x102 76.2 x102 39.6 x102 Actinomycetes 39.3 x102 168.2 x102 124.3x102 Chennappady, Pathanamthitta (9.505287/76.799155) Major Plants : Arecanut, Pepper Bacteria 276.7x102 507.8x102 660.5x102 Fungi 71.6x102 13.9x103 91.1x102 Actinomycetes 68.8 x102 26.3x x103 85.6 x102 Location: Manimala Town (9.494585/76.75152) Major Plants: Tapioca, Coffee Bacteria 432x103 480 x103 338x102 Fungi 105 x102 870x102 539x102 Actinomycetes 250.8x102 872 x102 206 x102 Manimalakavu Devi Temple (9.485855/76.742971) Major Plants: Rubber, Nutmug

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Bacteria 602x102 460x103 244.4 x102 Fungi 115.8 x102 151 x103 526.6 x102 Actinomycetes 77.2 x102 273.6 x103 97.2 x102 Puthenpally, Pathanamthitta 9.464755/76.725675 Major Plants: Rubber, Tapioca Bacteria 108.5x103 601 x103 376 x103 Fungi 160 x103 310x103 182 x103 Actinomycetes 180 x103 285 x103 393 x103 Kulathor Devi Temple (9.455686/76.71190) Major Plants: Coconut, Arecanut, Banana Bacteria 169x103 831x 103 129x103 Fungi 320x103 745x103 930x102 Actinomycetes 153 x103 245 x103 165 x103 St Marys Old Church, Vaipar (9.450274/76.70140) Major Plants: Jackfruit, Coconut Bacteria 347 x103 1252 x104 132 x103 Fungi 176x103 828 x103 465 x103 Actinomycetes 483x103 142x105 507 x103 Mallapally East Bridge (9.444151/76.657662) Major Plants: Pepper, Coconut, Teak Bacteria 166x103 313 x103 71 x103 Fungi 131 x103 438 x103 116 x103 Actinomycetes 75 x103 686 x103 246x x103 Chakkulathukavu, , Alappuzha (9.369389/76.510298) Major Plants: Coconut Bacteria 180x104 374 x103 241 x103 Fungi 38 x103 90 x103 87 x103 Actinomycetes 135 x103 711x103 263x103 Thalavadi, Alappuzha (9.372549, 76.510219) Major Plants: Mango, Banyan, Arecanut Bacteria 555x104 205 x103 186 x103 Fungi 76 x103 76x103 46x103 Actinomycetes 187 x103 237x103 73 x103 Muttar, Kumaranchira (9.39487/76.51876) Major Plants: Coconut, Banana Bacteria >1000X103 >1000X103 676X103 Fungi 37x103 121X103 44 x103 Actinomycetes 203 x103 491 x103 135 x103 Kidangara (9.45236/76.39874) Major Plants: Bamboo, Acacia Bacteria 429 x103 574 x103 122 x103 Fungi 37 x103 268 x103 74 x103 Actinomycetes 105 x103 210 x103 45 x103 Veliyanad (9.45275/76.4673) Major Plants: Mango, Arecanut Bacteria 46 x103 154 x103 25 x103 Fungi 35 x103 46 x103 16 x103 Actinomycetes 64 x103 75 x103 34 x103 Ramankari, Alappuzha (9.2542/76.4606) Major Plants: Mango, Banyan Tree

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Bacteria 81 x103 16 x103 13x103 Fungi 706 x103 710 x103 212 x103 Actinomycetes 240 x103 >1000X103 589 x103 Vezhapra, Ramankari (9.42356/76.4607) Major Plants Acacia, Coconut Bacteria 197 x103 195 x103 70 x103 Fungi 68 x103 32 x103 70 x103 Actinomycetes 234 x103 220 x103 91 x103 Thekkekkara, Mankombu (9.35462/76.43621) Major Plants: Nutmug, Coconut Bacteria 192 x103 >1000X103 46 x103 Fungi 70 x103 77 x103 73 x103 Actinomycetes 68 x103 130 x103 112 x103 Thekkekkara Boat Jetty (9.43186/76.42918) Major Plants: Paddy, Banana Bacteria 22 x103 >1000X103 60 x103 Fungi 74 x103 89 x103 38 x103 Actinomycetes 60 x103 19 x103 52 x103 Chambakkulam, Powerhouse Road Thekkekkara (9.47653/76.425612) Major Plants: Paddy, Coconut Bacteria 167 x103 260 x103 98 x103 Fungi 22 x103 11 x103 16 x103 Actinomycetes 180 x103 350 x103 143 x103 Thirumoolapuram, Thiruvalla (9.22056/76.34) Major Plants: Banyan/ Cashew Bacteria 106 x103 406 x103 54 x103 Fungi 37 x103 44 x103 37 x103 Actinomycetes 88 x103 55 x103 22 x103 Vembalam, Kuttoor (9.22/76. 34) Major Plants: Paddy Nutmug Bacteria 73 x103 104 x103 68 x103 Fungi 37 x103 41 x103 22 x103 Actinomycetes 23 x103 61 x103 30 x103 Kallumankavu, ARS Junction, Thiruvalla (9.3674 /76 .56135) Major Plants: Banana, Coconut Bacteria 28 x103 74 x103 35 x103 Fungi 37 x103 74 x103 37 x103 Actinomycetes 20 x103 49 x103 12 x103 Iramallikkara, Thiruvalla (9.22/76.3) Major Plants: Acacia, Tamarind Bacteria 35 x103 38 x103 29 x103 Fungi 37 x103 74 x103 71 x103 Actinomycetes 89 x103 96 x103 30 x103 Valanjavattom, Kadapra, Travancore Sugar & Chemicals Ltd (9.21/76.32) Major Plants: Cashew, Banana Bacteria 51 x103 >1000X103 69 x103 Fungi 37 x103 40 x103 24 x103 Actinomycetes 108 x103 196 x103 57 x103 Pulikeezhu, Thiruvalla, Pathanamthitta (9.21/76.31) Major Plants: Elengi, Teak

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Bacteria 130 x103 >1000X103 65 x103 Fungi 50 x103 15 x103 40 x103 Actinomycetes 243 x103 35 x103 12 x103 Pattode, Thiruvalla (9.23/76.31 ) Major Plants: Ornamental plants , Cashew Bacteria 46 x103 48 x103 42 x103 Fungi 37 x103 61 x103 38 x103 Actinomycetes 34 x103 54 x103 22 x103 Manakkachira, Thiruvalla, Pathanamthitta (9.38/76.60842) Major Plants: Paddy, Banyan tree, Acacia Bacteria 82 x103 94 x103 79 x103 Fungi 37 x103 54 x103 47 x103 Actinomycetes 36 x103 41 x103 21 x103 Vallalamkulam Bridge (9.38/76.6154) Major Plants: Cashew Bacteria 105 x103 >1000X103 45 x103 Fungi 87 x103 102 x103 79 x103 Actinomycetes 47 x103 49 x103 32 x103 Elanthore, Thiruvalla, Pathanamthitta (9.39/76.6286) Major Plants Cashew, Paddy, Acacia Bacteria 37 x103 66 x103 18 x103 Fungi 37 x103 67 x103 48 x103 Actinomycetes 24 x103 46 x103 33 x103

PURE CULTURES OF DIFFERENT FUNGI ON SDA MEDIUM

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SLIDE CULTURE & MICROSCOPY OF FUNGAL ISOLATES The fungal colonies isolated from the flood affected regions were morphologically identified using slide culture method using Lactophenol Cotton Blue Stain. This procedure used primarily with pure cultures. Placed a drop of SDA media in the centre of slide and place a coverslip over it and kept in a sterilized petriplate. Then inoculated a loopful of fungal strain to it and to maintain the moisture for fungal growth add 1mL of sterile distilled water to the filter paper placed on the plate. Allowed it for maximum growth of 5 to 7 days for better microscopical analysis .After 7 days of incubation place a drop of Lactophenol Cotton Blue Stain in the center of the slide. Place the fragment in the drop of stain along the sides of coverslip. Examined the preparation under low and high, dry magnification for the presence of characteristic mycelia and fruiting structures (100X) and photographed.

Lactophenol Cotton Blue Phenol 200.0gm Cotton Blue 0.5gm Glycerol 400.0ml Lactic Acid 200.0ml Deionized Water 200.0ml

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Microphotographs of Few Fungal isolates

Penicillium sp. Gliocladium sp. Purpureocillium sp.

Beauveria sp. Fusarium sp. Aspergillus sp.

Colletotrichum sp. Mucor sp. Trichoderma sp.

Verticillium sp. Talaromyces sp. Monilia sp.

Cladophialophora sp. Bipolaris sp. Phaeoacremonium sp.

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SOIL NUTRIENT ANALYSIS Analysis of pH, Inorganic Phosphates, Nitrates and Water conductivity for collected soil samples were also done. The soil samples were analysed for their pH using Water proof field pH Testr-10 (Eutech instruments).

The collected samples were dried at 110 oC suing hot air oven and stones and other impurities were removed. The dried sample were sieved using a sieve shaker to 250 µm grain size and the samples are packed for acid digestion and subsequent elemental analysis using Atomic Absorption Spectrophotometer. AAS is a spectro-analytical procedure for the quantitative determination of chemical elements employing the absorption of optical radiation by free atoms in the gaseous state. The technique is used for determining the concentration of a particular element in a sample to be analyzed. Atomic absorption occurs when a ground sate atom absorbed energy in the form of light of a specific wavelength and is elevated to an exited state. The relation between the amount of light absorbed and the concentration of analytes present in know standards can be used to determine unknown sample concentration by measuring the amount of light they absorb.

Table Showing Different Physico-Chemical Properties of Soil (A-Flooded, B- Buffer Zone and C- Non-flooded Control)

No Sampling Spots Sample Conductivity Nitrate Inorganic pH Type (μS/cm) (mg/kg) Phosphate (mg/kg) 1 Adamattom, Vazhoor A 58 665 9.5 5.2 B 59 412 8.1 4.4 C 62 289 8.7 4.8 2 Cheruvally A 95 271 10.2 5.7 B 65 189 8.2 5.3 C 54 575 7.7 6.0 3 Chennappady A 94 357 15.4 4.8 B 73 171 14.5 5.1 C 65 58.6 19.2 4.4 4 Manimala Town A 102 404 9.6 6.1 B 61 267 9.5 4.9 C 57 238 9.6 4.2 5 Manimalakavu Devi A 47 307 10.1 6.0 Temple B 40 289 8.1 5.8 C 51 963 7.5 4.9 6 Puthenpally A 110 601 9.8 4.7 B 52 585 8.0 5.5 C 47 855 9.1 5.0

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7 Kulathor Devi Temple A 84 369 8.8 4.8 B 72 289 7.0 5.6 C 125 1031 7.2 6.2 8 St Marys Old Church, A 96 1105 9.1 4.7 Vaipar B 88 561 7.1 5.4 C 51 364 6.1 6.0 9 Mallapally East Bridge A 79 292 12.7 4.4 B 68 321 10.1 6.1 C 65 699 7.6 5.7 10 Chakkulathukavu A 68 43 55 5.2 B 51 47 51 6.1 C 47 195 49 5.5 11 Thalavadi A 87 253 40 6.4 B 59 277 39 5.9 C 54 443 38 6.9 12 Muttar, Kumaranchira A 119 158 28 6.6 B 111 278 5.4 C 148 1288 34 5.3 13 Kidangara A 107 304 27 5.2 B 88 223 24 6.8 C 37 103 20 6.3 14 Veliyanad A 198 151 23 5.5 B 142 148 21 6.2 C 58 265 52 5.7 15 Ramankari A 115 3120 48 4.9 B 98 1129 40 6.0 C 99 501 32 5.5 16 Vezhapra A 49 211 29 5.7 B 45 208 33 6.0 C 41 253 66 5.4 17 Thekkekkara, A 74 321 24 5.1 Mankombu B 63 311 23 4.7 C 62 356 60 6.6 18 Thekkekkara Boat A 43 85 31 5.8 Jetty B 36 89 34 6.3 C 39 203 37 6.9 19 Chambakkulam, A 33 192 64 6.2 Powerhouse Road B 30 142 57 5.6 C 33 119 51 6.7 20 Thirumool A 58 1283 310 4.9 apuram, B 43 872 110 7.0 Thiruvalla C 37 372 29 6.7

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21 Vembalam, Kuttoor A 53 54 27 6.0 B 41 52 21 5.8 C 39 63 42 6.5 22 Kallumankavu, ARS A 108 242 23 5.0 Junction, Thiruvalla B 101 119 22 5.4 C 103 102 23 4.7 23 Iramallikkara, A 47 87 101 6.6 Thiruvalla B 45 67 87 7.2 C 49 94 68 5.9 24 Valanjavattom, A 103 62 38 5.0 Kadapra, B 85 49 33 4.9 C 49 46 32 5.5 25 Pulikeezhu, Thiruvalla, A 149 74 36 6.9 Pathanamthitta B 88 119 37 5.8 C 70 199 45 6.1 26 Pattode, Thiruvalla A 206 129 50 6.3 B 112 231 78 5.7 C 66 425 148 6.4 27 Manakkachira, A 165 113 21 6.5 Thiruvalla, B 83 88 55 7.1 Pathanamthitta C 49 87 78 6.8 28 Vallalamkulam Bridge A 66 63 28 6.2 B 163 89 27 5.8 C 222 591 22 6.6 29 Elanthore, Thiruvalla, A 132 242 47 5.9 Pathanamthitta B 109 176 46 6.3 C 58 172 51 5.2 . Soil Enzyme Analysis Soil enzyme activities are very sensitive to both natural and anthropogenic disturbances, and show a quick response to the induced changes. The analysis of soil enzymes could be useful to identify positive or negative effects of residue management, soil compaction, tillage, crop rotation and soil contamination during reasonable time period. Here we analyse effect of soil enzymes after flooding and its responsible impacts on soil vulnerability and microbial biota. Among different types of enzymes studied from various objectives of investigations Dehydrogenase, Urease and Phosphatase are thoroughly studied enzymes due to their specific importance in organic matter transformation processes and phosphorus cycle in agricultural practices. Here we carried out the Dehydrogenase & Urease activity on collected soils from different flood affected areas.

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DEHYDROGENASE

Soil Dehydrogenase is an extra-cellular enzyme which is considered to be a good tool to measure microbial oxidative activity and as an indicator of any disruption caused by pesticide application, trace element discharge and soil management practices. Dehydrogenase plays an important role in the initial stages of the oxidation of soil organic matter by transferring hydrogen or electron from substrates to acceptors. Because of its importance in the organic matter transformation processes and its potential to indicate the available microbiological activity in the soil. Triphenyl tetrazolium chloride, TTC (2,3,5-triphenyl-2H-tetrazoliumchloride) is a redox indicator commonly used in biochemical experiments especially to indicate cellular respiration. The white compound is enzymatically reduced to red TPF (1,3,5-triphenylformazan) in living tissues due to the activity of various dehydrogenases (enzymes important in oxidation of organic compounds and thus cellular metabolism), while it remains in its unreacted state in areas of necrosis since these enzymes have either denatured or degraded.

Method

TTC reduction technique

One g of fresh soil was taken in a test tube. The soil was then mixed with 0.1 g of calcium carbonate (CaC03) and 1 ml of 1 % TTC solution. The mixture was then shaken and plugged with a rubber stopper and incubated at 30° C for 24 hours in an incubator. Three replicates were maintained in each case. The resulting slurry was transferred on Whatman filter paper No.1 and extracted with successive aliquots of concentrated methanol. The volume of the filtrate was made to 50 ml by adding methanol. The optical density of the filtrate was read at 485nm Spectrophotometer as methanol as blank. The activity was representing in terms of concentration of Formazan (nmol TPF/g of dry soil/day), which was calculated by a standard curve of triphenyl formazan in methanol.

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Dehydrogenase Activity of Soil 2.5 Flooded Area Buffer Zone Control

2

1.5

1 nmol TPF/g dry soil/day dry TPF/g nmol 0.5

0

Vaipar

Muttar

Vazhoor

Kulathor

Vezhapra

Thalavadi

Veliyanad

Kidangara

Cheruvally

Ramankari

Pulikeezhu

Puthenpally

Iramallikkara

Thekkekkara-…

Chennappady

Manakkachira

Valanjavattom

Vallalamkulam

Manimalakavu

Chambakkulam

MallapallyEast

ManimalaTown

Chakkulathukavu

Thirumoolapuram

Thekkekkara-Jetty

Pattode-Thiruvalla

Vembalam-Kuttoor Elanthore-Thiruvalla

Kallumankavu-Thiruvalla The dehydrogenase activity was analysed for the soil samples from flood affected area and the result indicated a higher enzyme activity in the buffer zone than in the flood affected and control zone. The average dehydrogenase activity in the flooded samples is 0.4179, buffer zone is 0.6193 and that of control is 0.4696 nmol TPF/g dry soil/day.

UREASE Bacteria can hydrolyse urea are common in soils. Soil urease, a key enzyme involved in soil N cycling, can catalyze the hydrolysis urea into carbon dioxide and ammonia. It has an applied importance in the Nitrogen economy of soil. So, the enzyme assay is an important in understanding mineralization process of Nitrogen and its response to the application of inorganic fertilizers and soil management systems. Urease activity is also an important factor for survival of ammonium fertilizers oxidized in forest and agricultural soils .

Method

McGarity and Myers Method (1967) was followed for urease analysis. One gram fresh soil was kept in 100 ml volumetric flask and to it 1 ml of toluene was added. It was then allowed to stand for 15 minutes to permit the complete penetration of toluene into the soil. Then 10 ml of buffer (pH 7) solution and 5 ml of 10% Urea solution were added. The flask was shaken and incubated

22 at 37°C for 3 hours in an incubator. The flask was shaken and incubated at 37°C for 3 hours in an incubator. Whereas in control, 10 ml of distilled water was added instead of urea solution. After incubation, the volume was made up to 100 ml by adding distilled water. The content in the flask was mixed thoroughly and was filtered through Whatman filter paper No. 5 and the ammonia released as a result of urease activity was analysed by indophenol blue method.

To 0.5 ml of filtrate in a 25 ml volumetric flask, 5 ml of distilled water was added. The mixture in the flask was treated with 2 ml of Phenolate solution + 1.5 ml of sodium hypochloride solution containing 5% of active chlorine. The final volume was made up to 25 ml by adding distilled water. The optical density was read at 630 nm.

Average Urease Activity

Flooded Area, Control , 8.22069 9.02069

Buffer Zone, 13.16552

Urease Activity of Soil

Flooded Area Buffer Zone Control

20

15

N mg/g/3h) -

10

Urease (NH3 5

0

Vaipar

Muttar

Vazhoor

Kulathor

Vezhapra

Thalavadi

Veliyanad

Kidangara

Cheruvally

Ramankari

Pulikeezhu

Puthenpally

Iramallikkara

Chennappady

Manakkachira

Valanjavattom

Vallalamkulam

Manimalakavu

Chambakkulam

MallapallyEast

ManimalaTown

Chakkulathukavu

Thirumoolapuram

Thekkekkara-Jetty

Pattode-Thiruvalla

Vembalam-Kuttoor

Elanthore-Thiruvalla Kallumankavu-Thiruvalla Thekkekkara-Mangombu

23

The unit urease activity was expressed as milligram of Ammoniacal nitrogen per gram soil per 3 hr (NH3-N mg/g/3h). The average urease activity was observed to be higher in the buffer zone soil (13.16552) indicating a better mineralisation and utilization of nitrogen, whereas soil in the flood affected zone (8.22069) and control zone (9.02069) shows lesser urease activity. The increased activity in the buffer zone may due to the deposition of more organic matter due to

flood.

Statistical Analysis Data were processed with Microsoft Excel software. Statistical analysis was carried out by using SPSS 12.0. All the values expressed are means ± S.D. (standard deviation) of four replicates. Data were analysed by one-way analyses of variance with Duncan’s multiple range test to separate means. Differences were considered significant at P < 0.05.

Metagenomics and microbiome analysis of Flood affected Soils The study of microbiomes, the genetic material of all microorganisms in a given sample, has recently attracted considerable attention, mainly due to the realisation that the microbial composition of environment can have a profound effect on our health. The sequencing of DNA recovered directly from environmental samples can reveal the presence of microbial communities without requiring prior laboratory cultivation. This approach, termed metagenomics, has exposed previously hidden microbial diversity in a range of different environments, from the open ocean, to complex soil samples, to the human microbiome. Accordingly, metagenomics is often used to determine the profile of the microbes that inhabit a given environment, to diagnose the presence of a microbial pathogen and to identify novel microbial lineages. There has also been a surge in research efforts focusing on analysing the microbiome of extreme environments, water, soil, etc.

Classical microbiome research relies on culturing, which is associated with long sample-to- result time and biases related to the different susceptibility of microorganisms to laboratory handling. The advent of modern sequencing technologies has brought significant advantages to the field. Speed and accuracy of microbial analysis (i.e. species identification and abundance) have increased substantially, with culturing no longer being necessary. Nonetheless, some challenges remain with traditional sequencing approaches.

24

Comparisons between microbial communities that inhabit different environmental sites can also distinguish differences in the identity and abundance of microbes. These approaches can identify microbes that confer specific environmental characteristics, or measure the impact of environmental variables on microbial communities, and have been used to discover host– microbe interactions, identify novel microbes with biotechnological value, and measure environmental health.

Long-read nanopore sequencing technology improves on the traditional gene-level shotgun metagenomic analysis provided by short-read sequencing approaches to enable unbiased assembly of complete, closed genomes and plasmids from clinical research and microbiome samples. Discriminate closely related species, resolve challenging repeat regions and structural variants, and delineate plasmid and genomic AMR genes. Data is delivered in real time, providing rapid sample insights.

Methods DNA Extraction For the present study, community genomic DNA was extracted directly from soil samples collected from flooded Manimala River Bank and also from non-flooded adjacent area as control. PowerSoil® DNA Isolation kits (MoBio, Inc.) are commonly used to extract high quality DNA from soils. Generally, the protocol was implemented as per the manufacturer’s instructions. Soil samples (0.25g) were of taken to the Power Bed tubes and gently vortexed to mix. Added 60μl of Solution C1 and inverted several times or vortex briefly. Secured Power Bed tubes horizontally using the MO BIO vortex adapter tube holder for vortex. Centrifuged tubes at 10,000g for 30seconds at RT. The supernatant was transferred to a clean 2ml collection tube. 500μl of Solution C2 was added and vortexed for 5seconds. Incubated at 4oC for 5 minutes and centrifuged the tubes at RT for 1minutes at 10,000g. Transfered 600μl of supernatant to clean 2ml collection tubes and added 200μl of Solution C3 and vortex briefly. This was further incubate at 4⁰C for 5minutes and again centrifuged at RT for 1minutes at 10,000g. Supernatant (no more than 750μl ) was transferred into clean 2ml collection tubes. Shaken to mix Solution C4 before use and added 1200μl of Solution C4 to the supernatant and vortex for 5 seconds.

Loaded approximately 675μl into a SPINFILTER and centrifuged at 10,000g for 1minutes at RT. The flow through was discarded and added an additional 675μl of supernatant to the Spin filter and centrifuged at 10,000g for 1minutes at RT. Loaded the remaining supernatant to the

25

Spin filter and centrifuged again at 10,000g for 1minutes at RT (three loads were provided). Added 500μl of Solution C5 and centrifuged at RT for 30 seconds at 10,000g and discarded the flow through and centrifuged again at RT for 1minutes at 10,000g. The Spin filters were carefully placed in a clean 2ml collection tubes, avoiding splash of any Solution C5 on to the Spin filter. Added 100μl of Solution C6 to centre of the white filter membrane and centrifuged at RT for 30seconds at 10,000g. The Spin filter was discarded and the the DNA in tubes are now ready and were stored at -20 oC.

End Repair and dA tailing About 1-1.5 microgram High molecular weight sheared DNA was subjected end-repair/dA tailing using 3 microlitres of UltraII End prep enzyme mix in a 60 microlitre reaction volume in a 0.5 ml PCR tube for 5 minutes at 20 oC and for 5 minutes at 65 oC. The modified DNA fragments were purified using AMPure XP beads. The DNA was eluted in a 25 microlitre volume of MQ water. Recovered DNA was quantified using Qubit reader (ThermoFischer Scientific). Barcode labelling Exactly 500 nanogram of end prepared DNA was coupled with barcodes numbered from 1-24 (NBD103 and 104). Each barcode represent one sample. Barcode was ligated using 2X ligation MasterMix supplied by NEB. Ligation reaction was carried out at room temperature for 10 minutes in a 50 microlitre reaction volume. The barcode ligated fragments were purified using AMPure XP beads. The DNA was eluted in a 26 microlitre volume of MQ water. Recovered DNA was quantified using Qubit reader (ThermoFischer Scientific). Barcode Adapter ligation To the barcode labelled DNA barcode adapter was ligated using 2X ligation MasterMix supplied by NEB. Ligation reaction was carried out at room temperature for 10 minutes. The ligation reaction was carried out using 750ng of pooled samples (750ng/24) in 50 microlitre using 20 microlitres of barcode adapter mix. After the reaction the DNA fragments were purified using AMPure XP beads. The DNA was eluted in a 15 microlitre volume of elution buffer. Recovered DNA was quantified using Qubit reader (ThermoFischer Scientific).

Priming and Loading the SpotON flow cell The Sequencer MinION was connected to SpotON flow cell and primed using 1ml of Primer buffer for 5 minutes at RT and into the primed flow cell 75 microlitres of pooled library (430ng) was added through the SpotON sample port. Closed the sample port and closed the MinION lid.

26

Sequencing run and base calling MinKNOW program was started after connecting the flow cell to the computer. The sequencing was done at FAST5 mode for 48 hrs. The basecalling was done using a software GUPPY till base-calling completes.

Data analysis After basecalling the DNA sequence data is obtained as fastq format. The files were uploaded to metagenome analysis online tool EPI2ME. The data obtained from different samples were tabulated. OmicBox software (OmicsBox version 1.1.135) also used for constructing taxonomic pie wheel diagram and calculating Shanon index.

Results Soil Metagenomic DNA isolated with Powersoil kit (MoBio) in 1% agarose Gel

Distribution of Different Superkigdom in Flooded and non Flooded Areas

Superkigdom - Distribution

0 5000 10000 15000 20000 25000 30000 35000

7482 21891 Bacteria 23,805 30,085 269 935 Eukaryota 740 824 43 156 Archaea 168 224 4 6 Viruses 8 8

Control (unflooded) Vazhoor (Flooded) Manimala (Fooded) Mallapally (Flooded)

27

Distribution of Microbiota (%) in flooded Distribution of Microbiota (%) in Non-flooded Area Area

The cumulative reads of Bacteria in the control area was 7482, Eukaryota 269, Archea 43 and Viruses 4. While in flooded the microbial load is comparatively very high. Bacteria was noted with an average cumulative read of 25,260.33 (Vazhoor 21,891, Manimala 23,805 and Mallapally 30,085). Eukaryota has an average value of 833 (Vazhoor 935, Manimala 740 and Mallapally 824). Archaea is found have low distribution with an average value of 182.66 (Vazhoor 156, Manimala 168 and Mallapally 224). The viral load is comparatively negligible with an average value of 7.33.

Taxonomic Classification Report computed following Kraken 1.0 with DB 2019.06 (archaea, bacteria, fungi, human, protozoa, viral)-Bowtie2 2.3.5.1 using OmicsBox shows that the flood samples with Shannon Index (H) of Vazhoor with 5.55725, Manimala with 5.608 and Mallapally 5.55725. The Simpsons Index (D-1) of all flooded samples was 0.99. The Shannon Index (H) of control sample with 5.552 and Simpsons Index was 0.99.

It was noted the presence of different anaerobic bacteria in the flood affected samples. An anaerobic organism or anaerobe is any organism that does not require oxygen for growth. It may react negatively or even die if free oxygen is present. The prolonged water submerged conditions may reduce soil oxygen content and this will promote the growth of anaerobes. The rapid depletion of soil oxygen causes a shift in nitrogen cycle soil micro-organisms that lead to the denitrification processes. This loss of soil oxygen is the greatest challenge to established natural and crop plants. Flood also results in soil oxygen depletion, fostering anaerobic conditions and microorganisms able to survive these conditions. Such changes in soil abiotic conditions can alter soil microbial community composition. Anaerobiac bacterial members of Anaeromyxobacteraceae was with highest cumulative reads in flooded samples (average of 248). Members of Anaerolineaceae, Bacteroidaceae, Clostrideaseae, Porphyromonadaceae (Porphyromonas gingivitis), Propionibacteriaceae (Propionibacterium freudenreichii) are also prevalent in flooded samples. Among these, Bacteroides, Clostridium species are potential pathogens.

28

DISTRIBUTION OF DIFFERENT TAXA AT PHYLUM LEVEL

Control Vazhoor (Flooded) Manimala (Flooded) Mallapally (Flooded) Taxon Cumulative Reads Taxon Cumulative Reads Taxon Cumulative Reads Taxon Cumulative Reads

Proteobacteria Actinobacteria Acidobacteria Fimricutes Bacteroidetes Planctomycetes Nitrospirae

Fusobacteria Mucoromycota Cyanobacteria

18243

13637

13494

6009

5005

4174

3842

1710

1500

1215

1204

649

588

572

459

440

378 378

372

355 359

348

322

304

291

275

183

168

162

120 120 115

113 113

102

85

79

70

25

22 18

16 15

2 0 0 0 2 0 2 4 0 1 0 1 0

CONTROL VAZHOOR MANIMALA MALLAPALLY

29

Representative Fungal classes identified in Flooded and Control Areas

Cumulative Reads of Diffrent Class of Fungi

Mallapally (Flooded) Wallemiomycetes Malasseziomycetes Orbiliomycetes Manimala (Flooded) Exobasidiomycetes Xylonomycetes Pucciniomycetes Microbotryomycetes Vazhoor (Flooded) Tremellomycetes Ustilaginomycetes Agaricomycetes Control (Unflooded)

0 50 100 150 200 Cumulative Reads

Representing Phyla of Fungi in Flooded Area Sordariomycetes is a class of fungi in the subdivision , are also known as Pyrenomycetes. Members of this group can grow in soil, dung, leaf litter, and decaying wood as decomposers, as well as being fungal parasites, and insect, human, and plant pathogens. Verticillium alfalfa and V. dahliae are fungal plant pathogen. It causes verticillium wilt in many plant species, causing leaves to curl and discolour. Chaetomium spp. are also encountered as causative agents of infections in humans. Most of the Colletotrichum spp. are plant pathogens. C. gloeosporioides is one of the most common fungal plant pathogens. Anthracnose, caused

30 by the C. gloeosporioides, is the most widespread and serious postharvest disease of many tropical fruits, and also cause bitter rot in variety of crops worldwide. C. orchidophilum causes orchid Anthracnose. Fusarium wilt, is a widespread plant disease caused by many forms of the soil-inhabiting fungus Fusarium F. oxysporum f.sp. lycopersici is a fungal plant pathogen. It is a big pathogen to the tomato plants.

KINGDOM Fungi SUBKINGDOM PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Sordariomycetes

TAXON READS

Beauveria bassiana ARSEF 2860 2

Chaetomium globosum CBS 148.51 4

Chaetomium thermophilum var. thermophilum 9

Colletotrichum fioriniae PJ7 5

Colletotrichum gloeosporioides Nara gc5 4

Colletotrichum graminicola M1.001 5

Colletotrichum higginsianum IMI 349063 4

Colletotrichum orchidophilum 3

Cordyceps militaris CM01 3

Eutypa lata UCREL1 9

Fusarium oxysporum f. sp. lycopersici 4287 1

Fusarium pseudograminearum CS3096 2

Fusarium verticillioides 7600 2

Gaeumannomyces tritici R3-111a-1 7

Grosmannia clavigera kw1407 6

Isaria fumosorosea ARSEF 2679 2

Magnaporthe oryzae 70-15 6

Metarhizium acridum CQMa 102 4

Metarhizium brunneum ARSEF 3297 2

Metarhizium majus ARSEF 297 3

Metarhizium robertsii ARSEF 23 3

Nectria haematococca mpVI 77-13-4 4

Neurospora crassa OR74A 2

Neurospora tetrasperma FGSC 2508 3

Pestalotiopsis fici W106-1 7

Phaeoacremonium minimum UCRPA7 4

Pochonia chlamydosporia 170 2

Podospora anserina S mat+ 3

Purpureocillium lilacinum 3

31

Scedosporium apiospermum 4

Sordaria macrospora 5

Sporothrix schenckii 1099-18 17

Thermothelomyces thermophila 10

Thielavia terrestris NRRL 8126 12

Trichoderma atroviride IMI 206040 1

Trichoderma gamsii 6

Trichoderma reesei QM6a 7

Trichoderma virens Gv29-8 3

Verticillium alfalfae VaMs.102 3

Verticillium dahliae VdLs.17 6

The Eurotiomycetes are a class of ascomycetes within the subphylum Pezizomycotina. Some of them are true pathogens. benhamiae is a zoophilic dermatophyte transmitted to humans mostly from guinea pigs and occasionally other animals. Fonsecaea spp. are facultative parasites, it causes infections in humans and some animals. Histoplasma capsulatum is a global fungal pathogen. Histoplasmosis causes significant morbidity and mortality worldwide. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Eurotiomycetes

TAXON READS

Arthroderma otae CBS 113480 5

Aspergillus aculeatus ATCC 16872 6

Aspergillus bombycis 2

Aspergillus clavatus NRRL 1 7

Aspergillus fischeri NRRL 181 2

Aspergillus flavus NRRL3357 2

Aspergillus fumigatus Af293 4

Aspergillus glaucus CBS 516.65 7

Aspergillus nidulans FGSC A4 3

Aspergillus niger CBS 513.88 3

Aspergillus terreus NIH2624 1

Blastomyces gilchristii SLH14081 7

Capronia coronata CBS 617.96 5

Capronia epimyces CBS 606.96 6

Cladophialophora bantiana CBS 173.52 1

Cladophialophora carrionii CBS 160.54 4

32

Cladophialophora immunda 6

Cladophialophora psammophila CBS 110553 6

Cladophialophora yegresii CBS 114405 2

Coccidioides immitis RS 2

Endocarpon pusillum Z07020 6

Exophiala aquamarina CBS 119918 4

Exophiala dermatitidis NIH/UT8656 2

Exophiala oligosperma 2

Exophiala spinifera 4

Exophiala xenobiotica 5

Fonsecaea erecta 4

Fonsecaea monophora 3

Fonsecaea multimorphosa CBS 102226 4

Fonsecaea nubica 1

Histoplasma capsulatum NAm1 4

Nannizzia gypsea CBS 118893 2

Paracoccidioides brasiliensis Pb18 4

Paracoccidioides lutzii Pb01 4

Penicilliopsis zonata CBS 506.65 6

Penicillium arizonense 3

Penicillium digitatum Pd1 1

Penicillium expansum 1

Phialophora attae 2

Rasamsonia emersonii CBS 393.64 2

Rhinocladiella mackenziei CBS 650.93 5

Talaromyces atroroseus 2

Talaromyces marneffei ATCC 18224 2

Talaromyces stipitatus ATCC 10500 3

Trichophyton benhamiae CBS 112371 2

Trichophyton verrucosum HKI 0517 2

Uncinocarpus reesii 1704 2

Dothideomycetes 72 is the largest and most diverse class of ascomycete fungi. It comprises 11 orders 90 families, 1300 genera and over 19,000 known species. Some are pathogenic. Bipolaris is a dematiaceous, filamentous fungus. B. oryzae is the causal agent of brown spot disease of rice. Pseudocercospora fijiensis causes black leaf streak disease (BLSD) or black Sigatoka, the most economically important disease of bananas. Pyrenophora teres is a necrotrophic fungal pathogen of some plant species, the most significant of which are economically important agricultural crops such as barley (net blotch disease). P. tritici- repentis causes yellow spot.

33

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Dothideomycetes

TAXON READS

Aureobasidium namibiae CBS 147.97 6

Aureobasidium subglaciale EXF-2481 1

Baudoinia panamericana UAMH 10762 2

Bipolaris maydis ATCC 48331 3

Bipolaris oryzae ATCC 44560 2

Bipolaris zeicola 26-R-13 2

Coniosporium apollinis CBS 100218 3

Diplodia corticola 5

Leptosphaeria maculans JN3 3

Neofusicoccum parvum UCRNP2 5

Paraphaeosphaeria sporulosa 3

Parastagonospora nodorum SN15 3

Pseudocercospora fijiensis CIRAD86 4

Pyrenophora teres f. teres 0-1 2

Pyrenophora tritici-repentis Pt-1C-BFP 2

Setosphaeria turcica Et28A 5

Sphaerulina musiva SO2202 4

Verruconis gallopava 4

Zymoseptoria tritici IPO323 7

The Agaricomycetes are a class of fungi in the division Basidiomycota. Mostly saprophytic in nature. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM CLASS Agaricomycetes

TAXON READS

Agaricus bisporus 1

Agaricus bisporus var. bisporus H97 1

Auricularia subglabra TFB-10046 SS5 6

Coniophora puteana RWD-64-598 SS2 2

Coprinopsis cinerea okayama7#130 1

34

Dichomitus squalens LYAD-421 SS1 1

Fomitiporia mediterranea MF3/22 2

Gloeophyllum trabeum ATCC 11539 1

Heterobasidion irregulare TC 32-1 7

Laccaria bicolor S238N-H82 3

Moniliophthora roreri MCA 2997 1

Phanerochaete carnosa HHB-10118-sp 4

Postia placenta Mad-698-R 4

Punctularia strigosozonata HHB-11173 SS5 6

Schizophyllum commune H4-8 4

Serpula lacrymans var. lacrymans S7.9 1

Stereum hirsutum FP-91666 SS1 2

Trametes versicolor FP-101664 SS1 2

Saccharomycetes belongs to the kingdom of Fungi and the division Ascomycota. It is the only class in the subdivision , the budding yeasts. Saccharomycetes contains a single order: Saccharomycetales. The genus Candida that includes several human pathogens. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Saccharomycotina CLASS Saccharomycetes

TAXON READS

Ascoidea rubescens DSM 1968 5

Candida albicans SC5314 3

Candida dubliniensis CD36 2

Candida orthopsilosis Co 90-125 1

Candida tropicalis MYA-3404 1

Clavispora lusitaniae ATCC 42720 1

Debaryomyces hansenii CBS767 2

Eremothecium cymbalariae DBVPG#7215 1

Eremothecium sinecaudum 2

Hyphopichia burtonii NRRL Y-1933 1

Kazachstania africana CBS 2517 1

Kazachstania naganishii CBS 8797 1

Kuraishia capsulata CBS 1993 1

Lachancea thermotolerans CBS 6340 2

Lodderomyces elongisporus NRRL YB-4239 6

Metschnikowia bicuspidata var. bicuspidata NRRL YB-4993 2

Naumovozyma castellii CBS 4309 1

Ogataea parapolymorpha DL-1 1

35

Pichia membranifaciens NRRL Y-2026 3

Saccharomyces cerevisiae S288C 1

Saccharomyces eubayanus 1

Saccharomycetales 1

Sugiyamaella lignohabitans 2

Tetrapisispora blattae CBS 6284 1

Torulaspora delbrueckii 1

Vanderwaltozyma polyspora DSM 70294 1

Wickerhamomyces anomalus NRRL Y-366-8 1

Zygosaccharomyces rouxii 2

The Leotiomycetes are a class of ascomycete fungi. Many of them cause serious plant diseases. The class Leotiomycetes contains numerous species with an anamorph placed within the , that have only recently found their place in the phylogenetic system. Botrytis cinerea infects over 200 plant species, causing grey mould, evident on the surface as grey fluffy mycelium. Worldwide, it causes annual losses of $10 billion to $100 billion. Marssonina brunnea is an important pathogen that causes Marssonina leaf spot disease. Sclerotinia sclerotiorum is a plant pathogenic fungus and can cause a disease called white mold if conditions are conducive. S. sclerotiorum can also be known as cottony rot, watery soft rot, stem rot, drop, crown rot and blossom blight. Common hosts of white mold are herbaceous, succulent plants, particularly flowers and vegetables. It can also affect woody ornamentals occasionally, usually on juvenile tissue. This can affect their hosts at any stage of growth, including seedlings, mature plants, and harvested products.

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Leotiomycetes

TAXON READS

Botrytis cinerea B05.10 4

Glarea lozoyensis ATCC 20868 2

Marssonina brunnea f. sp. 'multigermtubi' MB_m1 9

Phialocephala scopiformis 2

Pseudogymnoascus destructans 20631-21 2

Pseudogymnoascus verrucosus 5

36

Sclerotinia sclerotiorum 1980 UF-70 4

Ustilaginomycetes is the class of true smut fungi. They are plant parasites with about 1400 recognised species in 70 genera. The smut genus Anthracocystis has been described for a causative agent of head smut of millet. Ustilago maydis is known as Corn smut that causes smut on maize and corns. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM CLASS Ustilaginomycetes

TAXON READS

Anthracocystis flocculosa PF-1 13

Kalmanozyma brasiliensis GHG001 3

Moesziomyces antarcticus 1

Pseudozyma hubeiensis SY62 1

Ustilago maydis 521 10

The Tremellomycetes are a class of dimorphic fungi. Some species have a gelatinous fruiting body or a sacculate parenthesome.

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM Agaricomycotina CLASS Tremellomycetes

TAXON READS

Cryptococcus amylolentus CBS 6039 1

Cryptococcus neoformans var. grubii H99 1

Cryptococcus neoformans var. neoformans 1

Cutaneotrichosporon oleaginosum 4

Tremella mesenterica DSM 1558 1

Trichosporon asahii var. asahii CBS 2479 5

37

Tsuchiyaea wingfieldii CBS 7118 2

The Microbotryomycetes are class of fungi in the subdivision of the Basidiomycota. Some species of Rhodotorula can cause infections in animals. There have been reports of skin infections in chickens and sea animals and lung infections and otitis in sheep and cattle. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM Pucciniomycotina CLASS Microbotryomycetes

TAXON READS

Rhodotorula graminis WP1 8

Rhodotorula toruloides NP11 3

The Pucciniomycetes are a class of fungi in the Pucciniomycotina subdivision of the Basidiomycota. It includes several important plant pathogens causing forms of fungal rust. Cereal rusts are caused by Puccinia graminis and are a significant disease affecting cereal crops. Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide.

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM Pucciniomycotina CLASS Pucciniomycetes

TAXON READS

Puccinia graminis f. sp. tritici CRL 75-36-700-3 7

Melampsora larici-populina 98AG31 4

38

Orbiliomycetes are a class of fungi in the Ascomycota. It includes the single order Orbiliales, which in turn includes the single family Orbiliaceae.

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Orbiliomycetes

TAXON READS

Arthrobotrys oligospora ATCC 24927 7

Dactylellina haptotyla CBS 200.50 2

Xylonomycetes is a fungi class in the unranked group of the Pezizomycotina. Xylona heveae is endophyte of Hevea brasiliensis (rubber tree).

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Xylonomycetes

TAXON READS

Xylona heveae TC161 6

Pezizomycetes are a class of fungi within the division Ascomycota. Pezizomycetes are apothecial fungi. Tuber melanosporum, is called as black truffle, is one of the most expensive edible mushrooms in the world. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM Pezizomycotina CLASS Pezizomycetes

TAXON READS

Tuber melanosporum 4

39

The Mixiomycetes are class of fungi in the Pucciniomycotina subdivision of the Basidiomycota. The fern pathogen Mixia osmundae produce exogenous blastospores on fern leaves. KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM Pucciniomycotina CLASS Mixiomycetes

TAXON READS

Mixia osmundae IAM 14324 3

The Exobasidiomycetes are a class of fungi sometimes associated with the abnormal outgrowths of plant tissues known as galls.

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM Ustilaginomycotina CLASS Exobasidiomycetes

TAXON READS

Tilletiaria anomala UBC 951 2

The Pneumocystidomycetes are a class of ascomycete fungi. It includes the single order Pneumocystidales, which contains the single monotypic family Pneumocystidaceae, which in turn contains the genus Pneumocystis, causative agent of Pneumocystis pneumonia. P. murina was first isolated from lab mice and hence the name.

KINGDOM Fungi SUBKINGDOM Dikarya PHYLUM Ascomycota SUBPHYLUM CLASS Pneumocystidomycetes

TAXON READS

Pneumocystis murina B123 1

40

The Wallemiomycetes are a class of fungi in the division Basidiomycota. It consists of the single order Wallemiales, containing the single family Wallemiaceae, which in turn contains the single genus Wallemia.

SUBKINGDOM Dikarya PHYLUM Basidiomycota SUBPHYLUM Agaricomycotina CLASS Wallemiomycetes

TAXON READS

Wallemia ichthyophaga EXF-994 1

Wallemia mellicola CBS 633.66 1

Chytridiomycetes is a class of fungi. Members are found in soil, fresh water, and saline estuaries. Spizellomycetalean chytrids have beneficial roles in the soil for nutrient recycling and as parasites of organisms that attack plants, such as nematodes and oospores of downy mildews. On the other hand, they also have detrimental roles as parasites of arbuscular mycorrhizae, symbiotic fungi that help plants gain essential nutrients.

KINGDOM Fungi PHYLUM Chytridiomycota CLASS Chytridiomycetes

TAXON READS

Spizellomyces punctatus DAOM BR117 1

41

42

43

44

Different Phyla and representative Genera of Fungi in Non-flooded Area

TAXON READS Sordariomycetes

Chaetomium globosum CBS 148.51 2

Chaetomium thermophilum var. thermophilum 1

Colletotrichum gloeosporioides Nara gc5 1

Colletotrichum graminicola M1.001 4

Colletotrichum orchidophilum 1

Cordyceps militaris CM01 3

Eutypa lata UCREL1 1

Fusarium oxysporum f. sp. lycopersici 4287 1

Fusarium pseudograminearum CS3096 1

Fusarium verticillioides 7600 2

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Gaeumannomyces tritici R3-111a-1 3

Grosmannia clavigera kw1407 1

Isaria fumosorosea ARSEF 2679 3

Magnaporthe oryzae 70-15 4

Metarhizium majus ARSEF 297 1

Metarhizium robertsii ARSEF 23 1

Nectria haematococca mpVI 77-13-4 1

Pochonia chlamydosporia 170 2

Podospora anserina S mat+ 1

Purpureocillium lilacinum 2

Scedosporium apiospermum 1

Sordaria macrospora 1

Sporothrix schenckii 1099-18 3

Thielavia terrestris NRRL 8126 2

Trichoderma reesei QM6a 2

Trichoderma virens Gv29-8 1 Eurotiomycetes

Aspergillus aculeatus ATCC 16872 2

Aspergillus bombycis 1

Aspergillus clavatus NRRL 1 1

Aspergillus glaucus CBS 516.65 1

Aspergillus nidulans FGSC A4 1

Aspergillus nomius NRRL 13137 1

Aspergillus oryzae RIB40 1

Blastomyces gilchristii SLH14081 1

Capronia coronata CBS 617.96 1

Cladophialophora immunda 1

Coccidioides immitis RS 1

Endocarpon pusillum Z07020 1

Exophiala aquamarina CBS 119918 1

Exophiala dermatitidis NIH/UT8656 2

Exophiala oligosperma 1

Exophiala spinifera 1

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Exophiala xenobiotica 1

Fonsecaea multimorphosa CBS 102226 1

Fonsecaea pedrosoi CBS 271.37 2

Paracoccidioides brasiliensis Pb18 1

Paracoccidioides lutzii Pb01 1

Penicilliopsis zonata CBS 506.65 1

Penicillium expansum 1

Rasamsonia emersonii CBS 393.64 1

Rhinocladiella mackenziei CBS 650.93 1

Talaromyces stipitatus ATCC 10500 2

Trichophyton benhamiae CBS 112371 2

Trichophyton rubrum CBS 118892 2

Uncinocarpus reesii 1704 2 Dothideomycetes Bipolaris maydis ATCC 48331 1 Bipolaris oryzae ATCC 44560 2 Diplodia corticola 4 Paraphaeosphaeria sporulosa 1 Parastagonospora nodorum SN15 3 Pyrenophora teres f. teres 0-1 2 Setosphaeria turcica Et28A 2 Sphaerulina musiva SO2202 1 Verruconis gallopava 2 Zymoseptoria tritici IPO323 1 Saccharomycetes Ascoidea rubescens DSM 1968 2 Babjeviella inositovora NRRL Y-12698 1 Candida dubliniensis CD36 3 Candida orthopsilosis Co 90-125 1 Cyberlindnera jadinii NRRL Y-1542 1 Eremothecium cymbalariae DBVPG#7215 1 Kuraishia capsulata CBS 1993 1 Naumovozyma dairenensis CBS 421 2

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Scheffersomyces stipitis CBS 6054 1 Spathaspora passalidarum NRRL Y-27907 1 Wickerhamomyces ciferrii 1 Agaricomycetes Gloeophyllum trabeum ATCC 11539 1 Laccaria bicolor S238N-H82 1 Phanerochaete carnosa HHB-10118-sp 1 Postia placenta Mad-698-R 1 Punctularia strigosozonata HHB-11173 SS5 1 Trametes versicolor FP-101664 SS1 1 Tremellomycetes Cryptococcus amylolentus CBS 6039 1 Kwoniella pini CBS 10737 1 Tremella mesenterica DSM 1558 1 Trichosporon asahii var. asahii CBS 2479 3 Ustilaginomycetes Anthracocystis flocculosa PF-1 2 Pseudozyma hubeiensis SY62 2 Ustilago maydis 521 1 Orbiliomycetes

Dactylellina haptotyla CBS 200.50 3 Xylonomycetes Xylona heveae TC161 2 Microbotryomycetes Rhodotorula graminis WP1 2 Schizosaccharomycetes Schizosaccharomyces pombe 1 Mixiomycetes Mixia osmundae IAM 14324 1

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OmicsBox Krona Pie Wheel Metagenome Taxa Analysis of Flooded Area – Vazhoor

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OmicsBox Krona Pie Wheel Metagenome Taxa Analysis of Flooded Area – Manimala

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OmicsBox Krona Pie Wheel Metagenome Taxa Analysis of Flooded Area – Mallapally

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OmicsBox Krona Pie Wheel Metagenome Taxa Analysis of Non-Flooded Area (Control)

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Conclusions The flooding has affected the natural microbiota of the soil as revealed by culture dependant and independent (metagenome) analysis. There is an increase in the anaerobic microorganisms and pathogenic fungal species in the flood affected soil than that of non-flooded areas. There is a depletion in soil enzyme activity compared to buffer and non-flooded areas indicating that there is a decrease in beneficial organisms in the soil (nitrogen fixing and phosphate solubilizing).

Recommendation

• Biological control measures to be taken to reduce phytopathogenic organisms before planning for eco-restoration plans. • Plants able to fix atmospheric nitrogen to be included as intercrops in the eco- restoration programmes and farming activities in the river basin. • Biofertilizers with nitrogen fixing and phosphate solubilizing microorganisms may be used for cultivation in the farmland near to River. • Any eco-restoration programmes in the flood affected river basins may take a support from soil microbiologists. • Similar Studies may extended to other flood affected River basins.

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Contributors and Acknowledgements

Dr. Shiburaj Sugathan, Senior Scientist and Head, Division of Microbiology, KSCSTE- JNTBGRI, who is the Principal Investigator of this project was responsible for the research planning and execution of different experiments. data analysis and interpretation. Dr. Rachana Mol, Post Doctoral Fellow, Division of Microbiology, KSCSTE-JNTBGRI, who is the Co- Principal Investigator of the project done the field collection and experimental works with support from Ms. Aparna B., Project Fellow. The field works were supported by Mr. Nitish, Project Assistant, JNTBGRI.

The Investigators acknowledge Kerala State Biodiversity Board, Govt of Kerala for financial assistance. Also thankful to the honourable Biodiversity Board Members (especially Prof. Swapna T.S. and Dr. Satheeshkumar K.), Member Secretary (Dr. Balakrishnan) and Chairman of KSBB for giving valuable suggestions and support during the course of study. Also acknowledges Dr. R. Prakashkumar, Director, JNTBGRI for providing facilities and necessary support.

Investigator also acknowledges all the lab members of Division of Microbiology, KSCSTE- JNTBGRI, for all the support endured. The Project Coordination Cell (PCC) of JNTBGRI for administrative support.

Investigator also thankful to Phytocom Pharmaceuticals (P) Ltd, KRIBS BIONEST, KINFRA Hi-Tech Park, Kalamassery, Cochin, for providing services for metagenomics analysis.

Investigators also thankful to the natives of Manimala river side, who have affected and suffered by 2018 flood. They have provided a whole hearted support for sample collection and field studies.

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