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Chapter II
REVIEW OF RELATED LITERATURE AND STUDIES
Review of Related Literature
Fresh water Fungi
Freshwater ascomycetes take place in both lentic and lotic habitats, originally as parasites and endophytes of aquatic macrophytes and algae, and saprophytic organisms. As with aquatic and wetland plants (Correll and Correll 1975), an accurate definition of what constitutes a freshwater ascomycete is problematic. Presence in water, alone, may not be an appropriate definition. Some ascomycetes reported from freshwater habitats also occur in terrestrial habitats. Whether their occurrence in freshwater is fortuitous or whether they are able to grow, to produce something that is the same and sustain their populations without nonstop immigration from land is not known for species.
Fungi play a key role in decomposition of submerged wood in streams, breaking down lignocelluloses and releasing nutrients, and are important in ecosystem functioning. These wood decay fungi are known as freshwater lignicolous fungi and are usually studied by collecting submerged woody litter, followed by incubation in a moist chamber. This review explains what are freshwater lignicolous fungi, their decay mechanisms, roles and physiological attributes. Asian/Australasian lignicolous freshwater fungi have been relatively well-surveyed and enable an account of their distribution along a latitudinal transect. Unlike freshwater leaf-dwelling fungi their diversity in water bodies is greater towards the Equator which suggests they are important for decaying submerged wood in the tropics. Riparian vegetation, disturbances such as pollution, streams drying and study methods, may all affect the diversity of freshwater lignicolous fungi, however, the overall trend is a higher diversity in the tropics and subtropics. Climate changes together with increasing deposition of woody debris from human activities, and alteration of environmental factors (such as water pollution, and dam building) will impact freshwater lignicolous fungi. Changing diversity, structure and activities of freshwater fungal communities can be expected, which will significantly impact on aquatic ecosystems, particularly on nutrient and carbon cycles. There is a great opportunity to monitor changes in freshwater fungi communities along latitudinal (north to south) and habitat gradients (from human disturbed to natural habitats), and study ecological thresholds and consequences of such changes, particularly its feedback on nutrient and carbon cycles in freshwater systems. (https://www.sciencedirect.com/science/article/pii/S1754504815000860)
Research on freshwater fungi has focused on their role in plant litter breakdown in streams. There is a higher fungi control over bacteria in terms of biomass, production and enzymatic substrate degradation. Microscopy-based studies suggest the prevalence of aquatic hyphomycetes, characterized by tetra radiate or sigmoid spores. Molecular studies have consistently demonstrated the presence of other fungal groups, whose contributions to decomposition are largely unknown. Molecular methods will allow quantification of these and other microorganisms. The capability of aquatic hyphomycetes to resist or mitigate anthropogenic stresses is becoming progressively important. Metal avoidance and tolerance in freshwater fungi connect a complex network of mechanisms involving exterior and intracellular detoxification. Investigating adaptive responses under metal stress will unravel the dynamics of biochemical processes and their ecological consequences. Freshwater fungi can metabolize organic xenobiotics. For countless such compounds, terrestrial fungal activity is characterized by cometabolic biotransformation’s involving initial attack by intracellular and extracellular oxidative enzymes, further metabolization of the primary oxidation products via conjugate formation and a considerable adaptability as to the range of metabolized pollutants. The same potentiality occurs in freshwater fungi. This proposes a largely ignored role of these organisms in weakening pollutant loads in freshwaters and their potential use in environmental biotechnology. (https://www.ncbi.nlm.nih.gov/m/pubmed/21276025/) Freshwater fungi contribute to the bulk of functional biodiversity in freshwater ecosystems (Geist 2011). They are also beneficial to the overall health of freshwater reservoirs as they function as decomposers, commensals, parasites, and predators (Wurzbacher et al. 2010; 2011).

In the study of Carascal et al. (2017), an entire of 28 fungal morph species were reported in this study: 12 morph species were derived from surface waters and 16 morph species came from submerged woods (tbl. 1). Of these, 26 morph species belonged to 20 fungal genera: Acremonium, Amphisphaeria, Aspergillus, Aureobasidium, Campylocarpon, Cladophialophora, Cladosporium, Corollospora, Cylindrocarpon, Fusarium, Gliomastix, Lecythophora, Neodeightonia, Ophioceras, Penicillium, Pestalotiopsis, Phialemonium, Prosthecium, Sclerotium, and Trichocladium. The noted species were mostly Ascomycetes. Two fungal isolates out of the 28 morph species unsuccessful to produce spores in MEA after lengthy incubation and were recorded as mycelia sterilia in the study. Since these two isolates differed in their colonial morphologies, they were well-thought-out as distinct records.
Bartolome et. Al (2016), in their study Butachlor Utilization Potential of the Morphologically Freshwater Fungal Isolates, showed that the fungal isolate taken from the submerged rice hay was asymmetrical, wavy, flat and white with slightly yellow mycelia while the waterlogged twigs fungal isolate was irregular, wavy, flat and white.

Goh ; Hyde (1996), in their study Biodiversity of Freshwater Fungi, there are more than 600 species of freshwater fungi with more known from temperate, as compared to tropical regions. These includeca 340 ascomycetes, 300 deuteromycetes, and a number of lower fungi which are not discussed here.Aniptodera, Annulatascus, Massarina, Ophioceras andPseudohalonectria are common freshwater ascomycetes, which appear to be well adapted for this lifestyle either in their ascospore types or their competitive-degradative characters. The most common genera of wood-inhabiting deuteromycetes includeCancellidium, Dactylaria, Dictyosporium andHelicomyces. They are categorized into four groups depending on their form and life style: the ingoldian hyphomycetes; the aero-aquatic hyphomycetes; the terrestrial-aquatic hyphomycetes; and the submerged-aquatic hyphomycetes. The adaptations of aquatic fungi for their dispersal and subsequent attachment to new substrates are discussed.

ButachlorButachlor (N-butoxymethyl-2-chloro- 2,6-diethyl acetanilide) is one of the most widely recommended herbicides. It is a preemergence herbicide belonging to chloroacetanilide group used widely in oriental countries for the control of annual grasses for rice cultivation (Jena, 1987; Yu et al., 2003). It also affects the various other metabolic processes and redox homeostasis adversely, in addition to lipid biosynthesis (Agrawal et al. 2014).

Butachlor was originally developed by Monsanto Co. (USA) in 1968 and commonly used as a post-emergence herbicide in Asia and Africa (Liu et al. 2011). Butachlor primarily enters the environment through various agricultural, horticultural and forestry practices, where inappropriate water management and rainfall contribute to butachlor runoff from the agricultural fields to the watersheds and aquatic ecosystems (Ok et al. 2012). The half-life of butachlor ranges from 1.65 to 2.48 days in field water and 2.67–5.33 days in soil (Huarong et al. 2010). The consumption of butachlor is approximately 4.5 9 107 kg per year in Asia alone (Ateeq et al. 2002). In India, nearly 6750 metric tons of butachlor are applied annually, as it was the first rice herbicide to be introduced (Verma et al. 2014; Tilak et al. 2007). The recommended field dosage of butachlor ranges from 10 to 150 lM (Alla et al. 2008; Chen et al. 2007).

Butachlor is the most commonly used herbicide on paddy fields in Taiwan and throughout Southeast Asia. Since paddy fields provide habitat for pond breeding amphibians, we examined growth, development, time to metamorphosis, and survival of alpine cricket frog tadpoles (Fejervarya limnocharis) exposed to environmentally realistic concentrations of butachlor. We documented negative impacts of butachlor on survival, development, and time to metamorphosis, but not on tadpole growth. The 96 h LC50 for tadpoles was 0.87 mg/l, much lower than the 4.8 mg/l recommended dosage for application to paddy fields. Even given the rapid breakdown of butachlor, tadpoles would be exposed to concentrations in excess of their 96 h LC50 for an estimated 126 h. We also documented DNA damage (genotoxicity) in tadpoles exposed to butachlor at concentrations an order of magnitude less than the 4.8 mg/l recommended application rate. We did not find that butachlor depressed cholinesterase activity of tadpoles, unlike most organophosphorus insecticides. We conclude that butachlor is likely to have widespread negative impacts on amphibians occupying paddy fields with traditional herbicide application. (https://link.springer.com/article/10.1007/s10646-010-0589-6)
In Region II, Nueva Vizcaya, there were 22 brands of insecticides being used in the area, 14 brands of fungicides, 3 herbicides, 2 molluscides, and 1 rodenticide. Soils in seven municipalities of Nueva Vizcaya are found positive of pesticide residues. These are Aritao, Bagabag, Bayombong, Diadi, Dupax del Sur, Quezon, and Villaverde. Among the pesticides detected are Monocrotophos (0.04-0.60 µg/g), Malathion (0.12 µg/g), Mancozeb (0.04 µg/g), Azinophos ethyl (0.04 µg/g) (http://www.pcaarrd.dost.gov.ph/home/momentum/philorgagri/index.php?option=com_content;view=article;id=87%3Apesticide-utilization-in-agricultural-production-in-nueva-vizcaya;catid=6;Itemid=21)
With the advent of the Green Revolution, there has been a quantum leap in the use of synthetic herbicides and pesticides throughout the world to sustain high-yielding crop varieties. Continuous use of these synthetic chemicals leads to loss of soil fertility and soil organisms. Histopathological studies may signal a damaging effect of organisms resulting from prior or ongoing exposure to toxic agents. A large number of studies have reported general histological changes in earthworms. A fewer studies have reported more specific types of histopathological studies in Eisenia foetida, Dendrodrilus rubidus, Lumbricus terrestris, Lumbricus rubellus and Octolasium transpandanum. In the result of the study of Muthukaruppan, worm growth was observed at various exposures over 60 days. While exposing the earthworm to the herbicide, no mortality was observed, not even at a higher dose. At the end of the experiment, the control group had a mean biomass of 0.0831±0.00 mg and, in the exposed group, at herbicide concentrations of 0.1973, 0.1315 and 0.0657 mg kg–1, the mean biomass was found to be 0.0497±0.00 mg, 0.0628±0.00 mg and 0.0781±0.00 mg, respectively. The mean earthworm biomass was found to be decreased with increasing herbicide concentration. Similarly, cocoon production was also reduced by the increasing herbicide concentration. All earthworms in the exposed group were found to have glandular cell enlargement and to be vacuolated.

In the developed countries, herbicides account for more than 70% of the total pesticides available on the market. During the period 1960 to 1985, herbicides had an annual worldwide growth rate of 16.7%. Herbicides are most widely used in the United States. In the Asia-pacific region, herbicide usage accounts for only 13.4%. They are mainly used for rubber, oil palm, tea, coffee and rice (ADB 1987). In the world market, the use of herbicides will continue to expand at 4.5% annually, which is the largest growth in the herbicide market so far (Adam 1976). In India, the use of herbicides has gradually increased. During the period from 1988 to 1997, herbicides had an annual growth rate of 13.7%.
Increased usage of herbicides on rice, tea, wheat, beans and other crops has destructive effects on beneficial organisms like earthworms and other non-target animals. The residues of herbicides in soil may create a variety of hazards. Many invertebrates take up herbicides from soil into their bodies and may accumulate herbicides several times greater in their tissues than those in the surrounding soil. The animal that feeds upon these invertebrates will be greatly affected by the concentration of these residues in the tissues, which will affect their normal activities.
Earthworms have been shown to be affected by the fate of herbicides in soil. Earthworms directly influence the persistence of pesticides in soil by metabolizing a parent compound in their gut (Gilman and Vardanis 1974, Stenersen et al. 1974), by transporting herbicides to depth and increasing the soil-bound (non extractable herbicide) fraction in soil (Farenhorst et al. in press) or by absorbing herbicide residues in their tissues (Edwards and Loffy, 1997). Numerous work done on the effects of pesticides and herbicides on earthworms mostly involve anatomical and population changes in Eudrilus, Eisenia and Lumbricus but the present study deals with both population and structural damages on Perionyx sansibaricu.

Lo Yc et al. (2008), in their study entitled Acute Alachor and Butachlor Herbicide Poisioning, most patients intentionally ingested the herbicides. The toxicities of alachlor and butachlor were largely similar. Twenty-eight out of 102 patients with oral exposure were asymptomatic, while the others developed vomiting, central nervous system depression, and other outcomes. Among patients using other exposure pathways, gastrointestinal effects were the main manifestation. Three patients died after manifesting profound hypotension and/or coma following alachlor ingestion.

In humans, Butachlor exposure may induce seizures, amnesia, central nervous system depression, and coma. It may cause pneumonitis and respiratory failure, hypotension, dysrhythmias, and sensitized myocardial tissue It raises the odds of liver and tissue necrosis, blood dyscrasias, renal failure, and thrombocytopenia. It primarily affects both the respiratory system and the reproductive health. It is detrimental to both the respiratory tract and the cardiovascular system. It may negatively affect the body’s overall blood circulation and renal health.

Degradation of ButachlorA fungus, Trichoderma harzianum, was found to degrade DDT, dieldrin, endosulfan, pentachloronitrobenzene, and pentachlorophenol but not hexachlorocyclohexane. The fungus degraded endosulfan under various nutritional conditions throughout its growth stages. Endosulfan sulfate and endosulfan diol were detected as the major fungal metabolites of endosulfan. Piperonyl butoxide, when added to the growth medium, completely inhibited the endosulfan degradation. Di?n?propyl malaoxon also inhibited the overall endosulfan degradation, but under such an inhibitory condition the formation of endosulfan sulfate was still observed. Using a cell?free preparation from Trichoderma harzianum, we could demonstrate that endosulfan metabolism in vitro was stimulated by exogenously added NADPH. Together with the evidence that the initial metabolic product of endosulfan was endosulfan sulfate, we concluded that the major enzyme system responsible in Trichoderma harzianum responsible for degradation of endosulfan is an oxidative system.

Rhodococcus sp. strain B1 could degrade 100 mg/L butachlor within 5 days. Butachlor was first hydrolyzed by strain B1 through N-dealkylation, which resulted in the production of butoxymethanol and 2-chloro-N-(2,6-dimethylphenyl)acetamide. Butoxymethanol could be further degraded and utilized as the carbon source for the growth of strain B1, whereas 2-chloro-N-(2,6-dimethylphenyl)acetamide could not be degraded further. The hydrolase designated ChlH, responsible for the N-dealkylation of the side chain of butachlor, was purified 185.1-fold to homogeneity with 16.1% recovery. The optimal pH and temperature of ChlH were observed to be 7.0–7.5 and 30 °C, respectively. This enzyme was also able to catalyze the N-dealkylation of other chloroacetamide herbicides; the catalytic efficiency followed the order alachlor > acetochlor >butachlor > pretilachlor, which indicated that the alkyl chain length influenced the N-dealkylation of the chloroacetamide herbicides. This is the first repx`ort on the biodegradation of chloroacetamide herbicides at the enzyme level.

Singh and Nandabalan (2018) showed in their study that indigenous bacterial species Ammoniphilus sp. JF was isolated from the agricultural fields of Punjab and identified using 16S ribosomal RNA analysis. The bacteria utilized butachlor as the sole carbon source and showed complete degradation (100 mg/L) within 24 h of incubation. Two intermediate products, namely 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester and 2,4-bis(1,1-dimethylethyl)-phenol were observed at the end of butachlor degradation. 
Zhang et al. (2011) isolated a butachlor-degrading bacterium, Paracoccus sp. FLY-8, from a rice field soil. They found that this strain degraded butachlor to DEA when its degradation products of butachlor were identified by GC-MS. As this strain mineralized butachlor, they analyzed some possible enzyme activities such as aniline dioxygenase and catechol 1,2-dioxygenase to infer the mineralization pathway. The cellular lysates of Paracoccus sp. FLY-8 exhibited these two enzyme activities, and thus, they proposed the plausible mineralization pathway of butachlor through alachlor, 2-chloro-N-(2,6-dimethylphenyl) acetamide, DEA, aniline, and catechol. 
Related Studies
According to Abd-Alrahman S.H. & Salem-Bekhit M.M. (2012)., in their research Microbial Degradation of Butachlor Pollution, six bacterial strains were isolated from an agricultural soil and found to be actively utilized butachlor, as a sole source of carbon and energy.

Few researches revealed that fungi on fresh water such as Trichoderma viride and Pseudomonas alcaligens quickly degraded butachlor and reached nearly 98% and 75% in a medium containing 50 mg/kg of butachlor after 15 and 21 days, respectively. These results can conclude that these two organisms can be used to degrade the obsolete butachlor formulation ( Abd-Alrahman et al, 2012). Trichoderma viride and Psedomonas alcaligens are examples of fungi that can be isolated and used to degrade butachlor in soil.

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