Molecular Characterization of Fusarium oxysporum G30 Isolated from Vegetable Wastes in Imphal, Northeast India

A new strain of Fusarium oxysporum (Schl.) emend. Synder & Hansen, viz. Fusarium oxysporum G30, is reported from seven different decomposing vegetable wastes and a mixture of the same in equal proportions, in Imphal, Northeast India. This strain was isolated by surface sterilization technique, followed by re-culturing till pure culture was obtained. The pure culture was characterised morphologically based on the colony features: colour, texture, colony diameter; conidial features: macro and microconidial shape, size, septation, presence of chlamydospores and foot-shaped basal cells. The strain was also identified at molecular level by partial sequencing using universal primers, ITS4 and ITS5. The ITS data reveals that isolated culture (F. oxysporum G30) match the existing isolates in Gen Bank (Gen Bank accession No. GQ497156.1) by 99% and it has got a code segment particular to this strain. This strain showed ubiquitous nature with mean percent frequency of occurrence ranging from 39.5 to 68% at various stages of decomposition of the selected vegetable wastes.


Introduction
Fusarium oxysporum (Schl.) emend. Synder & Hansen is an ubiquitous saprophyte found predominantly in the environment. This species has been reported to be responsible for a number of diseases in plants. F. oxysporum has been assigned as the most economically important species of the genus because of the numerous host range and the level of loss caused as a result of their infection in plants. Vegetable wastes which contribute 50-60 t/day of municipal solid wastes in Imphal (Anonymous, 2009) are known to be good supporters of microbial growth, especially of fungi, during the decay stages. Recent studies (Sharma and Pandey, 2012) reveal that this species occurs in maximum population in decaying vegetable wastes. Identifying this particular strain at molecular level will be useful for formulating effective biological treatment of vegetable wastes which have already become a problem for dumping in city areas. Such beneficial application of this fungus has been given less attention so far. Therefore, in the present study, the most common F. oxysporum strain, having the highest frequency of occurrence in decaying vegetable wastes, have been isolated and characterized at molecular level.

Decomposition of vegetable wastes
Decomposition studies of vegetable wastes were carried out both in litter bags by following nylon mesh bag technique (Bocock et al., 1960), as well as in experimental pots. Five gm air-dried vegetable wastes in each nylon net bag (10 x 15 cm; 1 mm mesh size) separately for each of the eight samples were randomly placed on soil surface in the experimental site located in the Department of Life Sciences, Manipur University. For each decomposing vegetable wastes at least three bags were recovered at monthly interval for a period of seven months (November-June) and brought to the laboratory in sterile polythene bags where the samples of each bag were brushed carefully to remove the adhering soil particles for the isolation of fungi. Similarly, 5 gm air-dried sample were also allowed to decompose in plastic pots placed in tin roofed experimental shade adjacent to the experimental plot. Sterilized water was sprayed at 4 days interval (1 ml/pot). As in the above case, contents of three pots for each sample were recovered at monthly interval for the isolation of fungi.

Isolation of fungi
The technique of surface sterilization using 15% H2O2 and 70% Ethanol (Kinkel and Andrews, 1988) was employed for the isolation of fungi inhabiting the decomposing of vegetable wastes. Bits of samples (3 x 3 mm 2 ) were cut using sterilized scissors, immersed in 70% ethanol for 1 minute. These were then transferred to 15% H2O2 for 1 minute and again to 70% ethanol for 1 minute, followed by washing with changes of sterilized distilled H2O for 5 minutes and air-dried in-between folds of sterilized blotting paper for 30 minutes. Five surface sterilized samples bits were then placed equidistant in each Petri dish containing 20 ml PDA medium supplemented with streptomycin (100 mg/l). The plates were incubated at 25 ± 1 °C for 7 days and fungal colonies developing on the bits were purified to identify and isolate F. oxysporum present. Three replicates were maintained in each case.

Molecular characterization
Molecular characterization of the culture isolate was performed at NFCCI, ARI, Pune by partial sequencing (ITS).
Genomic DNA was isolated in pure form from the culture. Nearly 500 dp rDNA fragments were successfully amplified using universal primers-ITS4 (5'TCC TCC GCT TAT TGA TAT GC3') and ITS5 (5'GGA AGT AAA AGT CGT AAC AAG G3'). The sequencing PCR was set up with ABI-BigDye Terminator v3.1 Cycle Sequencing Kit (Part No. 4337455). PCR reaction was performed in a 25 µL volume containing 10 X PCR buffer, 200 mM dNTP, 1U Taq polymerase, 10 pM of each primers and 10 ng genomic DNA. While the amplification cycle consisted of an initial denaturation step of 95 °C for 5 mins, followed by 30 cycles of (a) denaturation (95 °C for 1 min), (b) annealing (56 °C for 1 min) and (c) extension (72 °C for 1 min) further followed by a final extension step of 10 mins at 72 °C. The sequence data was aligned with publically available sequences and analyzed to reach identity (Table 1).

Data analysis
Percent frequency of occurrence was calculated by the formula: Frequency of occurrence (%) = (Number of sample bits on which fungal species occurred/total number of bits observed) × 100.

Results and discussions
In both decomposition studies a particular isolate of Fusarium oxysporum G30 was obtained from all the study samples throughout the study period with mean percent frequency of occurrence (F.O. %) ranging from 39.5% (M. paradisiaca) to 68.0% (B. arundinaceae) in litter bag decomposition and 44.0% (B. arundinaceae) to 63.5% (mixed sample) in pot decomposition (Fig. 1). This result is another evidence to show the ubiquitous nature of F. oxysporum. Earlier, ubiquitous nature of F. oxysporum was reported by Sharma et al. (2011) while working on the diversity of microfungi associated with surface soil and decaying leaf litter of Quercus serrata. It has also been reported that F. oxysporum is the most widely dispersed Fusarium species and can be recovered from most soils -arctic, tropical or desert and cultivated or not (Leslie and Summerell, 2006). In fact, the near ubiquity of this fungus in soil worldwide has led to its inclusion in what has been termed the global mycoflora (Gordon and Martyn, 1997

Morphological characterization
Culture media differentially influence the growth, colony characters and sporulation of fungi (Sharma and Pandey, 2010) hence use of more than one culture media has been recommended for morphological characterization of this fungi also. Seven days old colonies (Fig. 2a) of F. oxysporum G30 on PDA measured 52.0 ± 3.1 mm in diameter, floccose, magenta pink with concentric zones of dark and light reddish colouration; reverse magenta pink turning violet with age. Colonies on CYA measured 85.7 ± 0.3 mm in diam., white, lacking zonations; reverse colourless, while colonies on LCA exhibited 79.7 ± 0.3 mm diam. in growth, light cottony in texture, hyaline; reverse colourless after seven days of incubation. Microconidia and macroconidia were produced in all three culture media. Microconidia 1 to 2 celled, hyaline, fusiform to kidney shaped, produced singly from the tips of phialides, 0 Septate 4-14 × 2-4 µm, 1-Septate 9-33 × 2slightly curved usually with three to five septa, rarely more; no foot-shaped basal cells observed, 3-Septate 20 54 × 2 Septate 30 -65 × 3 -6 µm. Chlamydospores present, abundant in older cultures, terminal and intercalary, single in majority of the cases measuring 10 -15 µm in diameter (Fig. 2b).

Molecular characterization
Morphological characterization alone is not enough as genetic information is not provided for identification of a species since the process is emphasized on differences of the morphological features which could readily be altered by environmental and cultural factors (Edel et Sharma GL et al. / Not Sci Biol, 2015, 7(2):184-187 zonations; reverse colourless, while colonies on LCA n growth, light cottony in ess after seven days of incubation. Microconidia and macroconidia were produced in all three culture media. Microconidia 1 to 2 celled, hyaline, fusiform to kidney shaped, produced singly from the tips of phialides, 0--5 µm. Macroconidia slightly curved usually with three to five septa, rarely more; no Septate 20 54 × 2 -5 µm, 5-6 µm. Chlamydospores present, abundant ary, single in majority of 15 µm in diameter (Fig. 2b).
Morphological characterization alone is not enough as genetic information is not provided for identification of a species asized on differences of the morphological features which could readily be altered by et al., 1995). In fact, fungal taxonomy is in a state of reflux because of the recent researches based on molecular approaches. Despite of the advancements and their considerable value, molecular approaches to fungal identification still remains  (Pawar et al., 2007). O characterization this particular strain, oxysporum G30, has got the following characters compared to other already defined isolates available in the Gen Bank database (Fig. 3).
The ITS data reveals that the isolated culture oxysporum G30 match the existing isolates in Gen Bank (Gen Bank accession No. GQ497156.1) by 99% and has got a code segment in the Quary 481 to 491 of the sequence which is particular to this strain (Fig. 3). Fusarium oxysporum is a species complex consisting of morphologically indistinguishable strains. Therefore, molecular identification will serve the gene level documentation of this strain. In India, about 320 million tonnes of agricultural wastes are generated annually (Suth et al., 2005) of which vegetable wastes alone is in major proportion. These vegetable wastes are either burnt or dumped elsewhere causing environmental pollution. Biological treatment of these wastes appears to be cost effective and carry a less negativ (Paraskeva and Diamodopoulos, 2006). The filamentous fungus, Fusarium oxysporum has attracted a lot of scientific interest due to its ability to produce lignocellulolytic enzymes which are required for degradation of the lignin and cellulose that form the main components of such wastes. Infact, Fusarium oxysporum powerful lignin degraders isolated during a screening of lignolytic microorganisms in forest soil (Rodriguez 1996). In this connection, the present might have potential significance in biodegradation with the following properties: high F.O. percent (63.5%) in mixed vegetable in pot, ubiquitous nature of the strain for universal use and ability to grow in different detailed investigation may be carried out on assessing the Sequence analysis (491 bases) with (NCBI sequence Accession )/ G30 Sequences producing significant alignments untouched in Northeastern region of India, which forms an important portion of the Indo-Burma biodiversity , 2007). On molecular characterization this particular strain, Fusarium has got the following characters compared to other already defined isolates available in the Gen Bank database (Fig. 3).
The ITS data reveals that the isolated culture F. G30 match the existing isolates in Gen Bank (Gen Bank accession No. GQ497156.1) by 99% and has got a code segment in the Quary 481 to 491 of the sequence which is particular to this strain (Fig. 3).
is a species complex consisting of indistinguishable strains. Therefore, molecular identification will serve the gene level documentation of this strain. In India, about 320 million tonnes of agricultural wastes are generated annually (Suthur , 2005) of which vegetable wastes alone is in major proportion. These vegetable wastes are either burnt or dumped elsewhere causing environmental pollution. Biological treatment of these wastes appears to be cost effective and carry a less negative environmental impact (Paraskeva and Diamodopoulos, 2006). The filamentous has attracted a lot of scientific interest due to its ability to produce lignocellulolytic enzymes which are required for degradation of the lignin ellulose that form the main components of such Fusarium oxysporum is one of the most powerful lignin degraders isolated during a screening of lignolytic microorganisms in forest soil (Rodriguez et al., this connection, the present strain identified might have potential significance in biodegradation with the following properties: high F.O. percent (63.5%) in mixed vegetable in pot, ubiquitous nature of the strain for universal use and ability to grow in different media. Future iled investigation may be carried out on assessing the biodegradability of this fungal strain in terms of weight loss and lignocellulose contents.

Conclusions
Morphologically this particular strain of Fusarium oxysporum G30 is hardly distinguishable from the other known strains viz. Fusarium oxysporum strain KKK 19 (GQ497156.1), Fusarium oxysporum f. sp. gladioli isolate UAS007 (FJ158124.1), Fusarium sp. UAS011 (FJ158128.1), Fusarium oxysporum f. sp. vasinfectum strain Anyang City (EU849584.1) and Fusarium sp. 14018 (EU750682.1). However, molecular identification of this particular strain showed 99% sequence similarity with the above mentioned known strains. The difference lies in Quary 481 to 491 of the sequence. Thus, the present strain is a distinct strain of Fusarium oxysporum and reported for the first time as a new strain.