Isolation, Identification and Characterization of Fungi ...

International Journal of Horticultural & Crop Science Research. ISSN 2249-4243 Volume 2, Number 1 (2012), pp. 1-6 © Research India Publications http://www.ripublication.com/ijhcsr.htm

Isolation, Identification and Characterization of Fungi from Rhizosphere Soil of Barleria Cristata L. Jeyanthi Rebecca*, V. Dhanalakshmi1, S. Sharmila1, G. Susithra1, Santosh Kumar2 and Sashi Bala2

*Corresponding Author & Professor and Head, Dept. of Industrial Biotechnology, Bharath University, 173, Agaram Road, Selaiyur, Chennai-600 073, India. E-mail: [email protected] 1 Senior Lecturers, Dept. of Industrial Biotechnology, Bharath University, 173, Agaram Road, Selaiyur, Chennai-600 073, India. 2 B.Tech Students, Dept. of Industrial Biotechnology, Bharath University, 173, Agaram Road, Selaiyur, Chennai-600 073, India.

Abstract The rhizosphere region in the soil has a rich source of microorganism. These microorganisms may be either pathogenic or non-pathogenic to the plants and humans. The present study is specially focused on the fungal flora present in the rhizosphere region of the ornamental plant Barleria cristata. The soil was carefully processed and inoculated on SDA plates. Total six isolates were isolated from the soil on the SDA plates. Out of six fungi species four were identified and they belonged to division Ascomycota (genus Sporothrix sp, Aspergillus sp, Arthirinium sp, Fusarium sp), while two sample remained unknown. Based on the preliminary morphology identification and the literature consulted, the colony obtained was pathogenic to humans and causes diseases. Each of the four sample identified were described separately based on the literature found. Keywords: Barleria cristata, fungi, rhizosphere

Introduction Fungi are a member of large group of eukaryotic organism that include microorganism such as yeast and mould as well as several mushrooms. The fungi

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species can be used a biocontrol agent where they act as antagonists by developing the condition of generating competition for nutrient and space with pathogens to be killed. Certain fungi, particularly white rot fungi can degrade toxic compounds such as uranium oxides and azo dyes by the secretion of extracellular enzymes thus enabling in the process of bioremediation. The rhizosphere is the narrow region of soil that is directly influenced by root secretions and associated soil microorganisms. The rhizosphere microorganisms predominantly help in metabolizing the root exudates. Many organisms like phosphate solubilizing microorganisms and mycorrhizae help in mobilizing phosphates and other nutrients to the plants. Both pathogenic and symbiotic fungi are present in the rhizosphere. Zygomycetes and hyphomycetes establish most readily in the rhizosphere because they metabolize simple sugars (Sylvia et al., 2005). Microorganisms in the rhizosphere complete both chemical and physical modifications to the soil profile in and around the rhizosphere that affect plants. They can be beneficial to the plant (by pathogen suppression) or detrimental (by competition for nutrients). Chemical changes occur as a result of humification of organic matter. The resultant mineralization of various organic compounds (phosphorous, sulphur, and nitrogen, for example) provides plants with forms of nutrition that are readily available for uptake. The turnover of microbial populations also results in the release of nutrients. The process of nitrogen fixation by both asymbiotic and symbiotic bacteria results in increase of the available nitrogen in the rhizosphere region (Sylvia et al., 2005). Symbiotic Mycorrhiza causes an increase in the effective root area of plants, thereby providing added nutrient mining capabilities to the plant. Rhizosphere microbes can also release plant growth regulators. Physical changes occur primarily through the production of extracellular polymeric substances such as polysaccharides and glomalin, which improve soil aggregation and soil texture. The presence of mucigel in the rhizoplane is crucial to the water relations of plants, providing a bridge that prevents desiccation by maintaining the water column during water stress events. (Sylvia et al., 2005). Fungi are also often an important source of phosphorous. The microbes can also help the plant to survive in an otherwise inhospitable environment. For example, specific endophytes have been shown to confer heat resistance to grasses that grow in a hydrothermal area, or salt tolerance to coastal grasses. Rodriguez, (2008); Miglia et al., 2007 explored that Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. For plants, there are two influential specific plant–microbe associations: mycorrhizaes and root nodulation. Mycorrhizae demonstrate symbiosis between plant roots and soil fungi (Smith and Read, 1997; Connell et al. 2006). The impact of fungi is considerable and many of them are serious pathogens of maturing seed and reduce the yield impair quality. Other fungi include saprophytes and weak pathogens which lower the quality of seeds by causing discoloration and reducing germination. The microflora around the rhizosphere varies from genus to genus and it is specific to the plant environment.

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In the present study the rhizosphere soil of the ornamental plant Barleria cristata was analyzed for the presence of associated fungal flora. Barleria cristata (commonly called Philippine violet, Bluebell barleria or Crested Philippine violet or the December flower as it is called in Tamil Nadu, India), is a plant belonging to the Acanthaceae family. It is native to a wide area ranging from Southern China to India and Myanmar. It grows as a shrub 60 -100 cm tall. The leaves are dark green on the upper surface and pale green on the lower surface. They are elliptic to narrowly ovate. The flowers are about 5 cm long, funnel-shaped in violet, pink, or white color. The fruits are about 1.5 cm long ellipsoid capsules. They become glabrous and glossy at maturity.

Materials and Methods Collection of sample Samples were collected from the locality around the campus of Bharath University, Chennai. Plant was safely taken out from the soil and its bulk soil was discarded. Soil associated with the roots was isolated carefully scrapped in the sterile tube with the help of sterile blades in the laminar air flow chamber. Plating The samples were serially diluted upto 10-7. Sabouraud dextrose agar (SDA) plates were prepared and 100 µl of the samples ranging from 10-1 to 10-7 dilutions were plated on sterile SDA plates using spread plate technique. Individual colonies from the mixed or mother culture were subcultured on fresh SDA plates for obtaining pure culture of each isolates. The pure culture was isolated for further identification through LPCB staining technique. Preparation of Lacto- phenol Cotton Blue Slide Mounts The lacto-phenol cotton blue (LPCB) wet mount preparation is the most widely used method of staining and observing fungi and is simple to prepare. The preparation has three components: phenol, which will kill any live organisms; lactic acid which preserves fungal structures, and cotton blue which stains the chitin in the fungal cell walls. The samples were immersed in a drop of alcohol and it was viewed under the microscope after the addition of two drops of the lacto-phenol/cotton blue mount/stain.

Results and Discussions The fungi isolates from the rhizosphere soil were examined with the help of the lactophenol cotton blue (LPCB) wet mount preparation method. Out of six isolates, four were identified as belonging to the division Ascomycota and belonging to the genus as shown in Table 1. Microbial isolate1, 3, 4 and 5 were identified as Sporothirix sp., Aspergillus flavus, Arthirinium sp and Fusarium sp, respectively. The microbial isolates 2 and 6 are yet to be identified (Fig 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B).

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L. Jeyanthi Rebecca et al Table 1: Identification of microbial isolates. S.No 1 2 3 4 5 6

Sample name Fungal isolate 1 Fungal isolate 2 Fungal isolate 3 Fungal isolate 4 Fungal isolate 5 Fungal isolate 6

Name of the fungal isolate Sporothirix sp unknown Aspergillus flavus Arthirinium sp Fusarium sp unknown

Fig 1A: Aerial mycelium of the fungal isolate 1

Fig 1B: Vegetative mycelium of the fungal isolate 1





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Fig 4B: Vegetative mycelium of the fungal isolate 5.

Soil is the complex system containing flora of various microorganisms including bacteria, algae, moulds and mushrooms. Fungi are one of them, it is estimated to have 1.5 million species of fungi in the world at different surroundings with about 5% of these identified formally. In the present study, the fungal isolates obtained from the rhizosphere soil, belonged to the division Ascomycota, the largest phylum of Fungi, with over 64,000 species. The species present in the soil can be specifically pathogenic to the plants as well as to the human beings. The results in this study resemble the isolation of Aspergillus flavus by Schmidt et.al, (1976) from the rhizosphere soil in California. The soil was screened carefully to avoid any air contaminant. Out of six samples, three identified one are found to have history of causing diseases in human beings such as microbial isolate 1 identified as the Sporothirix sp. which is known to cause rose handler disease in the people working in gardens. Microbial isolate 3, identified as Aspergillus flavus causes aspergillosis of the lungs sometimes causing corneal and naso-orbital infections. A. flavus spores are allergenic. A.flavus is the second most common agent of aspergillosis, the first being Aspergillus fumigatus. Microbial isolate 4 identified as Arthirinium sp. There are no infections so far reported due to Arthrinium in humans or animals and is cosmopolitan. It grows rapidly in laboratory. Sample five identified as the Fusarium spp. Infections due to Fusarium spp. are collectively referred to as fusariosis. The root of Barleria cristata is also associated with arbuscular mycorrhizal fungi which are beneficial to the plant growth (Zhao and Zhao, 2007).

Conclusions In this study, we have found six cultures of the fungi in the rhizosphere soil which was collected from the locality around Bharath University, Selaiyur, Chennai. Out of six, four were identified with the help of the literature available for identification, while two remained unknown. According to the literature, the fungi isolates which is identified are pathogenic in nature for human beings except fourth sample which is identified as Arthirinium sp, which has no any history of causing disease in human or animals. Aspergillus flavus. Fusarium sp., Sporothirix sp. is pathogenic in nature and causes serious health problems.

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