This site uses cookies for learning about our traffic, we store no personal details. ACCEPT COOKIES DECLINE COOKIES What are cookies?
univerge site banner
Original Article | Open Access | Am. J. Pure Appl. Sci., 2022; 4(2), 36-40 | doi: 10.34104/ajpab.022.036040

Isolation and Identification of Fungal Pathogens from Five Flowering Plants in Jashore Region of Bangladesh

Shahidur Rahman Mail Img ,
Md. Khasrul Alam** Mail Img ,
Sabbir Ahmed Mail Img ,
Md. Mosharraf Hossen Mail Img ,
Mohammad Abu Hena Mostofa Jamal Mail Img ,
Md. Rezuanul Islam* Mail Img

Abstract

Bangladesh is a great market for the flower trade. It produces a lot of different types of flowers as well as imports from neighboring countries which cost 3 million BDT currencies every year. The Jashore district of plants viz. Gladiolus, Gerbera, Rose, Tuberose and Marigold. The infected part of the plant samples was collected from five separate flower gardens. The precisely prepared infected sample was cultured on Potato Dextrose Aga media at 28ºC in an incubator for 48 hours and sub-cultured several times of each sample of distinct features to get a fresh culture of fungal pathogens. The isolates were identified based on their morphological features of the colony and observation of mycelia structure. The infection by fungal pathogens is considered a great barrier to flower cultivation. Therefore, the present study was attempted to the isolation infecting fungal pathogen from five different flowering crystal violet dyes to analyze the spore structure, the shape of the tips, conidial structure, and we identified three different types of fungus from five flowering plants Aspergillus niger identified from gerbera and rose, Colletotrichum gloeosporioides (pinkish) from tuberose, Colletotrichum gloeosporioides (whitish) from gladiolus and Alternaria alternate from marigold. This study has provided the primary alarm of fungal infection by following a less expensive technique. This study will be helpful to identify and management of phyto-pathogen for floriculture.

INTRODUCTION

Flower cultivation is becoming very popular day by day. Low investment but higher rate of profit encour-ages the farmers for large scale cultivation of different flowers. Large-scale commercial production started from mid eighties in Jhikargacha upazila of Jashore district (Sultana, 2003). At present, 10,000 hectares of land covers flower cultivation taking the lead by the Jashore district (Nusrat, 2012). More than 5,000 resi-lient farmers are growing flower and foliage in the country and about 150,000 people are directly or indirectly involved in floriculture business as their sole livelihood (Chowdhury, 2010). The area coverage under commercial flower cultivation is approximately 10,000 hectares of land while commercial nurseries have covered approximately 2,000 to 2,500 hectares of land (Momin, 2006). Bangladesh has to spend roughly Tk. 2-3 million in importing flowers and ornamental plants from abroad. The flowers originated from other countries are higher susceptible for plant pathogens. The fungal pathogens are primary enemies of flower-ing plants. Generally bacterial infections are very less common incidents than fungal infection. A wide range of fungal pathogens are recorded for infection. The type of fungal infections widely depends on geogra-phical locations. Some of the changes in endemic fungal infections can be attributed to climate changes, extension of human habitats, ease of travel, and shifting populations (Jeannette et al., 2011).  

A wide range of fungal diseases are recorded like battling mildew, mold and black spot, powdery mil-dew, gray mold, black spot, cankers, etc. Each case, different microorganisms are involved for the deve-lopment symptoms. But the black spot may be gene-rated by different fungal species. The air born fungus is mostly involved for development of diseases. The mal-nutrition and lower immunized flowering plants are worse victim by air born fungus, because they can easily colonize and finally further reproduction. There are several techniques have been developed for identification, such as enriched morphological fea-tures, textures. The précised way for identification is to determine the specific agent present in the histo-pathologic specimen, including immunohistochemis-try, in situ hybridization, polymerase chain reaction (PCR) and DNA sequencing using bioinformatics tools. In addition, techniques such as laser micro-dissection will be useful to detect the now more fre-quently recognized dual fungal infections and the local environment in which this phenomenon occurs (Sharif et al., 2019; Jeannette et al., 2011). 

Management of fungal pathogens becomes an ongoing battle, the scientist have developed a new fungicide but becomes resistant after some generation of ap-plications. Fungus is very pathogenic for flowering plant. A very small colony can change the appearance of flower and fragrance. For proper management fungus infection, the identification process is very important. Our research carried out based on several aims and objectives.  My primary objective of research is to make well conscious of our farmer. It focuses some focuses to identify the unknown fungal patho-gens, to analyze the structure and function of a fungal mycelium and discuss its adaptive significance, to explore the nature and characteristics of the patho-genesis of fungal infections and find out the app-ropriate application of fungicide. 

MATERIALS AND METHODS

Sample Collection and Preparation - The different living and nonliving materials were used in this in-vestigation, different plant materials such as stems, leaves and flowers where we found visual charac-terized symptoms like rots, black spots, blights, wilts and anthracnose are used for isolation of fungus from different nurseries in the Jashore region of Bangla-desh. The investigation focused on flowering plants like gerbera, tuberose, rose, marigold and gladiolus. The infected parts were taken in the white transparent polythene bags in separately and tied each bag with cotton to prevent any type of contamination in these bags (Delhove et al., 2013, Rebecca et al., 2012, Shahen et al., 2019; Soni and Sharma, 2014).  

The preparation of sample was done in the laminar air flow cabinet of the microbiology laboratory. The un-infected parts of the leaves were discarded and in-fected parts are taken for further procedure. The infected parts were cleaned very carefully with 0.5 % hypochlorite solution (Larran et al., 2001, Amsalu Abera et al., 2016) and 0.5 % HgCl2. To perform this research in a clean room having provision for working space, free of dust and convection of current that carry spores of contaminating microorganisms. The prepar-ation of room was equipped with cabinet and shelf space for safe storage of chemical compounds and dust free storage facilities, transfer areas for aseptic mani-pulations, culture room where cultures incubated under controlled light and temperatures.

Preparation of Media - For the preparation of 250 ml of potato dextrose agar media 9.75 grams of powder weighted with top pan balance and taken in to a conical flask. Then 250 ml of autoclaved distilled water was added.  To mix properly, the gentle stirring was done.  For making a fine suspension, the conical flask was placed on water bath for 15 minutes around 70ºC and finally autoclaved at 121ºC for 15 minutes and sterilized media poured on Petri-dish.

Culture and Isolation - The prepared sample was inoculated in the surface of PDA media in the bio-safety cabinet. The plates containing sample were placed in an incubator for 3 to 5 days at 27±1ºC temperatures. After successful incubation period, the visual mycelial structure was found. That visualized mycelia was sub-cultured in fresh PDA media, 48 hours later, the pure fungus was found on the surface of the plate. Again re-subculture of fungus was done to obtain final pure fungal culture (Javadi et al., 2012; Jasuja et al., 2013; Gaddeyya et al., 2012).

Identification - The fungus was identified based on colony characteristics and micro-morphological and macro-morphological appearances both on agar surface, microscopic slide observation and shape of spore and nature of the sporulation, shape of the tips and mycelia structure. For measuring the diameter of mycelia, we used ocular and stage micro-meters. Pure cultures of the isolates were sub-cultured at-least for triple times of every sample for purification and a microscopic view of every slide also tabulated for confirmation and confirmed based on morpho-logical features with our existing fungal sample in microbiology laboratory, Department of Biotechno-logy and Genetic Engineering, Islamic University, Kushtia, Bangladesh and comparing with the features (Josep Cano et al., 2004, N. MacClenny, 2005 and Ramjegathesh et al., 2012). 

RESULTS AND DISCUSSION

We collected different infected parts of five flowering plants Gladiolus dalenii, Gerbera jamesonii, Rosa macdub, Polianthes tuberose and Tagete serecta. We confirmed two isolates as Aspergillus niger by mor-phological and cultural features by observing micros-copic slide and agar surface. We found typical features of hyphae of both isolates of Aspergillus niger from Gerbera jamesonii and Rosa macdub, where average width of hyphae was 2.6 to8 mm and tree like bran-ching and some slide acute angle branching, hyaline and septate as described by   N. McClenny. To differ-entiate between   A. fumigates and   Aspergillus niger, we monitored growth rate and color. We didnt ob-serve any grey-blue-green colonies and uniseriate conidial heads within 24 to 48 hours incubation periods (N. McClenny, 2005). 

For isolates of Tagete serecta we confirmed as Alter-naria alternata, where the conidia were muriform shaped and colour was light brown and the length of conidia was  31 to 35 μm and the mean diameter of mycelia growth on Potato Dextrose Agar (PDA) was 8.15 cm  as described by Ram-jegathesh et al. 2012. 

Fig. 1: A); Front view of Colletotrichum gloeosporioides from Polianthes tuberosa, B); Front view and F back view of Colletotrichum gloeosporioides from Polianthes tuberose. C); and D); are the front view and back view of Alternaria alternata and Tagete serecta. E); and G); are front view of plate and microscopic view of Aspergillus niger respectively and H is the mycelia structure of Alternaria alternata.

Two isolates were identified as Colletotrichum gloeo-sporioides both from Gladiolus dalenii and Polianthes tuberose. Colletotrichum gloeosporioides species have a complex genetically; morphological characteristics are more or less typical (Weir et al., 2012). All the cultures plate produced conidiogenous cells and aerial mycelium of the colony directly on the agar surface and conidiogenous cells were cylindrical shape and glassy and translucent in appearance, tapered and measured up to 20 by 3 to 4 μm.  The setae were dark brown, acicular, thick walled and up to 200 μm long as described by Josep Cano et al.  2004. Both isolates of Colletotrichum gloeosporioides were slow growing comparatively of all isolates. 

The Aspergillus niger, isolated from gerbera were very fast growing.  It was difficult to store this isolates in the refrigerator for long duration, because it turns into black within a very short period of time. As a result, a frequent subculture had to done.

Table 1: List of isolated fungus.

CONCLUSION

Commercial flower cultivation has increased drama-tically over the last few decades. A number of new farmers are getting involved every year. For making more economic of floriculture, the appropriate disease management should be practiced. Necessary inform-ation about fungal pathogens is integral part of disease management. Identification by phenotypic features and culture is quite easy and fast (N. McClenny, 2005). Our current research would be helpful for diagnosis of infecting fungus of phyto-pathogenecity. The morpho-logical diagnosis of fungi is considered as the powerful weapon for early diagnosis. It is the pre-condition for proper management of infecting fungal pathogens. The infecting fungus like Aspergillus niger, Colletotrichum gloeosporioides and Alternaria alternata have their own distinguished morphological features. 

ACKNOWLEDGEMENT

Special thanks to my colleagues in the workplace who were there to help me in every moment of this huge work for the successful study.

CONFLICTS OF INTEREST

The author(s) declare that there is no potential conflict of interest.

Article References:

  1. Amsalu Abera, Fikre Lemessa Girma Adunga. (2016). Morphological Characteristics of Colleto-trichum Species Associated with Mango (Mangi-fera indica L.) in Southwest Ethiopia, Food Sci-ence and Quality Management., 48, 106-115. https://core.ac.uk/download/pdf/234684196.pdf  
  2. B. S. Weir P. R. Johnston U. Damm. The Colleto-trichum gloeosporioides species complex (2012). Studies in Mycology, 73, 115–180. https://doi.org/10.3114/sim0011  
  3. Chowdhury S.Z. (2010). Produce more fruits and vegetables instead of rice. The Daily Independent, February 11, 2010, Dhaka.
  4. Delhove, R. and Vannière. (2013). New pests and invasive diseases. Mango Bacterial Disease. Available on www.coleacp.org/pip
  5. Gaddeyya G, Niharika PS, Bharathi P and Kumar PKR. (2012). Isolation and identification of soil mycoflora in different crop fields at Salur Mandal. Adv Appl Sci Res., 3, 2020-2026. https://www.primescholars.com/articles/isolation-and-identification-of-soil-mycoflora-in-different-crop-fieldsat-salur-mandal.pdf  
  6. Jasuja ND, Saxena R, Chandra S and Joshi SC. (2013). Isolation and identification of microor-ganism from poly house agriculture soil of Rajas-than. African J Microbiol Res., 7, 4886-4891. https://doi.org/10.5897/AJMR2012.2413  
  7. Javadi MA, Ghanbary MAT and Tazick Z (2012). Isolation and molecular identification of soil inhabitant Penicillia. Ann of Biol Res., 3, 5758-5761.
  8. Jeannette G, and Mary E. (2011). Histopathologic Diagnosis of Fungal Infections in the 21st Century. Clin Microbiol Rev., 24(2), 247–280. https://pubmed.ncbi.nlm.nih.gov/21482725/  
  9. Josep Cano, Josep Guarro, & Josepa Gené, (2004). Molecular and Morphological Identification of Colletotrichum Species of Clinical Interest. J Clin Microbiol. 42(6), 2450–2454.  
  10. Larran, S., Mónaco, C. & Alippi, H. (2001). Endo-phytic fungi in leaves of Lycopersicon esculentum Mill. World Journal of Microbiology and Biotech-nology, 17, 181–184. https://doi.org/10.1023/A:1016670000288 
  11. Momin M. A. (2006). Floriculture Survey in Bangladesh. A Consultancy Report, FAO. UNDP. (IHNDP/BGD/97/06).
  12. N. McClenny. (2005). Laboratory detection and identification of Aspergillus species by micros-copic observation and culture: the traditional approach. Medical Mycology Supplement 1, 43, S125_/S128. 
  13. Nusrat H. M. (2012). Profitability of Flower Pro-duction and Marketing System of Bangladesh. Bangladesh J. Agril. Res., 37(1), 77-95. https://doi.org/10.3329/bjar.v37i1.11179  
  14. R. Ramjegathesh and E.G. Ebenezar, (2012). Mor-phological and Physiological Characters of Alter-naria alternata Causing Leaf Blight Disease of Onion. International Journal of Plant Pathology, 3, 34-44. https://doi.org/10.3923/ijpp.2012.34.44 
  15. Rebecca L J, Dhanalakshmi V, Sharmila S, Susi-thra G, Kumar S and Bala S. (2012). Isolation, identification and characterization of fungi from rhizosphere soil of Barleria Cristata. Inter J Hort Crop Sci Res., 2, 1-6.
  16. Sharif IH, Haque MA, Jamal MAHM, and Uddin ME. (2019). Assessment and biomonitoring of the effect of rapeseeds oil on wister rat organs. Am. J. Pure Appl. Sci., 1(4), 20-29. https://doi.org/10.34104/ajpab.019.0192029 
  17. Shahen MZ, Uddin ME and Alam MS. (2019). Effect of antibiotic susceptibility and inhibitory activity for the control of growth and survival of microorganisms of extracts of C. officinalis, Eur. J. Med. Health Sci. 1(1), 1-9. https://doi.org/10.34104/ejmhs.0190109 
  18. Soni RK and Sharma K. (2014). Isolation, Scree-ning and Identification of Fungi from Soil. Inter J Sci Res., 3, 472-473.
  19. Sultana N. (2003). Floriculture exports from Bangladesh. A paper presented in International Floriculture Conference on 6th November, 2003, BARC, Farmgate, Dhaka.

Article Info:

Academic Editor

Dr. Phelipe Magalhães Duarte, Professor, Department of Veterinary, Faculty of Biological and Health Sciences, University of Cuiabá, Mato Grosso, Brazil.

Received

March 1, 2022

Accepted

April 1, 2022

Published

April 15, 2022

Article DOI: 10.34104/ajpab.022.036040

Corresponding author

Md. Khasrul Alam**

Associate Professor,  Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh and Authors are contributed equally.

Md. Rezuanul Islam*

Professor, Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh.

Cite this article

Rahman S, Alam MK, Ahmed MS, Hossen MM, Jamal MAHM, and Islam MR. (2022). Isolation and identification of fungal pathogens from five flowering plants in Jashore region of Bangladesh. Am. J. Pure Appl. Sci., 4(2), 36-40. https://doi.org/10.34104/ajpab.022.036040 

Views
273
Download
547
Citations
Badge Img
Share