MYCOFLORA ASSOCIATED WITH LEMON (CITRUS LEMON ) FRUTS IN STOTAGE
A total of six species of fungi were isolated from lemon fruits Citrus lemon (L.) Brum. f. during the period of April 2013 to December 2013. The isolated fungi were Aspergillus niger, Candida krusei, Fusarium sp., Rhizopus stolonifer, Penicillium digitatum and Trichoderma sp. Prevalence of P. digitatum and Trichoderma sp. was 100%. Prevalence of C. krusei, Fusarium sp, A. niger and R. stolonifer was respectively 66.60%, 53.3, 46.66 and 13.33%. Pathogenicity of each associated fungus was determined following detached fruit inoculation technique. All the associated fungi were found to be pathogenic to cause fruits rot.
Lemon (Citrus limon) is widely cultivated throughout Bangladesh. Botanically, the citrus plant belongs to the family of Rutaceae, in the genus, Citrus (Julia 1987). Fruit of lemon plays a vital role in human nutrition by supplying the necessary growth factors such as vitamins and essential minerals in human daily diet and helps to keep a good health. Its ellipsoidal yellow fruit is used for culinary and non-culinary purposes throughout the world, primarily for its juice, which has both culinary and cleaning uses. The pulp and rind (zest) are also used in cooking and baking. The juice of the lemon contains 5 to 6% citric acid, which gives a sour taste. The distinctive sour taste of lemon juice makes it a key ingredient in drinks and foods such as lemonade and lemon meringue pie. The fruit, juice, and peel are used to make medicine. Lemon is used to treat scurvy, a condition caused by not having enough vitamin C. (Andrea 2007).
Lemon is also used for the common cold and flu, ringing in the ears (tinnitus), Meniere’s disease, and kidney stones. It is also used to aid digestion, reduce pain and swelling (inflammation), improve the function of blood vessels, and increase urination to reduce fluid retention.
Considerable research has been done on ethnobotanical and medicinal properties of the plant and fruit (Kristina et al. 2008, Ghani 2003, Ahmed et al. 2009). Research reports on fungal diseases of lemon fruits in market is limited (El-Sayed et al. 2013, El-Gali and Ibrahim 2014). El-Sayed et al. (2013) reported Alternaria citri, Botryodiplodia theobromae, Fusarium sp., Penicillium digitatum and Penicillium italicum on lemon from Egypt. In Libya El-Gali and Ibrahim (2014) worked on lemon and found Alternaria alternata (Fr.) Keissler pathogenic to the fruit.
The present piece of research work was undertaken to identify the fungi associated with lemon fruits in market and their pathogenic potentiality to cause fruit rot disease.
Fresh fruit samples of lemon were collected from different markets of Dhaka city and stored in a refrigerator at 4C. Some of the fruits showed symptoms of fungal infestation after 5 to 7 days of storage. The fungi associated with the rotted fruits were isolated following tissue plating method on potato dextrose agar (PDA) medium. The pH of the medium was adjusted to 6.0. Infected tissues of the fruits were placed on PDA in Petri plates and incubated at 25-28C (Anon. 1968, Mian 1995). Fungi grew from the tissues were transfer to fresh PDA plates. Pure culture of the isolated fungi were prepared and preserved in a refrigerator at 4C for further use. The fungal isolates were identified based on morphological characteristics recorded under a compound light microscope using standard literature (Barnett and Hunter 2000, Booth 1971, Ellis 1971, Ellis 1976, Ellis and Ellis 1998).
To perform pathogenicity test, healthy and matured citrus fruits were collected, surface sterilized with 1.0% chlorox and rinsed with sterilized distilled water. Two holes were made on the sterilized surface of fruits with a sharp cork borer (5mm diameter) at a depth of 4 mm.
To prepare inocula, 5 mm mycelium blocks were cut from the young culture of the isolated fungi with a 5 mm cork borer. For inoculation, the blocks of each fungus were placed inside the hole with a sterilized scalpel at one block per hole. In the controls, 5 mm fresh PDA blocks were placed inside the holes of lemon. Three replications were maintained for each treatment as well as control. Ten fruits were used per replications. Inoculated fruits were incubated at 25 C. Inoculated fruits were observed regularly and characteristic symptoms of fruit rot appeared on inoculated fruits after 5-7 days of incubation. The fungi isolated from naturally infected fruits were also isolated from artificially inoculated fruits.
During the pathogenicity test, the extent of damage due to rot caused by each fungi was estimated using a standard procedures (Shamsi et al. 2010).
RESULTS AND DISCUSSION
Fruits collected from the market and stored in a refrigerator at 4C were infected with fungi causing rots. A total of six species of fungi were isolated from infected fruits. The isolated and identified fungi were Aspergillus niger, Candida krusei, Fusarium sp., Rhizopus stolonifer, Penicillium digitatum and Trichoderma sp. (Plate I). In pathogenicity tests performed following detached fruit technique, all the isolated fungi infect citrus fruits causing fruit rot. Their symptoms were the same as observed after storage in at 4C (Plate II). The prevalence of P. digitatum and Trichoderma sp. was 100% and that of C. krusei, Fusarium sp., A. niger Rhizopus stolonifer was respectively 66.66, 53.30, 46.66 and 13.33% (Fig. 1).
Ezeibekw and Unamba (2009) reported four pathogens on lemon. These were identified as Phytophthora citrophthora, Botryodiplodia theobromae (Pat), F. oxysporum and F. equiseti. They found that the rot caused by B. theobromae was highly severe followed by P. citrophthora rot, F. oxysporum rot and F. equiseti rot. Adisa and Fajola (1982) reported P. digitalum and P. citrinum as pathogens of citrus fruit rots in Nigeria. They also considered B. theobromae as the most important fruit pathogen in South Western Nigeria. Nzekwe (1996) found out that Fusarium spp, Curvularia spp., Aspergillus spp. and Penicillum spp. cause citrus fruit rots in Abia State of Nigeria. Nosheen et al. (2013) studied fungal association of stored citrus fruits collected from cold storage houses of major citrus growing areas of the Punjab, Pakistan and reported association of Aspergillus niger, A. flavus, A. fumigatus, A. terreus, Penicillium verrucosum, Rhizopus arrhizus, R. stolonifer, A. parasiticum, Fusarium oxysporum, P. citrinum, A. awamorii, Alternaria alternata, F. solani and Mucor sp. from fruit samples. They found that prevalence of P. italicum was 29.75% and that of A. niger was 14.87% and Dreshlera sp. was only 0.82%.
-Sayed et al. (2013) reported that in Egypt citrus fruits are attacked by a number of fungal pathogens namely Alternaria citri, Botryodiblodia theobromae, Fusarium sp., Penicillium digitatum and Penicillium italicum from bloom to harvest causing fruit rot.
Results of the present piece of research reveal that A. niger, C. krusei, Fusarium sp., R. stolonifer, Penicillium digitatum and Trichoderma sp. are common fungal pathogens of citrus fruit rot in storage. Association of A. niger, Fusarium sp., R. stolonifer and P. digitatum has also been reported as causal fungi of citrus fruit rot throughout the world. Citrus fruit rot caused by Candida krusei and Trichoderma sp. has been reported first time from Bangladesh.
Adisa, V. A and Fajola, A. O. 1982. Post harvest fruit rot of three species of citrus in South Western Nigeria. Indian Phytopath. 35(4):595-603.
Andrea, L. 2007. Fruit of the lemon- A Novel. Picador. USA. 352 pp.
Anonymous. 1968. Plant pathologists Pocket Book. The Commonwealth Mycological Institute, Kew Surrey, England. 267 pp.
Anonymous 2015 From Wikipedia, the free encyclopedia. https://en.wikipedia.org/wiki/Lemon.
Barnett, H. L. and Hunter, B. B. 2000. Illustrated Genera of Imperfect Fungi. (4th edn.), Burgess Pub. Co. Minneapolis. 218 pp.
Booth, C. 1971. The Genus Fusarium. The Commonwealth Mycological Institute. England 273 pp.
El-Sayed M., Embaby, M. Hazaa, Laila, F. Hagag, Talaat EL-Sayed Ibrahimd and Faten S. Abd El-Azem. 2013. Decay of Some Citrus Fruit Quality Caused by Fungi. J. Appli. Sci. Res. 9(11):5920-5923.
Ellis, M. B. 1971. Dematiaceous Hyphomycetes. The Commonwealth Mycological Institute, England, 608 pp.
Ellis, M. B. 1976. More Dematiaceous Hyphomycetes. The Commonwealth Mycological Institute, England. 507 pp.
Ellis, M. B. and J. P. Ellis. 1997. Micro fungi on Land plants. An Identification Handbook. 86 pp.
Ezeibekwe I. O and Unamba C. I. N. 2009. Pathogens Associated with Citrus Fruit Rots in Imo state of Nigeria. J. American Sci. 5(4):129-132.
Julia, F. M. 1987. Lemon in Fruits of Warm Climates. Purdue University. 160-168 pp.
Kristina, L., Penniston, Stephen, Y., Nakada, Ross, P., Holmes and Assimos D. G. 2008. Quantitative Assessment of citric acid in Lemon Juice, Lime Juice, and Commercially Available Fruit Juice Products. J.Endo.. 22(3): 567-570.
Mian, I. H. 1995. Methods in Plant Pathology. Published by IPSA-JICA Project. IPSA, Salna Gazipur.
Nosheen, A., Anjum, T. and. Jabeen, R. 2013. Isolation and Identification of storage fungi from citrus Samples from major growing areas of Punjab, Pakistan. Intl. J. Agric. Biol. 15(6): 1283.
Nzekwe, 1996. Diseases of Citrus sinensis fruit. Project work, Department of Botany, Abia State University. Pp. 11-29.
Shamsi, S., Nahar, N and Momtaz, S. 2010. First report of Lasiodiplodia pod rots disease of cacao-(Theobromae cacao L.) form Bangladesh. Bangladesh J. Plant Pathol. 26(1&2):81-82.