Relative Production of Lipase and Chitinase Enzymes Isolated from Cyst Nematode in Sugar Beet in Vitro

Document Type : Research Article

Authors

Ferdowsi University of Mashhad

Abstract

Introduction: Sugar beet is adaptable to be cultivated in different weather conditions and regions. In 2015, the overall area for sugar beet cultivation was estimated about 105,000 hectares which 19000 of this was in Khorasan Razavi province. Sugar beet is considered as a valuable agricultural crop both for economy and employment. Nevertheless, producing this crop faces many challenges including the high number of pests. One of these pathogenic factors is the nematodes. Among plant-parasitic nematodes cyst nematodes are a large group with economic importance in different countries. These nematodes cause much damage to agricultural crops. Among the different cyst nematode genera Globodera and Heterodera have species which are important due to economic damage.
Materials and Methods: During the year of 2016, 22 samples of soil and roots of sugar beet in cyst nematodes contaminated field in Khorasan Razavi were gathered. Cyst nematodes were extracted by the use of a small clip and a binocular and put in petri dish with some water. White materials were also taken from the root by a delicate needle and put in sterile distilled water after washing. Separating and purifying fungi were done in 3 parts: separating fungi from cysts and materials, separating fungi from eggs and larvae nematode, and making single spore fungi and pure culture. The cysts and separated materials were washed by distilled water for several times and antisepticised for 1, 2 or 3 minutes in 10% Sodium Hypochlorite, and 10% and 20% Ethyl alcohol. Cysts and materials were washed again with sterile distilled water and sterile sifter was used. The cysts and materials in petri dish containing PCA, PDA, CMA and MEA culture mediums were separately taken and cultured by a needle under laminar. Four cysts were placed at the 4 sides of each 8cm petri dish containing the above mentioned culture mediums. 16 brown cysts from each soil sample were cultured. Two petri dishes from each sample were kept and checked consistently in two hot and cold temperatures in incubator with 20-25 and incubator with 8-10. After 7-14 days the grown fungi were taken to a new culture medium of PDA for a better development and in later stages they were purified on WA culture medium by single spore or hyphal tip methods. Purified fungi were kept in test tubes containing PCA food environment and 4 temperature and also on sand for later studies. Cyst shells were destructed by cyst crusher (homogenizer) and their eggs and mash were released. Released eggs from cysts were formed into suspension in sterile water. 0.5-1 ml of suspension of eggs and larvae were taken by sterile micropipette and diffused on petri dish containing water agar culture medium. These culture mediums were kept and checked regularly in dark in the incubator with 20-25. Grown fungi from these eggs and larvae were taken to a new culture medium and purified by single spore and hyphal tip methods. WA culture medium was used for single spurring and purifying fungi. PCR based methods: morphological and molecular identification, were used to identify the fungi isolates. Mycelium fungi growth phases, fungi DNA extraction, PCR reaction and electrophoresis were done to identify molecular fungi. Lipase and chitinase assays were performed on the isolates.
Results and Discussion: For Lipase test, each fungal isolate was cultured on peptone agar media and after 7 days isolates were examined. Around the colony of the isolates produced by the lipase enzyme formed a precipitate or a colorless aura, which is due to the formation of calcium salts from free lauric acid by the lipase enzyme, which indicates the positive activity of lipase. Among the isolates, Colletotrichum gloeosporioides had the highest sediment content, indicating the activity of lipase enzyme and the highest isolate in this test. After that, Neonectria macrodidyma and Penicillium chrysogenum showed the highest activity of lipase enzyme activity. Fusarium oxysporum showed no sediment and the lowest level of lipase enzyme activity was observed in this fungus. For chitinase test, isolates were cultured on colitic kitein medium. Around the colony and also the color of the culture medium, the chitinase-producing isolates changed the violet color, which indicates the positive activity of the chitinase enzyme in these isolates. Relative analysis of chitinase activity showed that the isolate of P. chrysogenum had the largest and fastest change in color to violet, indicating the highest production of chitinase enzyme by this fungus. The lowest chitinase production was by C. gloeosporioides, which showed the slightest and slowest changes in color compared with other isolates and the weakest isolate was introduced for the production of chitinase enzyme.
Conclusion: In this research, different fungi were isolated from sugar beet cystc. Most fungal isolates belonged to Torbat-e-Haidiriyah and Fariman, and the least isolates belonged to Khaf. The highest frequency of Fusarium isolates was found to be 37.35%. Isolation of Simplicillium lanosoniveum fungi, P. chrysogenum, C. gloeosporioides was the first reported cyst nematode in sugar beet. In the relative analysis of lipase activity, it was found that P. chrysogenum and C. gloeosporioides fungi exhibited the highest amount of lipase production, which was the highest marker in this test. Relative analysis of chitinase activity showed that P. chrysogenum had the largest and fastest color change to purple, indicating the highest production of chitinase enzyme by this fungus. The lowest chitinase production was obtained by isolate C. gloeosporioides, which showed the slightest and slowest changes in color compared with other isolates. The fungus P. chrysogenum showed the best results in both tests, this fungus is, therefore, recommended for further research with the observation of the necessary points.

Keywords


Agrawal T., and Kotasthane A.S. 2012. Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India. SpringerPlus 1(1): 73.
2- Arbabi M. 2014. Sugar Beet Handbook.
3- Bailey J.A. 1992. Colletotrichum; biology, pathology and control.
4- Benitez T., Rincon A.M., Limon M.C., and Codon A.C. 2004. Biocontrol mechanisms of Trichoderma strains. International Microbiology 7(4): 249-260.
5- Bird A.F., and McClure M. 1976. The tylenchid (Nematoda) egg shell: structure, composition and permeability. Parasitology 72(1): 19-28.
6- Brown R., and Kerry B. 1987. Principles and practice of nematode control in crops, Academic Press.
7- Chaverri P., Salgado C., Hirooka Y., Rossman A., and Samuels G. 2011. Delimitation of Neonectria and Cylindrocarpon (Nectriaceae, Hypocreales, Ascomycota) and related genera with Cylindrocarpon-like anamorphs. Studies in mycology 68: 57-78.
8- El-Sayed S., and Mahdy M. 2015. Effect of chitosan on root-knot nematode, Meloidogyne javanica on tomato plants. International Journal of ChemTech Research 7(4): 1985-1992.
9- Ershad J. 2009. Iranian Mushrooms.
10- Evans K., and Rowe J. 1998. Distribution and economic importance. The cyst nematodes, Springer: 1-30.
11- Fenwick D. 1940. Methods for the recovery and counting of cysts of Heterodera schachtii from soil. Journal of Helminthology 18(4): 155-172.
12- Frisvad J.C., and Samson R.A. 2004. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Studies in Mycology 49(1): 1-174.
13- Harman G.E., Howell C.R., Viterbo A., Chet I., and Lorito M. 2004. Trichoderma species—opportunistic, avirulent plant symbionts. Nature reviews Microbiology 2(1): 43.
14- Hoseney R.C. 1994. Principles of cereal science and technology, American Association of Cereal Chemists (AACC).
15- Howell C. 2003. Mechanisms employed by Trichoderma species in the biological control of plant diseases: the history and evolution of current concepts. Plant disease 87(1): 4-10.
16- Jackson A., Walters D., and Marshall G. 1994. Evaluation of Penicillium chrysogenum and its antifungal extracts as potential biological control agents against Botrytis fabae on faba beans. Mycological Research 98(10): 1117-1126.
17- Jafarpoor B.S., and Jahanbakhsh V.M. 2012. Laboratory Methods in Plant Pathology.
18- Jatala P. 1986. Biological control of plant-parasitic nematodes. Annual review of phytopathology 24(1): 453-489.
19- Kavari M., Rouhani H., and Mahdikhani Moghaddam E. 2014. Investigating the relationship between the enzyme's ability of several isolates from Trichoderma fungi with biocontrol ability of this fungus against tomato root knot nematodes. Master's Thesis.
20- Kerry B., and Crump D. 1977. Observations on fungal parasites of females and eggs of the cereal cyst-nematode, Heterodera avenae, and other cyst nematodes. Nematologica 23(2): 193-201.
21- Khezri Nejad N., Gusta E., and Niknam G. 2006. Introducing fungi with sugar beet cyst nematode from western Azarbaijan fields (1). Rostaniha,7.
22- Khezri Nejad N., Gusta E., and Niknam G. 2009. Introducing fungi with sugar beet cyst nematode from western Azarbaijan fields (2). Journal of Sustainable Agricultural Science, Vol. 19/1.
23- Leslie J., and Summerell B. 2006. The Fusarium Laboratory Manual Blackwell Publishing. Ames, Iowa.
24- Mahdikhani Moghadam E., Rouhani H., and Rastgar Fallahi M. 2009. Biological control of Heterodera schachtii sugar beet nematode by Trichoderma fungus in laboratory and greenhouse. Journal of Water and Soil Science, Agricultural and Natural Resources Sciences- Isfahan University of Technology 13: 301-312.
25- Mireke K., Abdollahi Dehdari K., and Ashcbus. 2013. The reaction of eight tomato cultivars to the rootstock nematode producing Meloidogyne javanica in greenhouse conditions. Iranian Plant Protection Sciences.
26- Mokaram Hesar A. 2010. "Identification of heterodera species in sugar beet fields and Meloidogyne species in tomato fields of Khorasan Razavi province using morphological and molecular techniques." Master's thesis.
27- Mouchacca J. 1973. Deux Alternatria des sols arides d'Égypte: A. Chlamydosporum sp. nov. et A. Phragmospora Van Emden. Mycopathologia 50(3): 217-225.
28- Nigh E.A., Thomason I.J., and Van Gundy S. 1980. Identification and distribution of fungal parasites of Heterodera schachtii eggs in California. Phytopathology 70(9): 885-889.
29- Nonaka K., Kaifuchi S., Ōmura S., and Masuma R. 2013. Five new Simplicillium species (Cordycipitaceae) from soils in Tokyo, Japan. Mycoscience 54(1): 42-53.
30- Pitt J.I. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces.
31- Sasser J.N. 1989. Plant-parasitic nematodes: the farmer's hidden enemy. Plant-parasitic nematodes: the farmer's hidden enemy.
32- Statistics A.S., Publications 2014. Ministry of Agriculture.
33- Sunitha V., Nirmala Devi D., and Srinivas C. 2013. Extracellular enzymatic activity of endophytic fungal strains isolated from medicinal plants. World Journal of Agricultural Sciences 9(1): 1-9.
34- Sutton B.C. 1980. The Coelomycetes, Fungi Imperfecti with Pycnidia. Acervuli and stromata: 470-472.
35- Thiagarajan V., Revathi R., Aparanjini K., Sivamani P., Girilal M., Priya C., and Kalaichelvan P. 2011. Extracellular chitinase production by Streptomyces sp. PTK19 in submerged fermentation and its lytic activity on Fusarium oxysporum PTK2 cell wall. Int. J. Curr. Sci. 1: 30-44.
36- Whitehead A. 1986. Problems in the integrated control of potato cyst-nematodes, Globodera rostochiensis and G. pallida, and their solution. Aspects of applied Biology.