İn Vitro Kültür Tekniği Kullanılarak Arbusküler Mikorhizal Fungus (AMF) Glomus intraradices’in Yaşam Döngüsü’nün İncelenmesi

Yıl 2016, Cilt: 26 Sayı: 2, 161 - 167, 31.05.2016

Younes Rezaee Danesh Solmaz Najafı Semra Demır

Öz

Bu çalışmada, İran’ın Damghan bölgesinde bulunan buğday bitkisinin rizosfer bölgesinden izole edilen ve havuç (Daucus carota L.) bitkisinin saçak köklerine inokule edilen arbusküler mikorhizal fungus Glomus intraradices’in laboratuvar koşullarında yaşam döngüsünün incelenmesi amaçlanmıştır. Çalışmada AMF inokulumu olarak mikorhizal spor ve kökler kullanılmıştır. İnokulasyon ile birlikte yüzey sterilizasyonun yapılmasından 3-5 gün sonra, havuç bitkisinin saçak köklerinde spor çimlenmesi gözlenmiştir. İnokulasyondan 2-10 gün sonra ise fungal gelişimin olduğu kaydedilmiştir. Fungal çimlenmede, hifler dallanarak, 2.5 mm çapında kök şeklinde ağ oluşturmuşlardır. Fungus miselyumu ve bitki kökleri arasındaki ilk temas, fungus sporunun çimlenmesinden 1-3 gün sonra meydana gelmiştir. Fungus-konukçu temasından 7 gün sonra ise büyüklükleri dışında (20-30 µm çapında) diğer özellikleri gerçek sporlara benzeyen spor ve vesikel benzeri yapılar gözlenmiştir. İlk gerçek spor oluşumu, fungus-konukçu temasından 25 gün sonra gerçekleşmiş, daha sonra sayıları katlanarak artmıştır. Bu sporların rengi başlangıçta şeffaf ve beyazımsı iken daha sonra sarımsı kahverengine dönmüştür. İnokulasyondan 12 hafta sonra da her bir petrideki spor sayısı 1000-2500 olarak kaydedilmiştir.

Using In Vitro Culturing Technique for Studying Life Cycle of Arbuscular Mycorrhizal Fungus (AMF) Glomus intraradices

Öz

In this study, life cycle of arbuscular mycorrhizal fungus, Glomus intraradices isolated from wheat rhizospheres of Damghan region in the Iran was observed using carrot (Daucus carota L.) transformed hairy roots in vitro. Mycorrhizal spores and roots were used as inoculum. Spore germination was observed 3-5 days after surface sterilization as well as co inoculation with transformed roots. Fungal growth was also recorded 2-10 days after inoculation. Fungal germinating hyphae branched and produced radical shape network 2.5 mm in diameter. The first contact between fungus mycelium and roots occurred 1-3 days after germination. 7 days after fungus-host contact, several secondary spores or vesicle like structures observed were similar to true spores except of their size (20-30 µm diameter). The first true spore formed 25 days after contact and then number of spores increased exponentially. These spores were hyaline and whitish at first but then turned to brownish yellow. After 12 weeks, 1000-2500 spores could be recorded in each plate.

Kaynakça

• Bakhtiar Y, Iller D, Cavagnaro T, Smith S. (2001). Interactions between two arbuscular mycorrhizal fungi and fungivorous nematodes and control of the nematode with fenamifos. Appl. Soil. Ecol. 17: 107-117.
• Becard G, Fortin JA. (1988). Early events of vesicular arbuscular mycorrhiza formation on Ri T-DNA transformed roots. New Phytol. 108: 211-218.
• Becard G, Piche Y. (1989). Fungal growth stimulation by CO2 and root exudates in vesicular-arbuscular mycorrhizal symbiosis. Appl. Environ. Microbiol. 55: 2320–2325.
• Bethelenfalvay GJ, Schuepp H. (1994). Arbuscular mycorrhizas and agrosystem stability, pp. 171-131, In: Impact of Arbuscular Mycorrhizas in Sustainable Agriculture and Natural Ecosystems, Gianinazzi S and Schuepp H (eds), Birkhauser Verlag, Basel.
• Bever JD, Morton JB, Antonovics J, Schultz PA. (1996). Host dependent sporulation and species diversity of arbuscular mycorrhizal fungi in a mown grassland. J. Ecol.
• : 71-82.
• Bever JD, Schultz PA, Pringle A, Morton JB. (2001). Arbuscular mycorrhizal fungi: more diverse than meets the eye, and the ecological tale of why. Bioscience. 51: 923-931.
• Bever JD. (2002). Negative feedback within a mutualism: host-specific growth of mycorrhizal fungi reduces plant benefit. In: Proceedings of the Royal Society of London, B, Biological Sciences 269: 2595-2601.
• Brundrett MC, Abbott LK, Jasper DA. (1999). Glomalean mycorrhizal fungi from tropical Australia. I. Comparison of the effectiveness and specificity of different isolation procedures. Mycorrhiza. 8: 305-314.
• Chabot S, Becard G, Piche Y. (1992). Life cycle of Glomus intraradix in root organ culture. Mycologia. 84: 315-321.
• Danesh YR, Goltapeh EM, Alizadeh A, Modares Sanavi M. (2006). Optimizing carrot hairy root production for monoxenic culture of arbuscular mycorrhizal fungi in Iran. J. Biol. Sci. 6(1): 87-91.
• de Souza FA, Berbara RLL. (1999). Ontogeny of Glomus clarum in Ri T-DNA transformed roots. Mycologia. 91: 343–350.
• Declerck S, Cranenbrouck S, Dalpe Y, Seguin S, Grandmougin-Ferjani A, Fontaine J, Sancholle M. (2000). Glomus proliferum sp. nov.: a description based on morphological, biochemical, molecular and monoxenic cultivation data. Mycologia. 92: 1178–1187.
• Declerck S, Strullu DG, Plenchette C. (1996). In vitro mass production of the arbuscular mycorrhizal fungus, Glomus versiforme, associated with Ri T-DNA transformed carrot roots. Mycol. Res. 100: 1237-1242.
• Declerck S, Strullu DG, Plenchette C. (1998). Monoxenic culture of the intraradical forms of Glomus sp. isolated from a tropical ecosystem: a proposed methodology for germplasm collection. Mycologia. 90: 579-585.
• Douds D, Becard G. (1993). Competitive interaction between Gigaspora margarita and Gigaspora gigantea in vitro. In: Proceedings of the Ninth North American Symposium on Mycorrhizae, Guelph, ON, Canada, August 8-12.
• Douds DD, Schenck NC. (1990). Increased sporulation of vesicular arbuscular mycorrhizal fungi by manipulation of nutrient regimes. App. Environ. Microbiol. 56: 413-418.
• Fortin JA, Becard G, Declerck S, Dalpe Y, St. Arnaud M, Coughlan AP, Piche Y. (2002). Arbuscular mycorrhiza on root-organ cultures. Can. J. Bot. 80(1): 1-20.
• Gaspar L, Pollero RJ, Cabello MN. (1994). Triacylglycerol consumption during spore germination of vesicular-arbuscular mycorrhizal fungi. J. Am. Oil. Chem. Soc. 71(4): 449-452.
• Gemma JN, Koske RE. (1988). Seasonal variation in spore abundance and dormancy of Gigaspora gigantea and in mycorrhizal inoculum potential of a dune soil. Mycologia. 80: 211-216.
• Gerdemann GW, Nicolson TH. (1963). Spore of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Brit. Mycol. Soc. 46: 235- 244.
• Giovannetti M, Sbrana C, Avio L, Citernesi AS, Logi C. (1993). Differential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre-infection stages. New Phytol. 125: 587–593.
• Giovannetti M, Sbrana C. (1998). Meeting a non-host: the behaviour of AM fungi. Mycorrhiza. 8: 123–130.
• Gunasekaran P, Sundaresan P, Ubalthoose N, Lakshmanan M. (1987). Effect of pH, temperature and nutrients on the germination of a vesicular arbuscular mycorrhizal fungus, Glomus fasciculatum in vitro. In: Proceeding of Indian AS-Plant Science 97: 231-234.
• Hardie K. (1984). Germination of Glomus mosseae spores isolated from stock pots of different ages. Trans. Brit. Mycol. Soc. 83: 693-696.
• Hepper CM. (1984). Isolation and culture of VA mycorrhizal (VAM) fungi. pp. 95-112, In: VA Mycorrhiza, Powell CL and Bagyaraj DJ (eds). CRC Press, Boca Raton, FL.
• Hewitt EJ. (1966). Sand and water culture methods used in the studies of plant. Nutrition Technical Communications, Vol. 22. Commonwealth Agricultural Bureau, London, United Kingdom, p. 430–434.
• Jenkins WR. (1964). A rapid centrifugal technique for separating nematodes from soil. Pl. Dis. Rep. 48: 692.
• Klironomos JNN, Hart MM. (2002). Colonization of roots by arbuscular mycorrhizal fungi using different sources of inoculum. Mycorrhiza. 12: 181-184.
• Mosse B. (1962). The establishment of vesicular arbuscular mycorrhiza under aseptic conditions. Gen. Microbiol. 27: 509-520.
• Mugnier J Mosse B. (1987). Vesicular-arbuscular mycorrhizal infections in transformed Ri T-DNA roots grown axenically. Phytopath. 77: 1045-1050.
• Nagahashi G, Douds D, Buee M. (2000). Light-induced hyphal branching of germinated AM fungal spores. Pl. Soil. 219(1-2): 71-79.
• Paula MA, Reis VM,. Dobereiner J. (1991). Interactions of Glomus clarum with Acetobacter diazotrophicus in infection of sweet potato (Ipomoea batatas), sugarcane (Saccharum spp.) and sweet sorghum (Sorghum vulgare). Biol. Fert. Soils. 11: 111-115.
• Philips JM, Hayman DS. (1970). Improved procedures clearing root and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infections. Trans. Brit. Mycol. Soc. 55: 158-161.
• Sancholle M, Dalpe Y, Grandmougin-Ferjani A. (2001). Lipids of mycorrhizae. pp. 63–93. In: The Mycota IX fungal associations. Hock B (ed). Springer-Verlag, Berlin and Heidelberg.
• Sheikh NA, Sanders FE. (1988). Effect of temperature on germination of mycorrhizal spores and infection in roots. Biologia. 34: 223-236.
• Siqueira JO, Sylvia DM, Gibson J, Hubbell DH. (1985). Spores, germination and germ tubes of vesicular-arbuscular mycorrhizal fungi. Can. J. Microbiol. 31: 965-972.
• Smith SE, Read DJ. (1997). Mycorrhizal Symbiosis, 2nd ed., Academic Press, London.
• St Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA. (1996). Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots. Mycol. Res. 100: 328–332.
• Strullu DG, Romand C. (1986). Methode d’obtention d’endomycorhizes à vésicules et arbuscules en conditions axéniques. C.R. Acad. Sci. 303: 245–250.
• Sylvia DM, Schenck NC. (1983). Application of superphosphate to mycorrhizal plants stimulates sporulation of phosphorus-tolerant vesicular-arbuscular mycorrhizal fungi. New Phytol. 95: 655-661.
• Tommerup IC. (1983). Spore dormancy in vesicular-arbuscular mycorrhizal fungi. Trans. Brit. Mycol. Soc. 81: 37–45.
• Tsai SM, Phillips DA. (1991). Flavonoids released naturally from alfalfa promote development of symbiotic Glomus spores in vitro. Appl. Environ. Microbiol. 57: 1485–1488.