Nitrogen-fixing cyanobacteria in soils of Vojvodina province and their ultrastructural and genetic characterization

U radu je ispitana zastupljenost azotofiksirajućih cijanobakterija, ukupnog broja algi i ukupnog broja bakterija u različitim tipovima zemljišta na jedanaest lokaliteta u Vojvodini, od čega se sedam nalaze u zaštićenim prirodnim dobrima. Ispitana je brojnost u zavisnosti od dubine pedološkog profila, kao i od godišnjeg doba. Izvršena je izolacija sojeva azotofiksirajućih cijanobakterija, određena njihova taksonomska pripadnost i osnovne citološke karakteristike. Ispitana je ultrastruktura vegetativnih ćelija, heterocista i spoljnih struktura na ćelijama fimbrije i pili, transmisionim elektronskim mikroskopom. Izvršena je genetička karakterizacija izolovanih sojeva azotofiksirajućih cijanobakterija PCR metodom analizom STRR fragmenata DNA. Brojnost azotofiksirajućih cijanobakterija i ukupna brojnost algi je bila znatno veća kod hidromorfnih i halomorfnih zemljišta, nego kod automorfnih zemljišta. Najveća prosečna godišnja brojnost azotofiksirajućih cijanobakterija, u površinskom sloju 0-5cm, je utvrđena kod zemljišta fluvisol u SRP “Koviljsko petrovaradinskom ritu”, 150864 jedinki po gramu apsolutno suvog zemljišta. Zemljište sa najnižom brojnošću azotofiksirajućih cijanobakterija, je gajnjača u NP Fruška gora, 1582 jed./gr zemljišta u površinskom sloju. Kod svih ispitivanih zemljišta brojnost azotofiksirajućih cijanobakterija je bila najveća u površinskom sloju zemljišta, 0-5 cm dubine, opadala je sa dubinom zemljišta i bila najmanja u najdubljem sloju, 30 - 60 cm. Kod najvećeg broja ispitivanih zemljišta brojnost azotofiksirajućih cijanobakterija je bila najveća tokom zimskog perioda. Azotofiksirajuće cijanobakterije su dominantne u našim zemljištima i zastupljene sa 56.27% u odnosu na druge grupe algi.Izolovano je 30 sojeva azotofiksirajućih cijanobakterija: 19 sojeva Nostoc-a, 4 soja Anabaena, 4 Cylindrospermum, i po jedan soj Calothrix, Tolypothrix i Phormidium. Prosečna zastupljenost heterocista, ćelija koje vrše azotofiksaciju, kod roda Nostoc je iznosila 8.28%, Anabaena 4.25%, Cylindrospermum-a 2.93%, Calotrix elenkinii 6.19% i Tolypothrix 7.76%. Ultrastrukturnim ispitivanjem, TEM mikroskopom, vegetativnih ćelija azotofiksirajućih cijanobakterija uočili smo inkluzije redovnog pojavljivanja: karboksizome (Cs), cijanoficinkse granule (CG), polifosfatne granule (PG), ribozome (R), lipidne granule (ß –granule) i tilakoide (T), kao i inkluzije neredovnog pojavljivanja: membranom ograničene kristalne inkluzije. Koristeći TEM i tehniku bojenja ćelija sa RR i ultratankih preseka utvrdili smo prisustvo omotača od fimbrija kod tri soja (A.314, A.azollae i N.302) i tipične fimbrije kod dva soja (N.311 i N.9229). Metodom negativnog bojenja NS PTA uočili smo takođe tipične fimbrije, igličastog-dlakastog izgleda, jasnih granica niti kod tri soja (N.302, N.7901 i N.9229), međutim uočili smo i atipične sluzne fimbrije, koje nemaju jasno izražene granice, ali su veoma moćno raširene oko vegetativnih ćelija, kod tri soja (A.314, A.azollae, N.311). Kod simbiotskih-infektivnih sojeva N.7901 i N.9229 javljaju se samo tipične fimbrije iz prve klase, a kod diazotofnih sojeva i simbiotskog - neinfektivnog soja A.azollae javljaju se atipične-sluzne fimbrije iz druge klase. Za ispitivanje sličnosti cijanobakterija metodom PCR-a pomoću STRR konzervativnih sekvenci DNA genoma korišćeno je 39 sojeva azotofiksirajućih cijanobakterija i kod 38 je utvrđeno njihovo prisustvo. Svi sojevi se mogu podeliti u tri grupe, klastera. Prvi klaster je najveći i obuhvata 24 soja i deli se na dva podklastera: Ia koji obuhvata 12 sojeva gde dominiraju sojevi Nostoc-a (8), i podklaster Ib koji obuhvata takođe 12 sojeva, od čega 6 sojeva pripada rodu Anabaena. Podklaster Ia i podklaster Ib pokazuju različitost od 90%. Sva tri simbiozna, infektivna, soja Nostoc-a se nalaze u klasteru I: N.7901, N.9229 i N. 8001. Svaki simbiozni soj Nostoc-a ima genetske sličnosti sa po jednim diazotrofnim sojem Nostoc-a izolovanim iz zemljišta Vojvodine. Klaster II obuhvata sedam (7) sojeva među kojima dominiraju sojevi Cylindrospermum-a, dok klaster III obuhvata 7 sojeva od čega 6 pripadaju rodu Nostoc, a jedan rodu Rivularia.Detaljno poznavanje svojstava izolovanih azotofiksirajućih cijanobakterija doprineće njihovoj budućoj primeni kako u proizvodnji ratarskih i povrtarskih kultura, tako i u biotehnološkoj proizvodnjiIn this study examined is the frequency of nitrogen-fixing cyanobacteria, total number of algae and total number of bacteria in different soil types on eleven localities in the Vojvodina Province. Seven out of those eleven localities are found in protected nature reserves. Actually, studied was the number of the cyanobacteria and algae depending on the depth of pedological characterization as well as on season. First, isolated were the types of nitrogen-fixing cyanobacteria, determined was their taxonomic origin and basic cytological characteristics. Also examined was the ultrastructure of vegetative cells, heterocysts and other outer structures on fimbriae and pili cells using TEM, transmission electron microscope. Finally, performed was the genetic characterization of isolated types of nitrogen-fixing cyanobacteria using the PCR method and analyzing STRR fragments of DNA. The presence of nitrogen-fixing cyanobacteria and total number of algae was significantly higher with hydromorphic and halomorphic soils than with authomorphic ones. Highest annual average number of nitrogen-fixing cyanobacteria in the topsoil (0-5 cm) was reported with fluvisol soil in Special Nature Reserve „Koviljsko petrovaradinski rit” (Swamp) and there were 150864 units of bacteria per gram of absolutely dry soil. The soil with the lowest presence of nitrogen-fixing cyanobacteria recorded was cambisol in National Park “Fruska gora” with 1582 units per gram of soil in the topsoil. With all the researched types of soils the number of nitrogen-fixing cyanobacteria was in the topsoil, 0-5 cm of depth and decreased in line with the depth of soil and lowest was at the deepest layer, 30-60cm. The highest frequency of nitrogen-fixing cyanobacteria was found during the winter season with most of the examined soils. Nitrogen-fixing cyanobacteria are the dominant type of bacteria in our soils and are presented with 56, 27% compared to other types of algae. 30 strains of nitrogen-fixing cyanobacteria were isolated: 19 types of Nostoc sp., 4 of Anabaena sp. and one in each genus of Calothrix, Tolypothrix and Phormidium. Using ultrastructural examination and TEM microscope when studying vegetative cells of nitrogen-fixing cyanobacteria observed were the inclusions of regular frequency: carboxysomes (Cs), cyanophycin granules (CG) , polyphosphate granules (PG), ribosomes (R), lipid granules (SS -granule ) and thylakoids ( T ) as well as the inclusion of irregular occurrence: a membrane-bound crystal inclusions. Using TEM technique and staining the cells with the RR and ultra thin cross section, we determined the presence of depletion of the fimbriae with three strains (A.314, A.azollae and N.302) and typical fimbriae with the two strains (N.311 and N.9229). Applying the method of negative staining NS PTA also noticed were a typical fimbriae, needle-hairy like looks with clear boundaries with the three strains (N.302, N.7901, N.9229). However, also observed were atypical mucous fimbriae, which do not have clearly expressed borders, but they are very strongly spread around the vegetative cells, with the three strains (A.314, A.azollae, N.311). With symbiotic - infective strains N.7901 and N.9229 only typical fimbriae of first class occurred, and in diazotroph strains and symbiotic – non infectious strain A. azollae found were atypical mucous fimbriae of second class. To test the similarity of cyanobacteria by PCR method and using a STRR - conservative DNA sequence of the genome used were 39 strains fixing cyanobacteria and with 38 established was their presence. All strains can be divided into three groups of clusters. The first cluster is the largest and covers 24 strains, and is divided into two subclusters: Ia which includes 12 strains,where predominant are Nostoc strains ( 8 ) , and subcluster Ib , which also implies 12 strains , out of which 6 strains belong to the genus Anabaena. Subcluster Ia and Ib show a difference of 90 %. All three symbiotic , infectious Nostoc strains are classified in a cluster I: N.7901, N.9229 and N. 8001. Each symbiotic Nostoc strain has a genetic similarity with one di-nitrogen Nostoc strain isolated from a lot of different soils in Vojvodina. Cluster II includes seven (7) strains , including strains among which the predominant are Cylindrospermum ones , while cluster III includes 7 strains of which 6 belong to the genus Nostoc and one to genus Rivularia. Detailed knowledge of the properties of isolated fixing cyanobacteria could contribute to their future application both in the production of field crops and vegetables, as well as in biotechnological production