The alleviation of reforestation challenges by beneficial soil microorganisms

Surface mining causes major destruction of natural landscapes and ecosystems. The most fertile, surface soil layer is lost permanently, together with vegetation, wildlife, and micro flora. Post-mining areas are characterized with diverse edaphic, topographic, hydrographic conditions, which complicate land restoration. Successful establishment of forest ecosystems on such land depends mostly on selection of tree species. The chosen plants must be capable of tolerating a wide range of acidity, fertility, moisture, and have potential to ameliorate such substrates for more demanding species. But, reforestation of heavily damaged ecosystems, such as post-mining areas, demands a new approach in seedlings production. This new approach takes into account specific requirements of habitat and integrates them into “targeted production of planting materialâ€. A good strategy for successful reforestation of post-mining areas is the input of organic matter (compost, mulch). Also, current knowledge and experiences emphasize the potential of beneficial microorganisms such as, mycorrhizal fungi (MF) and plant growth promoting rhizobacteria (PGPR). The majority of studies that deal with beneficial interactions between trees and microorganisms are focused on the mycorrhiza, while plant growth promoting rhizobacteria are less present in silviculture. In this study, the focus is on the reforestation challenges of two mining basins, Majdanpek and Kolubara and suggests beneficial microorganisms as potential solution. The study presents results of several years’ researches on plant response to the presence of mycorrhizal fungi and PGPR. The substrates used for plant growth were Majdanpek and Kolubara mine deposals. Mycorrhizal seedlings were grown in Majdanpek mine deposal, and at the end of the experiment they had 30% higher biomass in comparison to control (seedlings without mycorrhiza). Seedlings linked with fungi had a higher survival rate. Deposals from Kolubara Mining Basin were used as a substrate for seedlings inoculated with PGPR. In the first experiment, Scots pine and Norway spruce were inoculated with Azotobacter chroococcum, Bacillus megaterium, B. circulans, B. licheniformis, B. pumilus, B. amyloliquefaciens. Inoculation resulted with higher biomass production (Scots pine 43%, Norway spruce 34%). Similar results were obtained in the second experiment where Scots pine and black locust were inoculated with Bacillus licheniformis, Aeromonas hydrophila, Pseudomonas putida and Burkholderia cepacia. Both species had higher biomass (around 20%) in comparison to un-inoculated control. The results confirmed the fact that early establishment and successful growth of vegetation on devastated areas depends on the presence and activity of soil microbes. Microorganisms as a “nature’s solution†pose the potential to alleviate reforestation challenges of anthropogenic devastated landscapes. Their presence and activity is crucial for ecosystem stability. In areas with compromised balance, their introduction is justified action for achieving the goal of long term ecosystem sustainability

Bakterijska inokulacija - postupak za stimulaciju rasta crvene deteline gajene u zagađenom zemljištu

Red clover (Trifolium pratense L.) seeds were inoculated with several plant growth-promoting bacteria (PGPB) and sown in the substrate contaminated with polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organometallic derivatives of tin (OT). The aim was to determine if selected PGPB strains can promote the growth of red clover in the substrate contaminated with several organic pollutants. The influence of bacteria on red clover growth (height, root length and biomass) was monitored during the three-month experimental period. The most significant improvements of seedling height were noted in the treatment with Bacillus amyloliquefaciens D5 ARV and Pseudomonas putida P1 ARV. Root growth was positively affected by Serratia liquefaciens Z-I ARV. The same isolates significantly affected biomass production. Those isolates caused total biomass increases of 70%, 48% and 33% compared to control. Bacterial strains used in this study were already confirmed as PGPB by biochemical testing, as well as by an in vivo test of mixed inoculums on several woody plants grown in the coal-mine overburden site. This work is the first-time record on their individual effects on one plant species. Obtained results confirmed that inoculation with several PGPB strains can enhance red clover growth in polluted soil.Seme crvene deteline (Trifolium pratense L.), inokulisno sa nekoliko bakterija stimulatora biljnog rasta (PGPB), posejano je u supstrat kontaminiran policikličnim aromatičnim ugljovodonicima (PAHs), polihlorovanim bifenilima (PCBs) i organometalnim derivatima kalaja (OT). Cilj je bio da se utvrdi da li selektovane PGPB mogu promovisati rast crvene deteline u supstratu kontaminiranom sa nekoliko organskih zagađujućih materija. Uticaj bakterija na rast crvene deteline (visina, dužina korena i biomasa) praćen je tri meseca. Najveća visina je zabeležena kod biljaka inokulisanih sa Bacillus amyloliquefaciens D5 ARV i Pseudomonas putida P1 ARV. Rast korena je stimulisan od strane Serratia liquefaciens Z-I ARV. Ovi izolati su značajno uticali i na produkciju biomase. Ukupna biomasa dobijena tokom celog ogleda je za 70%, 48% i 33% veća u odnosu na kontrolu. Bakterijski sojevi korišćeni u ovoj studiji su prethodno potvrđeni kao PGPB kroz biohemijske i in vivo testove mešanog inokuluma na nekoliko drvenastih vrsta gajenih u jalovini. Ovaj rad prvi put beleži njihove pojedinačne efekte na jednu biljnu vrstu. Dobijeni rezultati potvrđuju da inokulacija sa nekoliko PGPB sojeva može ubrzati rast crvene deteline u zagađenom zemljištu

Stimulacija zemljišne mikrobiološke aktivnosti sa klinoptilolitom - uticaj na rast biljke

Zeolites can mitigate ammonium losses to the environment by taking up the ions from sources such as farmyard manure and ammonium-bearing fertilizers. This ability can improve nitrogen fertilizing practice given that ammonium ions loaded onto zeolite are available for plant uptake. The main goal of this study was to assess the influence of zeolite (clinoptilolite) and ammonia-loaded clinoptilolite on growth and yield of red clover (Trifolium pratense L.), as well as their influence on the microbial dynamics in soil. Plants sown in pots were cut four times, and dry matter yield (DM) of each harvest was weighed. The number of culturable bacteria, moulds, ammonifiers, Azotobacter sp. and actinomycetes was determined at the beginning of the experiment, and after each harvest. Two control treatments (soil without fertilizer and soil supplemented with mineral fertilizer - CAN) were included in the study. The application of ammonia-loaded clinoptilolite positively and significantly affected the microbial activity and provided higher yields (from 4.9 g/pot in ammonia-loaded clinoptilolite treatment to 4.3 g/pot in soil with zeolite treatment). Our results suggest that the addition of clinoptilolite to organic fertilizers (manure, composts) could be recommended. The increase of microbial communities’ abundances and their activity represented the key benefit from clinoptilolite application.Zeoliti mogu ublažiti otpuštanje amonijaka u prirodu preko uzimanja jona iz izvora kao što su stajnjak i đubriva bogata amonijakom. Ova sposobnost može unaprediti đubrenje azotom preko zeolitom usvojenih amonijum jona koji bi tako postali dostupni za usvajanje biljkama. Cilj ovih ispitivanja je bio procena uticaja zeolita (klinoptilolita) i zeolita obogaćenog amonijakom na rast i prinos crvene deteline (Trifolium pratense L.), kao i na njihovu ulogu u mikrobiološkoj dinamici zemljišta. Biljke sejane u sudovima su košene četiri puta i meren je prinos suve materije (SM) svakog otkosa. Broj bakterija, plesni, amonifikatora, Azotobacter sp. i aktinomiceta je utvrđen na početku ogleda i nakon svakog košenja. U istraživanja su uključena dva kontrolna tretmana (zemljište bez đubriva i zemljište sa dodatim mineralnim đubrivom - KAN). Primena klinoptilolita obogaćenog sa amonijakom je pozitivno i značajno uticala na mikrobiološku aktivnost i povećala je prinos (od 4,9 g po sudu kod tretmana sa klinoptilolitom obogaćenim amonijakom do 4,3 g po sudu u zemljištu sa zemljištem i zeolitom zajedno). Naša istraživanja sugerišu da se može preporučiti dodavanje klinoptilolita organskim đubrivima (stajnjaku, kompostu). Ključna dobit od primene klinoptilolita bi se ogledala u povećanju zastupljenosti mikrobioloških zajednica i njihove aktivnosti

Izolacija i karakterizacija bakterija i kvasaca iz kontaminiranog zemljišta

Plant growth promoting (PGP) bacteria and yeasts play an important role in bioremediation processes. Thirty bacterial and ten yeast isolates were obtained from PAH and PCB contaminated soil with an aim of determining the presence of PGP mechanisms (production of ammonia, indoleacetic acid, siderophores and solubilization of inorganic phosphate). As a result, three bacterial (Serratia liquefaciens, Micrococcus sp. and Serratia sp.) and two yeast isolates (Candida utilis and Candida tropicalis) were recognized as PGP strains. Among them, Serratia sp. showed the highest indole production (25.5 μg/ml). Analyses of metal tolerance (Cu+2, Cr+6 and Ni+2) revealed that Serratia liquefaciens, Micrococcus sp., Serratia sp. and Candida tropicalis were capable to tolerate significant concentration of metals. As a result of this study several bacterial and yeast strains were attributed as potential plant growth promoters which can be applied in future remediation activities and environmental quality improvements.Zemljište predstavlja dinamičan ekosistem naseljen brojnim mikroorganizmima među kojima su bakterije najbrojnije. Najveći broj je skoncentrisan u uskoj zoni zemljišta koja okružuje koren i koja se naziva rizosfera. Procenjuje se da 1-2% bakterija koje naseljavaju ovu zonu imaju sposobnost da promovišu rast biljaka (engl. plant growth promoting bacteria - PGPB). Pored bakterija, sve više je podataka da i zemljišni kvasci poseduju ove sposobnosti. Mikroorganizmi koji stimulišu rast biljaka koriste različite mehanizme kojima povećavaju dostupnost nutrijenata biljkama, regulišu njihov hormonski status kao i odnos prema biljnim patogenima. Bakterije i kvasci koji stimulišu rast biljaka igraju važnu ulogu i u procesima bioremedijacije. Trideset bakterijskih i deset izolata kvasaca je izolovano iz kontaminiranog zemljišta (PAH i PCB) i testirano na prisustvo mehanizama kojima se pospešuje rast biljaka. Cilj je bio doći do sojeva koji su prilagođeni na život u zagađenom zemljištu, a istovremeno imaju i potencijalno stimulativno dejstvo. Nakon izvršenih biohemijskih analiza (produkcija amonijaka, indol-sirćetne kiseline, siderofora, rastvaranje neorganskog fosfora) u stimulatore biljnog rasta svrstana su tri bakterijska (Serratia liquefaciens, Micrococcus sp. i Serratia sp.) i dva izolata kvasaca (Candida utilis i Candida tropicalis). Među njima najveću sposobnost produkcije indol-sirćetne kiseline je pokazao izolat Serratia sp. (25,5 μg/ml). Analize tolerancije na prisustvo teških metala (Cu+2, Cr+6 i Ni+2) pokazale su da su izolati Serratia liquefaciens, Micrococcus sp., Serratia sp. i Candida tropicalis sposobni da podnesu više koncentracije. Rezultati ovih istraživanja mogu imati praktičnu primenu u budućim remedijacionim aktivnostima i unapređenju kvaliteta životne sredine

Use of overburden waste for London plane (Platanus x acerifolia) growth: the role of plant growth promoting microbial consortia

Overburden waste dumps represent a huge threat to environmental quality. The reduction of their negative impact can be achieved by vegetation cover establishment. Usually, this action is complicated due to site-specific characteristics, such as nutrient deficiency, elevated metal concentration, low pH value, lack of moisture and lack of organic matter. Establishment of vegetation can be facilitated by inoculation with plant growth promoting bacteria (PGPB) which improve the physicochemical and biological properties of degraded substrates and make them more hospitable for plants. In this study we selected several strains based on the ability to produce ammonia, indole-3-acetic acid, siderophores and lytic enzymes, and to solubilize inorganic phosphates. This selection resulted in microbial consortia consisting of Serratia liquefaciens Z-I ARV, Ensifer adhaerens 10_ARV, Bacillus amyloliquefaciens D5 ARV and Pseudomonas putida P1 ARV. The effects of PGPB consortia on one-year-old London plane (Platanus x acerifolia [Aiton] Willd.) seedlings replanted into overburden waste from Kolubara Mine Basin were examined. After seven months, inoculated seedlings were 32% higher with 45% wider root collar diameter and over 80% higher total dry biomass compared to uninoculated seedlings grown in Kolubara's overburden. Inoculation resulted in higher amounts of total soluble proteins, higher chlorophyll and epidermal flavonoids content and higher total antioxidative capacity in the leaves. This study represents a successful search for effective PGPB strains and shows that microbial consortia have an important role in enhancing the growth of seedlings in nutrient deficient and degraded substrates such as overburden waste from open-pit coal mines. Positive response of London plane seedlings suggest that inoculation may help widening the opus of species for reforestation of post mining areas and speed up natural succession processes and recovery of degraded landscapes

Raman spektroskopija i determinacija zemljišnih kvasaca

In this paper, two isolates of yeasts, members of the genus Candida, have been examined using Raman spectroscopy. The yeasts were isolated from soils sampled at the town park in Tivat (Montenegro) and experimental farm Radmilovac (Serbia), by the accumulation method. The pure cultures of yeasts were identified by the API 20C AUX system. The presence of Candida guilliermondii and Candida utilis was noted. The Raman spectra, originating from lipids, amides, proteins, carbohydrates, aromatic amino acids and the nucleotide bases, were analyzed in the wide range of the wave numbers from 500 to 3200 cm-1. It was noticed that peaks which correspond to the lipid fraction were more pronounced in the case of Candida guilliermondii compared to Candida utilis. This may represent a specific response of the yeast species to stress conditions.U ovom radu su Raman spektroskopijom ispitivana dva izolata kvasaca iz roda Candida. Izolacija kvasaca je izvršena iz zemljišta uzetog iz gradskog parka u Tivtu (Crna Gora) i sa oglednog dobra Radmilovac (Poljoprivredni fakultet, Beograd, Srbija), metodom nakupljanja. Dobijene čiste kulture kvasaca identifikovane su primenom API AUX 20C sistema (bioMerieux-Vitek). Konstatovano je prisustvo vrsta Candida guilliermondii i Candida utilis. Analizirani su Raman spektri koji potiču od lipida, amida, proteina, ugljenih hidrata, aromatičnih aminokiselina i nukleotidnih baza, u širokoj oblasti talasnih brojeva, od 500 do 3200 cm-1. Utvrđeno je da su kod kvasca Candida guilliermondii pikovi koji odgovaraju frakciji lipida bitno izraženiji nego kod Candida utilis, što može predstavljati specifičan odgovor date vrste kvasca na stres

In vitro antagonistic activity of Trichoerma spp. to Fusarium oxysporum and Fusarium graminearum

One of the major problems in agriculture are Fusarium species which cause fusariosis in wheat, corn, tomato, melon, watermelon, onion, peas, and beans. Also, Fusarium can synthesize thermostable mycotoxins which can lead to certain diseases if they were found in food. Currently, chemical fungicides are mostly used to prevent the occurrence of fusariosis disease, but the resistance of pathogens to such treatment is being more and more widespread. Soil microorganisms help in plant defense and growth. The rhizosphere fungi have an important role in the mutual exchange of nutrients with plants and they can establish specific interactions with plants. Such rhizosphere fungi are known as plant growth-promoting (PGP). These PGP fungi affect the better plant growth through the synthesis of certain phytohormones but they also have the function as biocontrol agents. They can inhibit the growth of phytopathogens through direct mechanisms of antibiosis, mycoparasitism, and competition. The goal of this research was an investigation of the antagonistic effect of Trichoderma harzianum TR1 and Trichoderma citrinoviride 1V on Fusarium oxysporum and Fusarium graminearum. The antagonistic effect was examined through two tests: Dual culture test (DUAL test) and the effect of volatile organic compounds (VOCs) of Trichoderma strains on the growth of Fusarium strains. Also, the cell-wall degrading enzymatic activity of T. citrinoviride and T. harzianum was examined. The results showed that antagonistic activity of Trichoderma strains varies from moderate to high. Mycelial growth inhibition by T. citrinoviride was 44% for F. graminearum and 67% for F. oxysporum. T. harzianum inhibited F. gramminearum growth for 59% and F. oxysporum for 66%. Based on the results, it was concluded that T.harzianum and T.citrinoviride can be considered biocontrol agents for F. oxysporum and F. graminearum

Changes in Pseudomonas sp. CY growth in the presence of atrazine

Microbial degradation, compared with many other degradation processes, is the most important pathway for the depletion of triazine herbicides in soil. The aim of this study was to determine the growth potential of Pseudomonas sp. CY in the presence of atrazine and additional carbon (sodium citrate) and nitrogen (ammonium-nitrate) sources. The experiment was performed with five treatments: i) 100 mg/L atrazine (control); ii) One hundred mg/L atrazine + sodium citrate (0.3 %, w/v); iii) One hundred mg/L atrazine + sodium citrate (0.3 %, w/v) + ammonium nitrate (0.6 %, w/v); iv) Atrazine (300 mg/L) + sodium citrate (0.3 %, w/v) and v) Atrazine (500 mg/L) + sodium citrate (0.3 %, w/v). The bacterial count was determined after incubation (7 days at 30°C) using the agar plate method, while atrazine degradation was determined by measuring the optical density at 221 nm. Pseudomonas sp. CY can partially utilize atrazine as the sole source of carbon and energy. The highest values of the bacterial count were determined at the highest initial atrazine concentrations; however, bacterial growth was not detected in these treatments. A significant impact of citrate on bacterial growth and atrazine degradation was observed, while the addition of nitrate decreased the atrazine degradation rate. This study confirmed that Pseudomonas sp. CY can be used as a prominent candidate for the remediation of atrazine-affected environments

Trichoderma spp. from Pine Bark and Pine Bark Extracts: Potent Biocontrol Agents against Botryosphaeriaceae

Pinus sylvestris bark represents a rich source of active compounds with antifungal, antibacterial, and antioxidant properties. The current study aimed to evaluate the antifungal potential of P. sylvestris bark against Botryosphaeria dothidea, Dothiorella sarmentorum, and Neofusicoccum parvum (Botryosphaeriaceae) through its chemical (water extracts) and biological (Trichoderma spp. isolated from the bark) components. The water bark extracts were prepared at two temperatures (80 and 120 °C) and pH regimes (7 and 9). The presence of bark extracts (30%) caused inhibition of mycelial growth of B. dothidea and D. sarmentorum for 39 to 44% and 53 to 60%, respectively. Moreover, we studied the antagonistic effect of three Trichoderma isolates originating from the pine bark. Trichoderma spp. reduced growth of B. dothidea by 67%–85%, D. sarmentorum by 63%–75% and N. parvum by 55%–62%. Microscopic examination confirmed typical mycoparasitism manifestations (coiling, parallel growth, hook-like structures). The isolates produced cellulase, β-glucosidase and N-acetyl-β-glucosaminidase. The volatile blend detected the emission of several volatile compounds with antimicrobial activity, including nonanoic acid, cubenene, cis-α-bergamotene, hexanedioic acid, and verticillol. The present study confirmed in vitro potential of P. sylvestris bark extracts and Trichoderma spp. against the Botryosphaeriaceae. The study is an important step towards the use of environmentally friendly methods of Botryosphaeriaceae disease control

Mikrobni diverzitet zemljišta kontaminiranog visokim sadržajem teških metala

Except of benefits for human population, several industrial activities have a negative consequences i.e. increasing of heavy metal content and changes in diversity of living organisms in environment. The aim of this work was to examinated the influence of high content of heavy metals on microbial diversity of soil. Researches was conducted at locations of former factory 'Rog' (Ljubljana, Republic of Slovenia), by taking of four samples of contaminated soil, while the control was uncontaminated soil sample. Determination of microbial diversity (abundance of bacteria, fungi and actinomycetes) was performed using the standard methodology. The results of research showed that abundance of microorganisms depends on location and presence/absence of contamination. In control treatment abundance of all examinated groups of microorganisms was higher compared with other samples. The bacterial number in contaminated samples was from 1.21 to 6.88x106 CFU/g, the number of fungi was from 1,08 to 30,30x103 CFU/g, while actinomycetes number from 302,15 to 381,82x103 CFU/g. In control treatment, bacterial number was 17,45x106 CFU/g, fungi number 54,02x103 CFU/g and actinomycetes number 481,91x103 CFU/g. These results confirms the importance of microbial diversity research on contaminated locations and potential applications of bioremediation technologies on locations of former factory 'Rog'.Mnogobrojne industrijske aktivnosti, osim koristi za ljudsku populaciju, imale su za posledicu povećanje sadržaja teških metala i promene diverziteta živih organizama u životnoj sredini. Cilj ovog rada bio je ispitivanje uticaja visokih koncentracija teških metala na mikrobni diverzitet zemljišta. Istraživanja su obavljena na području bivše fabrike 'Rog' (Ljubljana, Republika Slovenija), gde su uzeta četiri uzorka kontaminiranog zemljišta, dok je kontrolni uzorak predstavljao nekontaminirano zemljište. Ispitivanje mikrobnog diverziteta (brojnost bakterija, gljiva i aktinomiceta) obavljeno je primenom standardnih metoda. Rezultati istraživanja ukazuju da je brojnost mikroorganizama zavisila od lokacije i prusustva/odsustva kontaminacije. U kontrolnoj varijanti brojnost svih ispitivanih grupa mikroorganizama bila je veća u odnosu na ostale uzorke. Brojnost bakterija u kontaminiranim uzorcima iznosila je od 1,21-6,88x106 CFU/g, gljiva 1,08-30,30x103 CFU/g i aktinomiceta 302,15-381,82x103 CFU/g. U kontrolnoj varijanti broj bakterija iznosio je 17,45x106 CFU/g, gljiva 54,02x103 CFU/g i aktinomiceta 481,91x103 CFU/g. Ovi rezultati ukuzuju na značaj ispitivanja mikrobnog diverziteta na kontaminiranim lokacijama i predstavljaju osnovu za potencijalne aplikacije bioremedijacionih tehnologija na području bivše fabrike 'Rog'