Proizvodnja enzima i fiksacija dušika pomoću slobodnih, imobiliziranih i koimobiliziranih inokuluma plijesni Trichoderma harzianum i bakterije Azospirillum brasilense, te njihova uloga u stimulaciji rasta mladica rajčice

A plant growth-promoting rhizobacterium (Azospirillum brasilense strain Az) and a biocontrol fungus (Trichoderma harzianum strain T24) have been evaluated for their individual and combined production of hydrolytic enzymes, nitrogen fixation and their possible role in growth promotion of tomato seedlings. The studied organisms were inoculated as free or calcium alginate-encapsulated cells. All freshly prepared macrobeads showed high encapsulation capacity (EC/%) of inocula compared with dry macrobeads. Results of enzyme production did not exhibit consistent pattern of the effect of encapsulation process on enzyme production. Beads entrapping bacterial and/or fungal cells were used successfully in 3 repeated cycles in the presence of fresh sterile culture medium in each growth cycle. Enzyme production by immobilized bacterial and/or fungal cells increased as the growth cycles were repeated. Co-culturing of A. brasilense with T. harzianum (free or immobilized) in semisolid nitrogen deficient medium (N-free medium) enabled A. brasilense to fix nitrogen on pectin, chitin and carboxymethyl cellulose. The activity of nitrogen fixation by A. brasilense in the case of single and combined cultures with Trichoderma (using dry encapsulated beads) into the sterile soil increased with the addition of carbon source. Most of inoculations with free or alginate macrobead formulations of T. harzianum and/or A. brasilense showed significant increase in the growth parameters of tomato seedlings. The root system grew more profusely in the case of all seeds treated with A. brasilense. The growth parameters of Az/T24-treated seeds using dry coimmobilized macrobeads were higher than those of the untreated control. Moreover, the effect was improved significantly in soil enriched with different C sources. Enhanced tomato seedling growth after the co-inoculation could be due to the synergistic effect of both Trichoderma and Azospirillum. Finally, co-inoculation with Azospirillum and other microorganisms is one of the major frontiers of Azospirillum technology and perhaps the main area for future applications.Ispitan je utjecaj rizosferne bakterije Azospirillum brasilense Az, koja potiče rast biljaka, te plijesni Trichoderma harzianum T24, što se upotrebljava za biološko suzbijanje štetnika, na proizvodnju enzima, fiksaciju dušika te njihovu ulogu u stimulaciji rasta mladica rajčice. Mikroorganizmi su inokulirani u obliku slobodnih ili alginatom imobiliziranih zrna. Sva su svježe pripremljena zrna imala veću sposobnost enkapsulacije (EC/%) od suhih zrna, a enkapsulacija nije znatno utjecala na proizvodnju enzima. Zrna sa stanicama bakterija ili plijesni uspješno su korištena u tri uzastopna ciklusa rasta u svježoj sterilnoj podlozi, pri čemu se poboljšala proizvodnja enzima. Zajedničkim je uzgojem A. brasilense i T. harzianum (slobodnih ili imobiliziranih), u polučvrstoj podlozi bez dušika, omogućena fiksacija dušika i nakon dodavanja pektina, hitina ili karboksimetil celuloze. Dodatkom ovih izvora ugljika u sterilnu zemlju povećavala se fiksacija dušika, bilo uporabom suhih enkapsuliranih zrna A. brasilense, bilo primjenom A. brasilense uz plijesan Trichoderma. Primjenom svih vrsta inokuluma povećan je rast mladica rajčice, a prisutnost A. brasilense ubrzala je razvoj korijenja. Parametri rasta, ispitani nakon primjene suhih koimobiliziranih zrna Az/T24, poboljšali su se u usporedbi s kontrolnim uzorkom, a osobito nakon obogaćivanja tla ugljikom. Razlog tome je vjerojatno sinergistički učinak plijesni Trichoderma i bakterije Azospirillum. Koinokulacija bakterije Azospirillum s drugim mikroorganizmima ima veliku mogućnost primjene u obogaćivanju tla i pospješivanju rasta biljaka

Effect of Benomyl on Chitinase and β-1,3-Glucanase Production by Free and Alginate Encapsulated Trichoderma harzianum

On PDA-benomyl plates growth of Trichoderma harzianum was inhibited by 20 and 30 % at benomyl 1 and 2 μg/mL, respectively, and was completely inhibited at 5 μg/mL. In minimal synthetic medium (MSM) amended with different concentrations of benomyl (1.0, 3.0, 5.0, 7.0 and 10.0 μg/mL), fungal immobilisation improved chitinase and β-1,3-glucanase production at low benomyl concentrations (1, 3 and 5 μg/mL). Further increase in the production of both enzymes was obtained by immobilisation at higher benomyl concentrations (7 and 10 μg/mL). Fungal immobilisation increased bound chitinase by 15- to 30-fold at 3 and 5 μg/mL benomyl concentration, respectively. However, no effect was obtained on the bound β-1,3-glucanase. Different benomyl concentrations (0.3 to 1500 μg/mL) had no significant inhibitory effect on the activities of free or immobilised chitinase and β-1,3-glucanase. It could be suggested that either immobilised Trichoderma or immobilised chitinase and β-1,3-glucanase could be used in combination with benomyl to control plant pathogens

Improvement of Cell Wall Degrading Enzymes Production by Alginate Encapsulated Trichoderma spp.

Conidia of three Trichoderma isolates were formulated to make alginate pellets with or without 0.5 % chitin or dried fungal mycelium of Fusarium oxysporum as carbon source. The formulations were compared for their ability of in vitro chitinase and β-1,3-glucanase production with free fungal spore suspensions. Conidia entrapped in alginate with or without adjuvant showed high production of enzymes (especially for chitinase) even when repeated 4 times. The addition of chitin or dried fungal mycelium as adjuvant enhanced the enzyme production up to 5 and 2-fold for chitinase and β-1,3-glucanase, respectively. Alginate concentration and surface area of the beads affected the enzyme production. The optimum initial pH, incubation time and temperature were pH=6, 12 days and 40 °C for chitinase, and pH=7, 10 days and 35 °C for β-1,3-glucanase production. The improvement of cell wall degrading enzyme production by alginate encapsulated Trichoderma could explain the in vivo inhibitory effect of such formulations on the target phytopathogenic fungi

Enzyme Production and Nitrogen Fixation by Free, Immobilized and Coimmobilized Inoculants of Trichoderma harzianum and Azospirillum brasilense and Their Possible Role in Growth Promotion of Tomato

A plant growth-promoting rhizobacterium (Azospirillum brasilense strain Az) and a biocontrol fungus (Trichoderma harzianum strain T24) have been evaluated for their individual and combined production of hydrolytic enzymes, nitrogen fixation and their possible role in growth promotion of tomato seedlings. The studied organisms were inoculated as free or calcium alginate-encapsulated cells. All freshly prepared macrobeads showed high encapsulation capacity (EC/%) of inocula compared with dry macrobeads. Results of enzyme production did not exhibit consistent pattern of the effect of encapsulation process on enzyme production. Beads entrapping bacterial and/or fungal cells were used successfully in 3 repeated cycles in the presence of fresh sterile culture medium in each growth cycle. Enzyme production by immobilized bacterial and/or fungal cells increased as the growth cycles were repeated. Co-culturing of A. brasilense with T. harzianum (free or immobilized) in semisolid nitrogen deficient medium (N-free medium) enabled A. brasilense to fix nitrogen on pectin, chitin and carboxymethyl cellulose. The activity of nitrogen fixation by A. brasilense in the case of single and combined cultures with Trichoderma (using dry encapsulated beads) into the sterile soil increased with the addition of carbon source. Most of inoculations with free or alginate macrobead formulations of T. harzianum and/or A. brasilense showed significant increase in the growth parameters of tomato seedlings. The root system grew more profusely in the case of all seeds treated with A. brasilense. The growth parameters of Az/T24-treated seeds using dry coimmobilized macrobeads were higher than those of the untreated control. Moreover, the effect was improved significantly in soil enriched with different C sources. Enhanced tomato seedling growth after the co-inoculation could be due to the synergistic effect of both Trichoderma and Azospirillum. Finally, co-inoculation with Azospirillum and other microorganisms is one of the major frontiers of Azospirillum technology and perhaps the main area for future applications