Bacillus strains as potential agents for the biocontrol of phytopathogenic fungi Alternaria spp.

Plant diseases caused by fungal pathogens can lead to major economic losses in agriculture. Th e genus Alternaria includes saprobic, endophytic and pathogenic species that may cause various plant diseases such as blight disease. Symptoms of Alternaria blight include the presence of irregular, oft en circular, brown to dark brown coloured leaf spots. Alternaria spp. also cause collar rots, stem lesions, tuber and fruit rots of their respective hosts. In addition, bacteria are one of the most frequently biocontrol agents used to protect plants from diseases. Bacillus species control disease through a variety of mechanisms. Bacterial antagonists might act as inhibitors of growth, development and reproduction of pathogen, or as inducers of host resistance in plant. Th e objective of this study was to examine in vitro antifungal activity of ten antagonistic Bacillus strains from collection of Department for Microbiology of Institute of Field and Vegetable Crops, Novi Sad. Strains were originally isolated from the soil samples collected from several localities of Vojvodina Province. Antifungal activity of Bacillus strains against Alternaria padwickii, A. dauci and A. linicola, obtained from rice, carrot and linseed, respectively was tested using a dual plate assay. Th e results confi rmed that all tested Bacillus strains showed antifungal acivity against Alternaria spp. Th e highest antagonistic activity was exhibited by B. pumilus B11 (45.71% to 51.25%), B. subtilis B13 (45.71% to 49.37%) and B. subtilis B32 (45.71% to 52.08%), while B. pumilus B23 had the weakest antifungal activity. B. safensis B2 (35.24% to 48.33%), B. pumilus B21 (40.95% to 48.75%) and B. pumilus B22 (33.50% to 49.17%) also demonstrated good antifungal potential. A. dauci was the most sensitive fungus, while the most resistant was A. linicola. Obtained results indicate the possibility of usage of the most eff ective Bacillus strains as potential biocontrol agents of plant diseases caused by Alternaria sp

Antifungalna aktivnost prirodnih Bacillus spp. izolata iz zemljišta

Biocontrol using plant growth-promoting rhizobacteria (PGPR) represents an alternative approach to disease management, since PGPR are known to promote growth and reduce diseases in various crops. Among the different PGPR, members of the genus Bacillus are prefered for most biotechnological uses due to their capability to form extremely resistant spores and produce a wide variety of metabolites with antimicrobial activity. The objective of this research was to identify antagonistic bacteria for management of the plant diseases. Eleven isolates of Bacillus spp. were obtained from the soil samples collected from different localities in the Province of Vojvodina. The antifungal activity of bacterial isolates against five fungal species was examined using a dual plate assay. Bacillus isolates exhibited the highest antifungal activity against Fusarium proliferatum, Fusarium oxysporum f. sp. cepae and Alternaria padwickii, while they had the least antagonistic effect on Fusarium verticillioides and Fusarium graminearum. Molecular identification showed that effective bacterial isolates were identified as Bacillus safensis (B2), Bacillus pumilus (B3, B11), Bacillus subtilis (B5, B7) and Bacillus megaterium (B8, B9). The highest antagonistic activity was exhibited by isolates B5 (from 39% to 62% reduction in fungal growth) and B7 (from 40% to 71% reduction in fungal growth). These isolates of B. subtilis could be used as potential biocontrol agents of plant diseases.Biokontrola fitopatogena predstavlja alternativu primeni pesticida, s obzirom da bakterije označene terminom PGPR (Plant Growth Promoting Rhizobacteria) stimulišu biljni rast i štite biljke od bolesti. Zahvaljujući sposobnosti da formiraju veoma rezistentne endospore i produkuju širok spektar antimikrobnih supstanci, vrste roda Bacillus su veoma zastupljene u zemljištu i pogodne za primenu u biotehnologiji. Cilj istraživanja bio je da se utvrdi antifungalna aktivnost jedanaest Bacillus spp. izolata iz zemljišta s različitih lokaliteta u Vojvodini. Sposobnost bakterijskih izolata da inhibiraju rast pet izolata gljiva ispitana je metodom dvojne kultivacije. Izolati Bacillus spp. ispoljili su najveću antifungalnu aktivnost prema Fusarium proliferatum, Fusarium oxysporum f. sp. cepae, i Alternaria padwickii, dok je najmanji antagonistički efekat utvrđen prema Fusarium verticillioides i Fusarium graminearum. Efektivni izolati identifikovani su kao Bacillus safensis (B2), Bacillus pumilus (B3, B11), Bacillus subtilis (B5, B7) i Bacillus megaterium (B8, B9). Najveću antifungalnu aktivnost ispoljili su izolati B. subtilis B5 (39-62%) i B7 (40-71%). Ovi izolati mogu se koristiti kao potencijalni agensi za biološku kontrolu biljnih bolesti

Antifungalna aktivnost ekstrakata makrogljiva na fitopatogene sojeve gljiva rodova Fusarium sp. i Alternaria sp.

During the last decades, intensive application of synthetic fungicides in the agricultural crop protection practice caused growing concern for the existence of toxic chemical residues in food as well as in the whole environment. Instead of using synthetic fungicides, it is suggested that crop protection be carried out by using preparations based on compounds of natural origin (secondary metabolites of plants or microorganisms, including macrofungi from Basidiomycota) as biological control agents. The potential of macrofungal species as biocontrol agents was analyzed in this investigation of eight autochthonous species from different locations in Serbia. Both the terricolous species: Coprinus comatus, Coprinellus truncorum, Amanita strobiliformis, Hydnum repandum and the lignicolous species: Flammulina velutipes, Stereum subtomentosum, Trametes versicolor and Bjerkandera adusta were examined, with an aim to detect some novel sources of antifungal agents. This study surveyed antifungal activity of selected macrofungal extracts (MeOH, EtOH and CHCl3) against phytopathogenic Fusarium and Alternaria strains isolated from garlic, soybean and rice: F. proliferatum, F. verticillioides, F. proliferatum, F. graminearum and A. padwickii. Microdilution method in 96 well micro-plates was applied for the estimation of antifungal effects of macrofungi extracts in the range from 24.75 to 198.00 mg/ml and determination of minimal inhibitory (MIC) and minimal fungicidal concentration (MFC). EtOH extract of mychorhizal species H. repandum showed antifungal activity against all analyzed phytopathogenic strains, with the strongest effect on Fusarium strains (MIC 24.75 mg/ml; MFC 24.75 mg/ml). Among others, MeOH extracts of S. subtomentosum and C. micaceus showed similar effects while only B. adusta showed slight effect on Fusarium strains (MIC 24.75-99.00 mg/ml; MFC 24.75-99.00 mg/ml) and none effect on A. padwickii. The obtained results indicate the possibility of using examined extracts as efficient antifungal agents and provide the basis for the new formulations of biocontrol agents against phytopathogenic fungi in the future.Tokom poslednjih decenija intezivna primena sintetičkih fungicida u zaštiti poljoprivrednih useva izaziva sve veću zabrinutost ljudi zbog prisustva toksičnih hemijskih ostataka u prehrambenim proizvodima kao i celoj okolini. Umesto korišćenja sintetičkih fungicida, preporučuje se da se zaštita useva vrši korišćenjem preparata na bazi jedinjenja prirodnog porekla (sekundarni metaboliti biljaka ili mikroorganizama uključujući i makrogljive iz razdela Basidiomycota) kao agense biološke kontrole. U ovom radu istraživan je potencijal agenasa biološke kontrole za osam autohtonih vrsta makrogljiva s različitih lokaliteta u Srbiji. Obe, terikolne vrste: Coprinus comatus, Coprinellus truncorum, Amanita strobiliformis, Hydnum repandum i lignikolne vrste: Flammulina velutipes, Stereum subtomentosum, Trametes versicolor i Bjerkandera adusta su istražene s ciljem da se otkriju neki novi izvori antifungalnih agenasa. U okviru ovog rada istražena je antifungalna aktivnost odabranih ekstrakata makrogljiva (metanolni, etanolni i hloroformski) protiv fitopatogenih sojeva Fusarium i Alternaria izolovanih s belog luka, soje i pirinča: F. proliferatum, F. verticillioides, F. proliferatum, F. graminearum i A. padwickii. Za procenu antifungalnog efekta ekstrakata makrogljiva upotrebljena je mikrodiluciona metoda mikrotitar pločama s 96 velova u opsegu koncentracija od 24,75 do 198,00 mg/ml i determinaciju minimalne inhibitorne (MIC) i minimalne fungicidne koncentracije (MFC). Etanolni ekstrakt mikorizne vrste H. repandum pokazao je atifungalnu aktivnost prema svim analiziranim fitopatogenim sojevima, sa najjačim efektom prema sojevima Fusarium (MIC 24,75 mg/ml; MFC 24,75 mg/ml). Između ostalog, sličan efekat pokazali su i metanolni ekstrakti S. subtomentosum i C. micaceus, dok je samo B. adusta imala blagi efekat na sojeve Fusarium (MIC 24,75-99,00 mg/ml; MFC 24,75-99,00 mg/ml), ali ne i na A. padwickii. Dobijeni rezultati ukazuju na mogućnost korišćenja ispitanih ekstrakata kao veoma efikasnih antifungalnih agenasa i samim tim oni predstavljaju osnovu za nove formulacije biokontrolnih agenasa protiv fitopatogenih gljiva u budućnosti

Antifungal activities of ionic liquids against phytopathogenic Alternaria spp. strains

A modern sustainable approach in agricultural practice would be a replacement of synthetic and toxic substances in the treatment of crop with benign and environmentally friendly compounds, by using principles of green chemistry. One of the possible directions could be using of ionic liquids (ILs), salts with melting points below 100 °C, which are well-known for manifesting antimicrobial activity. ILs can be used in the development of novel sources of antimicrobial agents such as antiseptics, biocides and antifungal agents. Furthermore, ILs have already been reported as alternative ‘green’ solvents for a wide range of reactions and technological processes. Considering the importance of research in the area of bioactivity of ILs for discovery of new green ILs for diff erent purposes, the aim of this study was to examine antifungal activity of nine newly synthesized ILs against phytopathogenic Alternaria strains isolated from from rice (A. padwickii), carrot (A. dauci) and linseed (A. linicola). Antifungal eff ect was estimated by micro-plate microdilution method for establishing minimum inhibitory (MIC) and minimum fungicidal concentration (MFC). All ionic liquids showed strain specifi c but good antifungal activity on Alternaria strains with MIC and MFC detected at the range from 9.23 mg/ml to 75.89 mg/ml. Only in case of the 1-(4-hydroxy-2-oxy)butyl -3-methylimidazolium chloride [OHC2OC2mIm][Cl], no antifungal eff ect on A. dauci were observed. Comparing to A. padwickii and A. dauci., A. linicola showed higher sensitivity to all tested ILs. Th e obtained results indicate the possibility of usage of ILs in biocontrol of plant diseases, representing their application in crop protection. However, further research is necessary in order to examine their toxicity and biodegradability in the environment