mutation

Cellulases and xylanases are hydrolytic enzymes that randomly cleave the β-1, 4 backbones of the cellulose and arabinoxylans of wheat flour and are widely used in the bakery industry as a dough texture improver in the formulations of flour products. Creating novel sources of a microbial strain using induced gamma irradiation can increase enzyme production for bakery industrial usage. According to this, Co60 gamma irradiation has been used to develop a mutant strain of Trichoderma afroharzianum. Trichoderma mutants were isolated, and the qualitative and quantitative screening were used to evaluate the extracellular enzyme production with the wheat bran waste as a substrate. The best Trichoderma mutant isolate was identified using the DNA barcoding method. The highest xylanase activities were observed in the superior mutant isolate of Trichoderma afroharzianum NAS107-M82, which is approximately 3.3 times higher than its parent strain. The electrophoretic pattern of proteins showed that the exo-glucanase I, endo-glucanase III, and the xylanase I enzymes hydrolyzed the wheat bran, synergistically. Overall, gamma irradiation-induced mutation could be an expedient technique to access such superior mutants for the bioconversion of wheat bran wastes to xylanase enzyme.

Plant viruses may behave differently in glasshouse than in nature, especially those that are naturally transmitted by vector. Biological assays, as the oldest and still used virus detection methods, involve inoculation of host plant and monitoring symptoms in glass- or green-house. While maintaining Peanut stunt virus (PSV) in glasshouse we came across with symptom attenuation of PSV strain B (PSV-B) in Nicotiana benthamiana after eight successive passages. To determine if any changes have occurred in the PSV-B genome, the virus particles were purified from the plant and subjected to RNA extraction with phenol/ chloroform to remove the virus coat protein (CP), and followed by precipitation with ethanol to purify the virus RNAs. Then, electrophoresis of the virus RNAs on agarose gel showed that the RNA3 component of the PSV-B isolate was longer than that in the original reference PSV-B, suggesting occurrence of insertion(s) in the RNA3. To confirm this observation, the full length cDNA corresponding to the RNA3 was synthesized by RT-PCR with the use of the 5’ and 3’ terminal primers which were designed according to sequences of the PSV RNA3 terminal ends (accession PSU31366). The RT-PCR product was subjected to dideoxy cycle sequencing and the sequence data was compared with the corresponding PSV RNA3 sequences. This revealed occurrence of a 183- nucleotide repeat at the 3’ untranslated region of the RNA3 and suggested an association between this mutation (repeat) and the symptom attenuation. This emphasizes that in glasshouse studies, sequential passages of a virus can lead to emergence of mutants. This observation also has implication as to plant protection because the mutated variant may be used in cross protection.

Trichoderma is very important as a bio control agent and probably a good alternative for chemical fungicides. Fusarium oxysporum is a plant pathogen that causes wilt in a wide range of plants. The use of gamma irradiation can be employed to increase Trichoderma efficiency against F. oxysporum. The induced mutation provides genetic changes in Trichoderma and in some of the mutated isolates the efficiency of bio control may be improved. In this study the efficiency of mutated T. harzianum CS5 against the F. oxysporum f.sp. radicis-cucumerinum was evaluated. Among 16 wild isolates of Trichoderma, CS5 isolate was chosen based on synchronous and nonsynchronous mutual cultivation with pathogen. The spore suspensions of T. harzianum CS5 were irradiated in a cobalt- 60 γ- irradiator at a dose rate of 0.23 Gy/Sec in Nuclear Agriculture Research School, (NSTRI, AEOI). Then antagonist screening of 76 mutants was investigated in the experiments of synchronous and nonsynchronous dual culture with pathogen. The results showed that only 17 mutants were able to control the pathogen better than wild type, and YFTM80 isolate had the most prevention. Using the ERIC-PCR marker, the distinction was investigated between the wild type and the 17 selected mutants. The results showed that the gamma ray is able to improve biocontrol efficiency of Trichoderma and the ERIC-PCR marker can differentiate among derived mutants, but it does not have the ability to distinguish mutants from wild type. In terms of antagonistic superiority, mutations might have occurred in antagonistic sites, which have led to improved antagonistic efficiency. Probably the ERIC-PCR marker has failed to replicate these areas.