Effect of pH, water activity and temperature on the growth and accumulation of ochratoxin A produced by three strains of Aspergillus carbonarius isolated from Italian vineyards

Aspergillus carbonarius colonizes grapes and its derived products and produces ochratoxin A (OTA). In previous studies we screened 107 strains of A. carbonarius isolated from grapes for production of OTA, and we selected three high OTA-producing strains for this study (AC05, AC06, and AC07). The effect of different values of three conditions [temperature 15, 25, 30, 35°C; water activity (aw) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0] on A. carbonarius growth and OTA production was examined. A. carbonarius AC07 produced higher levels of OTA than AC06 and AC05 at all the variables tested. At 30°C the strains of A. carbonarius produced more OTA than at the other temperatures. A water activity of aw 0.98 produced the greatest mycelial growth and OTA accumulation for all three A. carbonarius strains. A pH of 4.0 produced the highest levels of OTA in all the strains. No growth was seen at aw 0.88 or at pH 10.0 in any strain, except AC07, which grew at pH 10.0. The optimal conditions for growth and production of OTA by A. carbonarius strains were 30°C, aw 0.98 and pH 4.0. When all the three factors (aw, pH, T) were combined, aw 0.98, pH 4.00 and 30ºC produced the highest levels of OTA in all the strains. Maximum amounts of OTA were found 7–9 ays after inoculation in all the strains. Temperature, water activity, and pH had a great impact on OTA production by A. carbonarius and these factors should be taken into account when developing management practices for future research programmes

Ochratoxigenic Black Species of Aspergilli in Grape Fruits of Northern Italy Identified by an Improved PCR-RFLP Procedure

A collection of 356 isolates of Aspergillus spp. collected during 2006 and 2007 from grapevines in northern Italy were identified through Internal Transcribed Spacer based Restriction Fragment Length Polymorphism (ITS-RFLP) and tested for ochratoxin A (OTA) production. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, HinfI and RsaI, distinguished five different RFLPs. From each pattern, three samples were sequenced and the nucleotide sequences showed different species corresponding to Aspergillus niger, A. carbonarius, A. tubingensis, A. japonicus and A. aculeatus. By comparing the sequences of the ITS regions, also the uniseriate species A. japonicus and A. aculeatus could be differentiated by HinfI digestion of the ITS products. Among the aspergilli, A. niger was the major species associated with grapes during 2006 (57.4%), while A. carbonarius was the major species during 2007 (46.6%). All the strains of Aspergillus were tested for their ability to produce OTA on Yeast extract sucrose medium (YES), as it was tested as an optimal substrate for the evaluation of OTA production by black aspergilli. Out of 356 isolates, 63 (17.7%) isolates produced OTA ranging from 0.05 to 3.0 µg mL−1. Most of the ochratoxigenic isolates were A. carbonarius (46) in both years, but also some strains of A. tubingensis (11) and A. japonicus (6) produced lower amounts of OTA

Assessing the Efficacy of Broad-Spectrum Antibiotics in Controlling Bacterial Contamination in the In Vitro Micropropagation of Ginger (Zingiber officinale Rosc)

Ginger (Zingiber officinale Rosc) (Zingiberaceae) is a livelihood and commercial crop in Ethiopia. But, the availability of clean and healthy planting materials has become a problem due to wilt disease, caused by Ralstonia solanacearum Biovar 3 Race 4. This problem obliged growers to seek for tens of millions of vigorous and disease-free planting materials very quickly via in vitro micropropagation of shoot tip explants. For this purpose, protocols of sterilizing shoot tip explants and controlling bacterial contamination of one Ethiopian ginger cultivar called Deribo were tested. Hence, this article reports the finding of a study that aimed at testing the (a) effectiveness of three sterilization agents, namely, 0.25% w/v RBK (composed of ridomile, bayleton, and kocide at 1 : 1 : 1 ratio), 0.50% v/v NaOCl, and 70% v/v ethanol at three different treatment times in combination with 0.25% HgCl2; (b) efficacy of four broad-spectrum antibiotics and their combinations in controlling bacterial contaminants of ginger shoot tip explants and in vitro micropropagation media; and (c) effects of the antibiotics on the shooting performances of the explants of the cultivar. A 0.50% v/v NaOCl at exposure time of 20 min followed by 0.25% HgCl2 has resulted in 80% contamination-free and 70% live explants after three weeks of incubation. Likewise, cefotaxime at 50, 100, and 200 mg/L and cefotaxime plus streptomycin at 25, 50, and 100 mg/L yielded 87 to 93% contamination-free microshoots after three weeks of culturing. The number of explants killed by the antibiotics increased with increasing the concentration of the antibiotics. Cefotaxime at 50 mg/L and cefotaxime plus streptomycin at 25 mg/L yielded significantly highest mean microshoots per explant (7.10 ± 0.36 and 7.51 ± 0.27, respectively) and mean shoot length (4.2 ± 0.26 and 3.56 ± 0.17 cm, respectively). Some of the microshoots showed some yellowing. But, they turned green and grew normal after subcultured into fresh, antibiotics-free culture media. These findings are important foundations towards developing more optimized protocols of sterilizing explants and controlling bacterial contaminants for large-scale in vitro micropropagation of the Deribo ginger cultivar

Effect of pH, water activity and temperature on the growth and accumulation of ochratoxin A produced by three strains of <I>Aspergillus carbonarius</I> isolated from Italian vineyards

Aspergillus carbonarius colonizes grapes and its derived products and produces ochratoxin A (OTA). In previous studies we screened 107 strains of A. carbonarius isolated from grapes for production of OTA, and we selected three high OTA-producing strains for this study (AC05, AC06, and AC07). The effect of different values of three conditions [temperature 15, 25, 30, 35°C; water activity (aw) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0] on A. carbonarius growth and OTA production was examined. A. carbonarius AC07 produced higher levels of OTA than AC06 and AC05 at all the variables tested. At 30°C the strains of A. carbonarius produced more OTA than at the other temperatures. A water activity of aw 0.98 produced the greatest mycelial growth and OTA accumulation for all three A. carbonarius strains. A pH of 4.0 produced the highest levels of OTA in all the strains. No growth was seen at aw 0.88 or at pH 10.0 in any strain, except AC07, which grew at pH 10.0. The optimal conditions for growth and production of OTA by A. carbonarius strains were 30°C, aw 0.98 and pH 4.0. When all the three factors (aw, pH, T) were combined, aw 0.98, pH 4.00 and 30ºC produced the highest levels of OTA in all the strains. Maximum amounts of OTA were found 7–9 ays after inoculation in all the strains. Temperature, water activity, and pH had a great impact on OTA production by A. carbonarius and these factors should be taken into account when developing management practices for future research programmes

Shoot Regeneration Is Not a Single Cell Event

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4&ndash;5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days