Characterization of Aspergillus section Flavi from pistachio soils in Iran

During 2012, soil samples from commercial pistachio orchards in three major production regions include Rafsanjan (Kerman Province, center of Iran), Damghan (Semnan Province, north-central Iran) and Feyz-Abad (Khorasan-e Razavi Province, northeastern Iran), were assayed on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) and Aspergillus flavus-parasiticus agar media to quantify populations of Aspergillus species from the section Flavi. The mean propagule density of Aspergillus species from the Flavi section [log10 (CFU/g soil)] was higher in Feyz-Abad (3.06, 2.88–3.24), compared to Damghan (2.55, 2.44–2.65) and Rafsanjan (2.40, 2.26–2.54). A. flavus (69.7, 65.3 and 57.9%), A. parasiticus (19.6, 25.4, and 29.3%), and A. nomius (10.7, 9.3, and 12.8%) were the predominant species in the regions of Rafsanjan, Damghan, and Feyz-Abad, respectively. There were significant differences among sclerotia producing isolates of A. flavus in the sampling regions (p < 0.05). The percentage of sclerotium-producing isolates of A. flavus from Rafsanjan (14.5%) was much lower than Damghan (39.5%) and Feyz-Abad (41.4%). The A. flavus isolates from Damghan, Rafsanjan, and Feyz-Abad were toxigenic at 53.7%, 61.6%, and 60.4%, respectively. In Rafsanjan, aflatoxin B1 (AFB1), and AFB1 + AFB2 (aflatoxin B2) ranged from 274 to 553 ppb (393±17.11) and 394 to 3745 ppb, respectively, while AFB1, and AFB1 + AFB2 ranged from 257 to 392 ppb (285±13.18) and 415 to 1658 ppb, respectively, in Damghan. We found 16 and 20 vegetative compatibility groups (VCGs) for 41 and 37 nit mutant producing isolates of A. flavus from Rafsanjan and Damghan, respectively. From Damghan the VCG diversity for A. flavus isolates was greater (54%) than from Rafsanjan (39%). Because there were a few number of sclerotium-producing isolates of A. flavus, we did not determine the relationships between sclerotium production with VCGs and/or geographical distribution in the three pistachio production regions. This study was the first to determine the strain and VCG diversity of A. flavus soil isolates from Iranian pistachio orchards

Evaluation of Colorimetric LAMP Assay for Visual Detection of Ralstoniasolanacearum in Potato Shipments at Quarantine Stops in Ira

Introduction: Race 3/ biovar 2 of this pathogen causes bacterial blight of solanaceous plants especially potato in both tropical and temperate regions and results in great economic losses worldwide. Infection is prevented via quarantine or incineration of infected plant materials. However, the use of healthy seed tubers is the most effective way to avoid dissemination of this harmful plant pathogenic bacterium to pathogen-free areas. Amplification of functional genes such as endoglucanase and hrpB and fliChas been used as an alternative to study R. solanacearumspecies complex. In order to facilitate detection of R. solanacearumin imported seed tubers and identify high-risk fields and stores where inoculums population is low, loop-mediated isothermal amplification (LAMP) reaction as a potentially fast and cost-effective method was used. The attention of the present study wason evaluation of latent infection in potato tubers with R. solanacearum bacterium targeting the fliC gene by colorimetric LAMP assay. The LAMP protocol was compared with the conventional PCR which routinely used at most quarantine stops. Materials and Methods: In this study, bacterial strains were isolated on tetrazolium chloride (TZC) agar medium. Pathogenicity assay was carried out on tomato and potato seedlings under greenhouse conditions. Total DNA of bacterial strains was prepared using Chen and Kao (1993) protocol. In some cases, the boiled filtrated potato extract was used directly in molecular experiments. Identification of R. solanacearum strains at species and phylotype levels and biovar determination were done based on literature. The PCR products were analyzed on 1.2 % agarose gels in TBE buffer and visualized with UV light. To detect R. solanacearumin symptomatic and symptomless tissues, conventional PCR and LAMP assay according to fliC gene were performed and compared with each other. In order to check amplified LAMP products in visual assessment, the existence of magnesium pyrophosphate precipitate in tested tubes was analyzed. Furthermore, change in colourdue to the reaction was evaluated bynaked eye and UV treatmentafter adding the calcein. Finally, the LAMP products were examined by electrophoresis through 2% agarose gel after staining with green viewer. To determine limit of the LAMP assay, seven dilution series (2×107 to 2×10 CFU/ml) were prepared and 2 μl of each dilution was used for LAMP. Results and Discussion: Bacterial colonies showed mucous and opaque appearance with red centre and whitish periphery on TZC agar medium were selected for further study.In plant bioassay two weeks after bacterial inoculation, different levels of wilting were observed on tomato and potato seedlings.The expected 281 and 372 bp PCR-amplified fragments was observed in all strains supporting species and phylotype identification, respectively. Moreover, utilization of carbon sources indicated that the strains were related to biovar 2. Furthermore, all strains from potato were screened using Ral-fliC and Rsol-fliC primers. A 400 bp PCR product specific to R. solanacearum was obtained from all strains. Sequencing three purified PCR products confirmed the right amplification of fliC gene specific to R. solanacearum. The amplified products were detected by visual observation which the white turbidity of the reaction mixture by magnesium pyrophosphate was seen after 55 min. An alternative indicator to visually check the positive reactionwas calceinwhich was based onobservation ofyellow (green) in colour at the absence (presence) of UV light in infected samples and clear colour in negative control. Detection limits in pure cultures and infected potato extract were also determined. In conventional fliC-PCR, the detection limit rangedapproximately from 10 3 to 10 4cfu ml−1in both infected potato extract and pure cultures. Moreover, the lowest amount of consistently tested positive through LAMP assay was 10 4cfu ml−1 for both cases. Although the sensitivity of the fliC LAMP assay wasequal or lower than that of the conventional PCR, the accuracy of fliC LAMP seems to be sufficient toreliably confirmthe presence of R. solanacearum in potato samples. In addition, LAMP protocol assay is time-consuming procedure, does not require expensive equipments, provides visually detection of positive reactions and can apply to survey possible infection in host plants. Conclusion: Consequently, LAMP assay with ashort nucleic acid extraction step like as boiling treatment and efficient visualization processes such as calcein provide suitable preliminary data for screening of pathogen–free tubers prior to storage and during transportation

Studies on Aspergillus flavus Link. isolated from maize in Iran

The Aspergillus flavus population structure from maize kernels was examined. During 2011, samples were collected from two main grain maize production areas in Iran (Fars and Ardebil provinces), shortly before harvest. One-hundred nine A. flavus isolates were recovered on Dichloran Rose Bengal Chloramphenicole (DRBC) agar and Aspergillus flavus/parasiticus medium (AFPA) and grouped into morphotypes and Vegetative Compatibility Groups (VCGs) based on morphological (e.g. sclerotia production), physiological (e.g. aflatoxin-producing ability) and genetic criteria (e.g. heterokaryosis). In general, morphotype and VCG composition were highly dissimilar in both provinces. In total, 43.8% and 44.3% of A. flavus isolates from Ardebil and Fars, respectively, produced sclerotia. Sclerotia producers were identified as A. flavus L and S strain morphotypes in Ardebil (66.7% and 33.3%, respectively) and Fars (29.6% and 70.4%, respectively). Furthermore, 71 isolates (65.1%) were able to produce aflatoxin (Ardebil 40.8%, Fars 59.2%). The aflatoxin values were categorized into four different classes ( 1,000 ppb). In total, 51 aflatoxin producing isolates of A. flavus (Ardebil n = 22, Fars n = 29) were assigned into 26 VCGs by complementation of nit auxotrophs on nitrate medium. None of the A. flavus isolates from Ardebil complemented with any isolates from Fars. Genetic diversity of A. flavus isolates was 59.1% and 41.8% for Ardebil and Fars, respectively. The different geographical adaptation and genetic make-up of A. flavus isolates may be due to different climatic conditions, soil types and crop sequences in both maize production areas