In Iran, native oak species are under threat from episodes of Charcoal Disease, a decline syndrome driven by abiotic stressors (e.g. drought, elevated temperature) and biotic components, ...Biscogniauxia mediterranea (De Not.) Kuntze and Obolarina persica (M. Mirabolfathy). The outbreak is still ongoing and the country's largest ever recorded. Still, the factors driving its' epidemiology in time and space are poorly known and such knowledge is urgently needed to develop strategies to counteract the adverse effects. In this study, we developed a generic framework based on experimental, machine-learning algorithms and spatial analyses for landscape-level prediction of oak charcoal disease outbreaks. Extensive field surveys were conducted during 2013-2015 in eight provinces (more than 50 unique counties) in the Zagros ecoregion. Pathogenic fungi were isolated and characterized through morphological and molecular approaches, and their pathogenicity was assessed under controlled water stress regimes in the greenhouse. Further, we evaluated a set of 29 bioclimatic, environmental, and host layers in modeling for disease incidence data using four well-known machine learning algorithms including the Generalized Linear Model, Gradient Boosting Model, Random Forest model (RF), and Multivariate Adaptive Regression Splines implemented in MaxEnt software. Model validation statistics Area Under the Curve (AUC), True Skill Statistics (TSS), and Kappa index were used to evaluate the accuracy of each model. Models with a TSS above 0.65 were used to prepare an ensemble model. The results showed that among the different climate variables, precipitation and temperature (Bio18, Bio7, Bio8, and bio9) in the case of O. persica and similarly, gsl (growing season length TREELIM, highlighting the warming climate and the endophytic/pathogenic nature of the fungus) and precipitation in case of B. mediterranea are the most important influencing variables in disease modeling, while near-surface wind speed (sfcwind) is the least important variant. The RF algorithm generates the most robust predictions (ROC of 0.95; TSS of 0.77 and 0.79 for MP and OP, respectively). Theoretical analysis shows that the ensemble model (ROC of 0.95 and 0.96; TSS = 0.79 and 0.81 for MP and OP, respectively), can efficiently be used in the prediction of the charcoal disease spatiotemporal distribution. The oak mortality varied ranging from 2 to 14%. Wood-boring beetles association with diseased trees was determined at 20%. Results showed that water deficiency is a crucial component of the oak decline phenomenon in Iran. The Northern Zagros forests (Ilam, Lorestan, and Kermanshah provinces) along with the southern Zagros forests (Fars and Kohgilouyeh va-Boyer Ahmad provinces) among others are the most endangered areas of potential future pandemics of charcoal disease. Our findings will significantly improve our understanding of the current situation of the disease to pave the way against pathogenic agents in Iran.
•Immersion of contaminated pistachio nut in citric acid solution and then ozonation plus UV-C exposure could be used to degrade aflatoxins in nuts.•The UV-C, had more effect on AFB2 and AFG2.•The O3 ...treatment degrades AFB1 and AFG1 more than AFB2 and AFG2.•Acid treatment had more effect on AFB1 and AFG1, against AFB2 and AFG2.
The effects of gaseous ozone (O3), UV-C radiation, and citric acid (CA) on aflatoxins degradation in contaminated pistachio samples were investigated. By the evaluation of different combinations, the optimum amounts of O3, UV-C, and CA were achieved. The best treatment was a combination of the immersion of the samples in 3 N CA, 30 minutes’ exposure to O3, and 36 hours exposed to UV-C radiation, by this combination more than 90 % of aflatoxin B1 and aflatoxin B2 and more than 99 % of aflatoxin G1 and aflatoxin G2 were degraded. No significant changes were observed in total fat content, protein content, acid and peroxide content, total phenolic compounds, soluble and insoluble carbohydrates of pistachios. Also there were no significant changes between sweetness, acidity, flavor, color, and overall quality of treated and non-treated samples. The results showed that the combination of O3, UV-C, and CA was much more effective than the effect of each of them alone.
Pistachio nuts are among the commodities with the highest risk of aflatoxin contamination in Iran. Aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins for humans and livestock. In nature, ...there are microorganisms which are capable of reducing aflatoxins contamination in food and feed products. In this study, Bacillus subtilis strain UTBSP1 was isolated from pistachio nuts and studied for the degradation of AFB1. The AFB1 contents were determined by the use of HPTLC and HPLC as well as multiple reactions monitoring (MRM) method in LC–MS/MS. The results indicated B. subtilis UTBSP1 could considerably remediate AFB1 from nutrient broth culture and pistachio nut by 85.66% and 95%, respectively. Cell free supernatant fluid caused an apparent 78.39% decrease in AFB1 content. The optimal conditions for AFB1 degradation by cell free supernatant appeared at 35–40 °C, during 24 h. Furthermore, the results indicated that AFB1 degradation is enzymatic and responsible enzymes are extracellular and constitutively produced. The destructive AFB1 differed from standard AFB1 chemically, and lost a fluorescence property.
► We find the beneficial strain of Bacillus subtilis in Iranian pistachio nut. ► It causes the reducing of AFB1content in liquid culture and pistachio nut. ► It destructs AFB1 by the extracellular constitutively produced enzyme. ► The destructive AFB1 differs from AFB1 chemically, and loses a fluorescence property.
Buxus sempervirens subsp. hyrcana (Pojark.) Takht. (boxwood) is an evergreen shrub/tree in Caspian hyrcanian forests covering the Alborz mountain range of northern Iran. During the summer of 2012, a ...sudden leaf and twig blight disease of boxwood was observed throughout the northern forests of Iran. Disease symptoms included circular dark spots on leaves leading to defoliation, and longitudinal brown-black streaks on the shoots. Diseased plant material was collected from the Guilan and Mazandaran areas, placed in moist chambers, and incubated at 20°C to induce sporulation. Single conidia were plated onto half-strength potato dextrose agar supplemented with 250 mg/L streptomycin and incubated at 25°C under near-ultraviolet light. Isolates were transferred to carnation leaf agar and incubated at 25°C under near-ultraviolet for morphological characterization, and representative isolates were deposited into the culture collection of the CBS-KNAW Fungal Biodiversity Centre under accession numbers CBS 134431 and CBS 134432. Gross morphological characters were determined by mounting fungal structures in lactic acid and 50 measurements at 1,000× magnification were made for all taxonomically informative characters. The observed macroconidiophores consisted of a stipe bearing a penicillate suite of reproductive branches and a stipe extension, terminating in a naviculate vesicle. The stipe extensions were septate, hyaline (85 to 160 × 2 to 4 μm), terminating in a naviculate vesicle, 6 to 11 μm in diam. Conidia were cylindrical, rounded at both ends, straight, with one septum (55 to 68 × 4 to 6 μm). These morphological observations agreed to those provided for C. pseudonaviculata Lombard, M. J. Wingf. & Crous (1,2). To confirm morphological identification, DNA sequence data were generated for the ITS1-5.8S-ITS2 region of the rDNA, and a fragment of the translation elongation factor 1-alpha gene region (3). These sequences were compared to other sequences of C. pseudonaviculata in GenBank (100% similarity for both loci), which confirmed the morphological observations. Sequences were submitted to GenBank under the accession numbers KC736850 and KC736851 for ITS, and KC736852 and KC736853 for TEF. Koch's postulates were proven by spraying a 3 × 10
conidia/ml conidial suspension of isolate CBS 134431 onto 1-year-old B. sempervirens subsp. hyrcana plants until run-off, and covering them for 24 h with a plastic bag to maintain high humidity. Control plants were sprayed with sterile water. Ten plants were used for each treatment and maintained in a greenhouse at 20 to 22°C with 95% relative humidity. Symptoms similar to those observed in nature developed within 4 days of inoculation and the test fungus was successfully reisolated from the inoculated plants. No symptoms were observed on the control plants. Boxwood blight caused by C. pseudonaviculata, was first reported in the United Kingdom in the mid-1990s and has since become widespread, causing epidemics globally (1,2,4). To our knowledge, this study represents the first report of boxwood blight in its native environment and in Iran. References: (1) P. W. Crous et al. Sydowia 54:23, 2002. (2) B. Henricot and A. Culham. Mycologia 94:980, 2002. (3) L. Lombard et al. Stud. Mycol. 66:31, 2010. (4) M. R. Saracchi et al. J. Plant Pathol. 90:581, 2009.
A series of 5‐arylidene derivatives 3a‐k, as potential antifungal agents, were synthesized in good to high yields by the reaction of 2‐benzimidazolylimino‐4‐thiazolidinone and corresponding aromatic ...aldehyde in a buffered medium. These compounds were evaluated for their antifungal activities against four agricultural fungi, Botrytis elliptica, Fusarium graminearum, Phytophthora nicotianae, and Rhizoctonia solani. Thereby, it was found that the compound 1 exhibits an antifungal effect against P. nicotianae and B. elliptica, comparable with carbendazim as a standard antifungal. Our results may provide some guidance for development of some novel benzimidazole‐based antifungal lead structures. J. Heterocyclic Chem., 2010.
Background: Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic compound mainly produced by the Aspergillus parasiticus, A. flavus, A. nomius, A. tamari, and A. pseudotamarii. AFB1 biodegradation is ...the most important strategy for reducing AFB1 in plant tissues. Bacteria can deactivate and biodegrade AFB1 for effective detoxification of contaminated products. The present study investigated the efficiency of AFB1 degradation by soil bacteria from the Southern Khorasan Province in Eastern Iran by thin-layer and high-performance liquid chromatography during 2014–2015. Methods: DNA was extracted from AFB1-degrading isolates by the cetyltrimethylammonium bromide method and the 16S rRNA gene was amplified with the 27f and 1492r general bacterial primers and the sequences were used to identify the isolates based on their similarity to Gene Bank sequences of known bacterial species. Results: We isolated five strains from four species of AFB1-degrading bacteria from Birjand plain, including Bacillus pumilus, two isolates of Ochrobactrum pseudogrigonens, Pseudomonas aeruginosa, and Enterobacter cloace, which had AFB1-degrading activities of 88%, 78%, 61%, 58%, and 51%, respectively. Conclusion: We provide the first demonstration of AFB1 degradation by B. pumilus in from Iran and the first report identifying O. pseudogrigonens and E. cloace species as having AFB1-degrading activity.
Summary
Zagros oak (Quercus spp.) forests (ZOF) cover approximately 4 million hectares of the Zagros Mountains in Iran. Oak charcoal disease caused by Biscogniauxia mediterranea and Obolarina persica ...has recently increased in some regions of ZOF. Detection of these fungi in host tissue and identification of the anamorphs by traditional methods have limitations and difficulties which were overcome using two primers, OP1 and OP2, based on rDNA sequences of O. persica and used along with the specific primers MED1 and MED2 for B. mediterranea to develop a multiplex PCR. This method was used to correctly identify 1 pg of fungal DNA per 1 mg of inner bark tissues of Quercus brantii, Q. infectoria and Q. libani.
Iran is the largest producer of pomegranate (Punica granatum) in the world, with more than 60,000 ha currently in production. In the spring of 2011, a decline and dieback of young pomegranate trees ...(7 to 10 years old) were observed in the Kheir area of Fars Province. Dieback and twig blight developed toward the lower part of the stem, resulting in death of aerial tree parts and growing suckers from roots. Surface-disinfected tissues of diseased plants were plated on potato dextrose agar (PDA) and malt extract agar media. Isolates were separated into two groups that had either pale green or white aerial mycelia and sporulated after 5 to 7 days at 25°C. Pycnidia were globose and black with thin, membranous, pseudoparenchymatic walls, 80 to 140 μm in diameter. Conidia were hyaline, one-celled, elongate to fusiform, straight, and 11 to 17 × 4 to 6 μm (average 14 × 4.7 μm). Cardinal minimum growth temperatures were 8 to 10°C, optimum at 27 to 30°C, and maximum at 35°C. Radial growth rate at 30°C was 8 to 9 mm per day. Representative isolates were deposited in the CBS-KNAW Fungal Biodiversity Centre, the Netherlands (CPC 19625 = CBS 130974 and CPC 19626 = CBS 130975; GenBank JN815312 and JN815313, respectively). Genomic DNA was extracted with the UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Inc., Solana Beach, CA) and the internal transcribed spacer (ITS) region of the nrDNA operon of two isolates were sequenced as described previously (1). On the basis of morphology (3), the causal organism was identified as Pilidiella granati Sacc. This identification was corroborated by the ITS sequence data, which was identical for both colony types to GenBank HQ166057 (identities = 614 of 614 100%). Pathogenicity tests were conducted using two representative isolates from each group on 5-month-old P. granatum trees with 10 replicates under greenhouse conditions; 5-mm mycelial plugs from the edge of 7-day-old colonies on PDA were placed under the bark of twig wounds. Uncolonized PDA plugs were used as noninoculated controls. Pathogenicity was also tested on nonwounded fruit by placing colonized 5-mm-diameter mycelial plugs on surface-disinfected pomegranate fruits; noncolonized PDA plugs were used as controls. All treated fruit were placed in plastic bags and maintained at 25°C for 10 days. Isolates were found to be pathogenic on twigs after 2 months, giving rise to brown lesions that were 2 to 5 cm long. No lesions were observed on the controls. Furthermore, the fungus was reisolated from all infected tissues, satisfying Koch's postulates. On pomegranate fruit, the fungus colonized the fruit after 5 to 8 days, followed by the appearance of fruit rot symptoms leading to the formation of abundant pycnidia covering the skin after 10 days. No decay was observed in control inoculations. Pilidiella granati has previously been reported as a pathogen of P. granatum fruit from Europe, Asia, and the United States (2). To our knowledge, this is the first report of this pathogen causing dieback and fruit rot of pomegranate in Iran. References: (1) J. Frank et al. Persoonia 24:93, 2010. (2) L. Palou et al. Online publication. doi:10.5197/j.2044.0588.2010.022.021. New Dis. Rep. 22:21, 2010. (3) J. M. Van Niekerk et al. Mycol. Res. 108:283, 2004.
The essential oil of Zataria multiflora Boiss. (ZEO) was prepared using a British Pharmacopeia-type hydrodistillation apparatus. The ZEO was encapsulated with a natural polymer, chitosan (CS) by ...oil-in-water emulsification, and ionic cross-linking techniques. The shape and size range of the encapsulated particles were evaluated by Atomic-Force Microscopy (AFM) and Dynamic Light Scattering (DLS) and FT-IR spectroscopy. The AFM analysis of the ZEO encapsulated nano-particles (CS-ZEO) showed spherical shape particles with inner cavities. The mean size of CS-ZEO particles was 293 ± 17 nm according to DLS analysis. The chemical constituent of the oil was analyzed by GC-MS and thymol acetate (32.4%), carvacrol acetate (19.0%), γ-terpinene (18.5%), and ρ-cymene (14.5%) were reported as its major constituents. The non-encapsulated ZEO in the synthesis process was determined to calculate the encapsulation efficiency and loading capacity of the prepared CS-ZEO which were 87.4% and 6%; respectively. In vitro antifungal activity of ZEO and CS-ZEO in growth media against Aspergillus flavus was evaluated with the decrease in fungal mycelial mass. On the other hand, the HPLC results showed the reduction of aflatoxins B1 levels by 95% and 92% at concentrations of 0.8 mg/mL of ZEO and 6.7 mg/mL of CS-ZEO, respectively. The above-mentioned effective concentrations of the ZEO and CS-ZEO, were applied to pistachio samples, which reduced 90.5% and 99.0%, of the aflatoxin B1 levels, respectively compared to non-treated samples. Therefore, we suggest here, the CS-ZEO as a possible natural fungicide with an effective mechanism of lowering aflatoxins production in the contaminated pistachio.
•Essential oil of Zataria multiflora (ZEO) was encapsulated into chitosan.•Physicochemical properties of the nanoparticles were determined using AFM, DLS, FT-IR, and GC.•Antifungal activity of the ZEO and its chitosan nanocarriers were evaluated against Aspergillus flavus.•The nanoparticles caused a significant reduction of aflatoxin B1 in a pistachio sample.
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