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The dry bubble disease, caused by Lecanicillium fungicola, is an important fungal disease of white button mushroom in Iranian mushroom production farms. Twenty-three isolates of the pathogen collected in Iran and identified as L. fungicola var. fungicola, were compared for genetic polymorphism, diversity in growth rate and virulence. Ten Universal Rice Primers (URP) were used to evaluate the genetic diversity of L. fungicola var. fungicola. URP analysis showed that the genetic diversity of Iranian isolates was low (average 10 % over the 10 primers used) and that they were almost clonal. Relative correlations between geographical origins of isolates and molecular grouping were observed but there was no correlation between mycelial growth rate, virulence assays and URP patterns. Significant differences were observed between isolates based on mycelial growth rate and virulence assays. The high level of genetic homogeneity is attributed to the effect of fungicides used for control of the mushroom diseases which might have imposed a significant selection pressure on the fungal populations.  

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Beet western yellows virus (BWYV), a species of the genus Polerovirus in the family Luteoviridae, is an agriculturally important virus infecting over 150 plant species in 23 dicotyledonous families worldwide. A survey of BWYV in canola fields in Golestan and Tehran provinces of Iran using indirect triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) indicated 8.3 % infection. The presence of BWYV was confirmed by amplification of the coat protein (CP) gene of the virus via running a reverse transcription-polymerase chain reaction (RT-PCR) on total RNA extracted from ELISA positive leaf tissues. DNA sequences of the BWYV coat protein (CP) gene of seven Iranian isolates were determined and compared at the nucleotide (nt) and amino acid (aa) levels with those of twelve BWYV isolates from different countries deposited in GenBank. Sequence analysis data showed that the identity of BWYV-CP at nt and aa levels among the Iranian isolates were 93.4 % to 100 % and 93.2 % to 100 %, respectively. The maximum similarity of isolates at nt and aa levels were 97.2 and 96.6 %, which occurred among two Iranian isolates (Ir 8 and Ir 100) and four isolates from France (L39967 and X13063) and England (L39973 and L39970). The recombination analysis among the nineteen isolates including seven Iranian isolates revealed that there was no distinct intra-specific recombination event among BWYV isolates. This is the first report of sequencing and analyzing of the BWYV CP gene of Iranian BWYV isolates.

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Mycosphaerella graminicola, the causal agent of septoria tritici blotch (STB), is a widespread and significant pathogen of wheat. To determine mating types, 89 isolates were collected randomly from wheat fields of Khuzestan, East Azerbaijan, Ardebil, Kermanshah and Golestan provinces of Iran, during 2006-7. DNA was extracted based on standard protocols. Multiplex PCR was conducted using two pairs of mating type-specific primers for MAT1-1 and MAT1-2. Sensitivity to strobilurin fungicide was determined using strobSNPrc7 and strobSNPrc1 primers. The results showed that in 35 isolates, a fragment of 340 bp amplified with MAT1-1 idiomorph specific primers and in 54 isolates a fragment of 660 bp was amplified with MAT1-2 idiomorph specific primers. While the mating type frequencies were highly unequal, the MAT1-1 was predominant. All isolates were sensitive to strobilurin and amplified a fragment of 639 bp. It is concluded that both mating types are present in Iran, although with different frequencies, which may affect genetic variation through sexual cycle. Meanwhile the studied isolates were not resistant to strobilurin fungicides which may be due to growing wheat cultivars resistant to STB rather than using strobilurin fungicides as a dominant control method.

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Soybean, Glycine max, is susceptible to a large number of disease agents such as seedling and root pathogens that cause serious damages to this crop plant. One of these soil-borne pathogens is Macrophomina phaseolina, the causal agent of charcoal root rot. In this study, two bacteria, Pantoea agglomerans and Bacillus sp. BIN, and a fungus, Trichoderma harzianum T100, as potential biocontrol agents, and maneb fungicide, were evaluated against soybean charcoal rot disease in In Vitro and greenhouse conditions. All antagonists inhibited growth of the pathogen in dual culture test by 73.8, 63.3 and 55.3 %, respectively. Data from greenhouse experiments showed that in the presence of pathogen all antagonists increased the growth indices of soybean in both pasteurized and non-pasteurized soil. Reductions of microsclerotia coverage on soybean root and stem by P. agglomerans, Bacillus sp. and T. harzianum were up to 62.5, 87.6 and 62.5 %, respectively and for maneb fungicide was 87.6 % in pasteurized soil. The overall results of this study show high capability of used antagonists in reduction of initial inoculums for next season of this monocyclic disease.

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Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is one of the most important diseases of canola (Brassica napus) in Golestan province, the leading canola producer in Iran. In order to assess the yield loss of canola caused by SSR, 80 fields were surveyed in four different regions of the province (Gorgan, Ali Abad, Kalaleh and Gonbad) during 2006-2007, and SSR intensity was recorded weekly in the fields. Study of yield loss-SSR severity relationships by linear, nonlinear and multiple regression analyses with final intensity (S_f), time to initial symptoms (t_is), Gompertz rate of disease progress (r_G), and standardized area under disease progress curve (SAUDPC) as independent variables indicate that single point and integral models were significant (P < 0.05) only in three cases. Results of multiple point models which were performed using weekly recorded SSR intensities (S₁, S₂, …), were significant in two cases and a general model for 2007 survey was developed using S₃ to S₆. Eventually, response surface models were developed for each region by integrating t_is with SSR intensity variables (S_f or SAUDPC).

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One of the important technical obstacles in the study of many filamentous fungi is the development of efficient transformation system. Transformation of filamentous fungi is difficult because they have a cell wall and for most frequently used approaches preparing of protoplast is necessary. Protoplast preparation is batch-dependent and often frustrating work. In this study, a modified method was introduced for gene transfer to the plant pathogenic fungus, Fusarium graminearum, the major causal agent of Fusarium Head Blight disease in small grains. This protocol was based on protoplast-PEG method. Age of mycelia, enzymes exposure time and mycelium/enzyme ratio were optimized for the purpose of protoplast preparation. The outcome showed that the best result for protoplast preparation was obtained when 1.5 × 10⁵ spores were let germinate for 6 h then exposed to 10 ml of enzyme solution for 3 h. The effect of other parameters that might enhance transformation yields including PEG concentration, DNA quantity and number of protoplasts was also examined. The most efficient condition for transformation involved the use of 10⁶ protoplasts, 20 µg DNA and 30% PEG (w/v). In the course of this study, a simple and appropriate modified protocol for transformation of F. graminearum was established. The method introduced is also more economical and faster than other current methods.

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Rice blast, caused by Pyricularia grisea, is one of the most important diseases of this crop in Iran and all over the world. To evaluate the relationship between spore population (SP) and meteorological factors, SP was measured daily using spore trap during growing seasons of 2006-2008 in Rasht and Lahijan regions (Guilan province, Iran). Weather data including precipitation, daily maximum and minimum temperatures, daily maximum and minimum relative humidity and duration of sunny hours were obtained from weather stations which were five kilometers away from the fields. The relationship between spore population and metrological factors was evaluated by Neurosolution 5.0 software. Weather data and spore population were considered as input and output data, respectively. In this study, multilayer perceptron neural network, regression model and Log(x + 1) transformation were performed. To evaluate the model efficiency, correlation coefficient and mean square error were used. The results showed that the correlation coefficient (r) and mean square error (MSE) parameters were 0.55 and 0.03 in Rasht and 0.1 and 0.03 in Lahijan, respectively. The results also showed the potential of this model for modeling SP using meteorological factors; however more data is needed for validation of this model. There has been no previous report on modeling the relationship between SP and meteorological data using artificial neural network in Guilan province (Iran).

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The severe epidemics of Fusarium head blight (FHB) as a devastating disease of cereal crops has occurred on wheat in North and Northwest Iran in recent years. The in vitro production of Deoxynivalenol (DON) was qualitatively evaluated in 41 Fusarium isolates collected from wheat heads associated with the scab disease, in Moghan area/Northwest Iran. Infected wheat heads were collected during 2004-2007. The isolation of causal agents was carried out using standard methods. According to morphological characteristics and using valid descriptions, all isolates belonged to Fusarium graminearum species complex and F. culmorum of which the former was dominant. In order to evaluate the potential of DON production in isolates, this mycotoxin was extracted and qualitatively examined by TLC method. The quantification of DON was achieved using HPLC method. TLC results indicated that 54.5% of studied isolates produced DON but there was no significant relationship between this property and cultivars or sub-regions or years. Also based on HPLC analysis, maximum content of DON was detected in F. graminearum isolated from cv. Izen green from Moghan Agro-industry company fields in 2004 at the rate of 5827.11 µgkg^-1. The results of present study show that DON mycotoxin is produced at various contents by F. graminearum isolates on different cultivars and from different origins/ years. Since DON plays a role in pathogenesis and is of paramount importance in contamination of wheat grains, these results give a better insight into the significance of this disease in Northwest Iran.  

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Macrophomina phaseolina is one of the major yield limiting factors of melons in tropical and subtropical regions. For eco-friendly and effective management of the disease, 24 gamma induced mutants from Trichoderma harzianum were evaluated against three isolates of the pathogen representing three geographically different regions viz. Khorasan (isolate 1), Garmsar (isolate 2) and Khuzestan (isolate 3). The isolates of Trichoderma (mutants and wild type) were evaluated against the pathogen in dual culture and through production of volatile and non-volatile inhibitors. Maximum growth inhibition was observed in Th1, Th4, Th15, Th9 and Th22 mutants after three days. In greenhouse evaluation against M. phaseolina (isolate 1) among the inoculated treatments minimum plant infection was observed in Th9 treatment (28% disease reduction) as compared to infected control and among the uninoculated treatments Th1and Th9 mutants resulted in maximum growth of roots and shoots of melon plants as compared to uninfected control. These mutants are introduced as potential candidates against M. Phaseolina. The results proved that gamma-mutagenesis by enhancing the antagonistic properties of T. harzianum 65 can be useful for the biocontrol of soil borne plant pathogens such as Macrophomina phaseolina.

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On the basis of preliminary in vitro screening tests, a competent strain of Pantoea agglomerans ENA1 (P. agg. ENA1) recovered from soybean nodule was evaluated for its antagonistic activity against Macrophomina phaseolina causal agent of charcoal rot of soybean. The results of various in vitro assays showed that P. agg. ENA1 is capable of exerting strong antagonistic effect against M. phaseolina inhibiting its mycelial growth up to 89% as compared to control. The results showed a significant reduction of the disease as measured in host-plant weight increase, reduced microsclerotial coverage of the host tissues and decreased population of the pathogen in soil. Soils treated with the antagonist in presence of the pathogen resulted in 40% increase in aerial fresh weight and 63% decrease in root and stem surface covered by microsclerotia as compared with control. Furthermore, a significant decrease in the pathogen population ranging from 73 to 76% was observed in sterile and non-sterile soils, respectively. P. agg. ENA1 is suggested as a potent biocontrol agentthat provides excellent rhizosphere colonization and control of M. phaseolina    

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Sheath blight, caused by Rhizoctonia solani AG1-IA, is one of the most destructive disease. Conventional methods of disease control using fungicides may develop new problems. Therefore, understanding molecular mechanisms of plant–pathogen interaction is necessary to adopt effective approaches for managing the disease. Here for the first time, by using bioinformatics tools and RT-PCR analysis and sequencing confirmed the presence of a Magnaporthe oryzae Avr-pita gene orthologous sequence designated as Rhiz-pita1 gene in three different geographic isolates of R. solani AG1- IA( A2,R1 and T2) genome. SignalP program predicted a secretion signal upstream of Rhiz-pita1 gene. Nucleotide sequences of 5' region of Rhiz-pita1 gene from geographical isolates showed 99% identity in exons and 100% in introns which are characteristics of fast evolving effector proteins. Also, 98% homology between Rhiz-pita and M.oryza-pita1gene suggests that Rhiz-pita encodes an effector protein. Howevere, more researchs are necessary to confirm of this suggestion. Keywords: Rhizoctonia solani, signal peptide Rice blast , Effector

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Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is one of the destructive diseases of wheat in the world. The fungal pathogen can infect 365 different grass and more than 70 Berberis species. DNA sequences for the ribosomal RNA internal transcribed spacer (ITS) have proven suitable to explore relationships at the species and subspecies levels. An isolate of Pgt which was collected from Iran and designated as TTSSK was used in this study. Three samples of the isolate were used. ITS region of the samples was amplified and sequenced. Consensus tree based on Maximum Parsimony clustering method was produced by Mega 6.0. Iranian isolate of TTSSK was placed in a clade with P. graminis which was isolated from Berberis sp. and Pgt isolate from Tajikistan. The results showed that more than one conserved genomic regions would be informative to identify phylogenetic relationship of Iranian Pgt isolates and samples from different parts of the world. Complementary studies with more sequence data from other genome loci are in progress.

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In vitro antifungal activity of silver nanoparticles, at concentrations of 6, 8, 10, 12, 14 and 16 ppm, was studied on five phytopathogenic fungi, and a biocontrol agent. Then effect of silver nanoparticle at 6 ppm (optimum concentration) was evaluated on Macrophomina phaseolina in greenhouse. For in vitro experiment, the fungal isolates were grown on potato dextrose agar medium amended with silver nanoparticles. Radial fungal growth was recorded after 1, 2, 3, 5 and 10 days and mycelial growth inhibition rates were calculated. The most sensitive fungus to nanoparticles was Pythium aphanidermatum, since all tested concentrations showed 100% inhibition during the 10 days of observation.The second most sensitive fungus was Sclerotinia sclerotiorum, since it was able to grow only at concentration of 6 ppm and M. phaseolina was the third in sensitivity since its growth was inhibited in all concentrations after three days. In greenhouse experiments, five treatments including no nanosilver-no pathogen (Negative control), no nanosilver +pathogen (Positive control), 6 ppm nanosilver– no pathogen, 6 ppm nanosilver +pathogen, Carboxin-Thiram (0.15%) +pathogen were compared. Four characters viz shoot and root fresh and dry weights were measured. Based on the greenhouse experimental results, treatments with nanosilver and fungicide gave higher yields than the positive control. The chemical control treatment had the highest measured parameters, while 6 ppm nanosilver +pathogen treatment had the same parameters as negative control. It may therefore be suggested to use nanosilver as a safer alternative to chemical fungicides for control of M. phaseolina.

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Culture filtrates (CF) of two species of the nematophagous fungi, Arthrobotrys oligospora and Arthrobotrys conoidesat three concentrations (25%, 50% and 100%) of stock, were tested on the mortality of second stage juveniles (J2) and egg hatching rate of Meloidogyne incognita and Meloidogyne javanica. Results showed that the percent juvenile mortality was directly proportional to concentration of the filtrates. Egg hatching rate of these nematodes was inversely affected by increasing concentrations. Also CFs had various impacts on the mortality of J2 and egg hatching rate. In case of M. incognita maximum J2 mortality (28.98%) occurred after 24 hours of exposure to A. conoides filtrate at concentration of 100%. The minimum toxicity (12.5% J2 mortality) was recorded for A. oligospora at 25% filtrate concentration. At the same time, the highest rate of J2 mortality of M. javanica (19.18%) belonged to the 100% concentration of A. conoides, while minimum toxicity belonged to 25% concentration of A. oligospora causing 9.09% mortality. Maximum egg hatching rate for M. incognita (30.75%) belonged to control and minimum hatching rate (1.25%) belonged to 100% concentration of A. conoides. The highest hatching rate of M. javanica (36.25%)belonged to control and minimum hatching rate (1.25%) occurred at 100% concentration of A. conoides.

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Fusarium proliferatum, as a toxigenic fungus, is one of the important agents of onion basal rot (FBR). Among the control methods of the disease, biological control is considered as one of the best options. In this study, Trichoderma harzianum strain T100 at the rate of 1 × 10⁶ cfu/g was mixed with pot soil artificially infested with F. proliferatum. Also Glomus mosseae was applied to each pot at the rate of three grams of soil containing 80 chlamydospores/ml. Combination of T100 and G. mosseae was used as well. Onion seeds were disinfected & planted in pots, arranged in completely randomized design in 4 replicates under greenhouse condition and finally, the individual or combined effects of these bioagents were assessed on FBR control and also root colonization by Glomus 23, 30 and 36 days after sowing. Inoculation of Arbuscular mycorrhizae improved onion growth effectively, but its biocontrol effect was not considerable. Trichoderma amended soil decreased disease incidence by 25% but its usefulness as biocontrol agent was reduced in the course of time. AM root colonization was decreased in plants in presence of Trichoderma. Nevertheless, the disease control in combination of Trichoderma and Glomus treatment was better than the treatments by each one of the agents singly. The disease control achieved by fungicide seed treatment was inferior to that of Trichoderma and Glomus in combination.  

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Many environmental and physiological factors affect plant temperature. The objective of this study was to use thermal imagery to investigate robust features for early diagnosis of Botrytis cinerea infection in cut rose flowers under the postharvest application of Methyl Jasmonate (MeJA). Three cases treated with different concentrations of MeJA (0.1, 0.2, and 0.3 µl.l⁻¹), a control (0 µl.l⁻¹ MeJA) and an ethanol-treated case (20 µl.l⁻¹ ethanol) were considered as five treatments in this study. Infrared images of MeJA-treated and non-treated flowers were captured during five consecutive days. Eight days after inoculation, disease severity in all concentrations of MeJA was significantly lower than that of control and ethanol treatments. Maximum temperature difference (MTD) index and median temperature could be used to diagnose the existence and growth of fungal pathogen, at least a day before any significant visual symptoms appear. To identify some robust features for classifying the infected and non-infected flowers, analysis of temperature frequency distribution was implemented. Laplace and normal distributions were considered as the best fitted probability distributions based on the shape of thermal histograms. Parameters of normal and Laplace probability density functions were estimated and the most effective attributes were selected. A radial-basis-function neural network with 60 neurons in the hidden layer was designed to classify and distinguish the infected flowers from the healthy ones. Results showed that the network can classify the infected and non-infected flowers with a 96.4% correct estimation rate.

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Surfactin is one of the most efficient biosurfactants excreted by Bacillus subtilis which displays the highest potential as induced systemic resistance elicitor among all metabolites produced by B. subtilis. Environmental factors have considerable effect on surfactin production. In this study surfactin production of two Bacillus subtilis strains were analyzed using high performance liquid chromatography (HPLC). C14 and C15 surfactins were detected in the ethanol extract from acid-precipitated supernatant. HPLC analyses of different media including Nutrient Broth (NB) medium, NB plus 40g/l glucose, NB plus 10% soil extract and NB plus 10% plant extract medium, clearly showed that these bacteria produced different amounts of surfactins C14 and C15 in these media. Surfactin production in NB/plant medium was relatively the highest in quantity. Microelements analysis of media containing plant and soil extract with atomic absorption spectrometry showed high amounts of Fe, Mn and Zn in medium containing plant extract compared with that of soil extract. Since these elements play an important role in surfactin production, high amounts of Fe, Mn and Zn in NB/plant extract medium compared to the NB/soil extract medium could be the possible reason for relatively higher amounts of surfactins C14 and C15 produced in NB/plant medium. So adding these important elements to soil may boost biocontrol effect of B. subtilis against plant pathogens.

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Huanglongbing (HLB) also known as citrus greening, is a destructive disease of citrus and now, is considered as a new emergence and spread out threat to the Middle East and North Africa (MENA) citrus production. In a survey conducted in southern Iran in 2013-2014, 77 citrus samples exhibiting symptoms of HLB were collected. Single-step and nested polymerase chain reactions (PCR) were employed to determine the presence of the phloem-limited bacterial pathogens ‘Candidatus Liberibacter asiaticus’(CLas), phytoplasma and Spiroplasma citri. Both CLas and phytoplasma were detected in HLB-affected citrus trees as co-infection (7.79%) and single infection (10.38% for phytoplasma and 42.85% for CLas). According to the proposed 16S rDNA-based phytoplasma classification scheme, the HLB-associated phytoplasma from this study was a member of peanut witches’broom (16SrII) phytoplasma group. This is the first report of association of a phytoplasma with HLB in sweet lime in the world and first record of association of CLas with sour orange (Citrus aurantium L.) and sweet lime in Iran.

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The thermophilic fungus Mycothermus thermophilus is one of the most important thermophilic fungi in mushroom composting process. Thirty nine isolates of M. thermophilus were collected from nine provinces of Iran and were identified as M. thermophilus based on morphological features and ITS regions. The studied isolates significantly increased the growth of Agaricus bisporus hyphae compared to control when used in vitro situation. Also the colony morphology of the mushroom changed when it grew on the colony of M. thermophilus. While the studied thermophilic isolates were morphologically different, no diversity was observed in terms of Random Amplified Polymorphic DNA (RAPD) finger-printing. The genetically clonal population of M. thermophilus collected from Iranian mushroom composting farms was attributed to lack of sexual reproduction, similar raw materials used in compost formulations, compost temperature, and concentration of ammonia during pasteurization as selection pressures.

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To assess the variability of Macrophomina phaseolina (Tassi) Goid, the causal agent of charcoal rot of Sesame, sixty isolates recovered from ten geographic regions, were analyzed using inter simple sequence repeat (ISSR) and universal rice primer (URP) markers. Isolates were grouped into eight clusters at 78% genetic similarity level. Our results showed that the five ISSR primers produced 105 bands of which 77.11% were polymorphic and eight URP-PCR primers generated 135 bands of which 66.84% were polymorphic. These methods showed a considerable genetic diversity among Iranian isolates, but no correlation was found between genetic diversity and geographical origins of the isolates. Analysis of molecular variance revealed that a large proportion of genetic variability resulted from the differences among isolates within regions. The findings of this study demonstrated that the low-genetic differentiation (G_ST) and high gene flow (N_m) among populations had a significant effect on the emergence and evolutionary development of M. phaseolina.