فصلنامه بیماریهای گیاهی
سال چهل و هفتم شماره 3 (پاییز 1390)

For taxonomic studies, 37 isolates of the genus Rhizopus obtained from various hosts in Tehran and west Azarbaijan provinces were investigated. Internal transcribed spacer (ITS) region and D1-D2 regions of the large subunit ribosomal DNA were amplified using ITS1/NL4 primer pair. Amplified products were digested with four restriction endonucleases HaeIII, RsaI, HinfI and MspI and seven representative isolates were selected and sequenced. These and 23 isolates from GenBank were analyzed to determine their phylogenetic relationship. In contrast to the extreme variation of the ITS region and D1/D2 regions of LSU rDNA proved to be valuable in the phylogeny of the genus Rhizopus. Based on the results, 37 isolates were divided into three groups, R. lyococcus, R. stolonifer and R. oryzae. R. lyococcus is a new record for Iran which is the first report of R. stolonifer on nectarines and R. oryzae on persimmons and nectarines. R. stolonifer on nectarines is a new record for the world.

Histopathology of fungus Ustilago maydis causal agent of common corn smut was investigated in exposure to both light and electron microscopy in cultivars SC301, SC647 and DC704 that were known before as sensitive, semiresistant and resistant cultivars, respectively. For providing fungal inoculum, mixture of teliospors were collected from Khozestan province, then cultured in CMA and PDA + 10% dextorose media and incubated 5 days at 25ºC. Fungal inoculums were inoculated under 2 different methods, injection and spray; then samples were collected and fixed 2, 4, 11 and 25 days after inoculation. 18 days after inoculation, disease symptoms were first observed in injection and then spray method. Fungal penetration in host by scanning electron microscopy showed that penetration is possible both in direct and indirect ways and fungus makes a swollen and brilliant appressorium. Light microscopy of fungal extension showed that infection cells were larger than intact ones and extension of fungal hyphes were first intercellular and then intracellular. Some intracellular branches were lobed and others terminated in pointed finger like or other kinds of swelling. At the site of teliospore formation, cavity of mycelium was formed that contained both mature and immature teliospores. These young teliospores were surrounded with gelatinus sheath. Corn infection happened easily under control conditions and made an appropriate system for investigation of some aspects of pathogen interaction.

The interaction of salt and water stress to infect the roots by Macrophomina phaseolina, and affect the ion composition and growth of sorghum (Sorghum bicolor) was studied in a greenhouse experiment (19-35˚C). Treatments consisted of 4 levels of salinity (0, 1400, 2100 and 2800 mg of NaCl kg-1 soil) and three water stress levels (3, 7 and 10 irrigation intervals). Infested soil containing 100 viable microsclerotia g-1 of a melon isolate of M. phaseolina and non-infested soil were used for all treatments. The experiment was arranged in a completely randomized design with four replications. Six-week-old sorghum seedlings after their transferring to infested and non-infested soil were exposed to salt stress, after which, water stress was started. Shoot dry weights were reduced by increasing salinity levels. This reduction was more pronounced in infested soil than in non-infested. Increasing irrigation intervals reduced salt injuries. Shoot and root colonization by M. phaseolina significantly increased by increasing salinity levels up to 1400 mg of NaCl kg-1 soil. Moreover, salinity and M. phaseolina interaction increased the concentrations of Na+ and Cl- compared to salt stress per se, but negatively correlated with increasing irrigation intervals. Concentration of K+ was in contrast with Na+ and Cl-. Also, disease symptoms appeared only in the highest irrigation intervals (7 and 10 days). Consequently, more infected crown and root were observed by increasing irrigation intervals and NaCl levels up to 1400 mg kg-1 soil.

RNA directed RNA polymerase 1 (RDR1) expression is induced by virus infection and appears to be involved in resistance to infection by some viruses. In the present study, reactions of two transgenic tobacco (Nicotiana tabacum cv. Samsun NN) lines in which the expression of their RDR1 gene was silenced were assessed against several plant viruses under greenhouse conditions. Silencing the RDR1 gene in tobacco led to no change in the response to virus infection but in others, as expected, an enhanced susceptibility to tobacco mosaic virus was observed. By contrast, lines of tobacco with silenced RDR1 gene showed enhanced resistance to infection by several viruses, viz., tobacco rattle virus (genus Tobravirus) and tomato bushy stunt virus (genus Tombusvirus). These results suggest that complex interactions occur between the innate resistance pathways, so inhibiting one component involved in resistance may show effects on other aspects of innate resistance. Nevertheless, suppressing RDR1 gene expression may provide novel, broad-spectrum resistance to a range of viruses.

Sleeping hibiscus (Malvaviscus penduliflorus) plants showing leaf spot symptoms have been observed since 2008 in Sari. The spots were 2-4 mm in diameter, necrotic angular to irregular brown to black, and surrounded by chlorotic halos. A levan positive, Gram and oxidase negative bacterium was isolated from the symptomatic leaves on sucrose nutrient agar (NAS). The bacterium produced fluorescent pigment on medium B of King and rotted potato tuber slices. Pathogenicity of selected strains was confirmed by inoculation of sleeping hibiscus, sweet orange and seville orange with bacterial suspension of strains. In electrophoretic profile of cell proteins the strains were, partially similar to the strains of Pseudomonas viridiflava. 16S rRNA gene of strains were sequenced at Millegen company(France) using standard primers, nucleotide sequence analysis in GenBank showed that the nucleotide sequence of 16s rRNA gene of sleeping hibiscus strains had 99% similarity to few reference strains of Pseudomonas viridiflava. In ERIC-BOX and REP- PCR, the fingerprints of the strains from sleeping hibiscus showed 45% and in Is50- PCR 46% similarity to the reference strain of Pseudomonas viridiflava. Based on phenotypic, pathogenicity and genomic properties, the causal agent of leaf spot of sleeping hibiscus (Malvaviscus penduliflorus) were identified as levan positive strains of Pseudomonas viridiflava.

The shallow bark canker of Persian walnut (Juglans regia L.) trees incited by the bacterium Brenneria nigrifluens affects the bark of trunk and scaffold branches. Fifty-nine isolates of bacterium were isolated on the basis of disease symptoms from 114 plant samples, collected from Mazandaran, Golestan, Kerman and Kermanshah provinces. Furthermore, 10 isolates from Fars province, one isolate from Kohgiluyeh-va-Boyerahmad province and one isolate from Kurdistan province were received. Based on physiological and biochemical characters, bacterial isolates were divided into two groups and identified as Brenneria nigrifluens or strains close to B. nigrifluens. The electrophoretic profile of the strains cell proteins also confirmed this grouping. Isolates were assessed by polymerase chain reaction (PCR) using a specific primer pair for B. nigrifluens. Twenty four isolates produced the expected band of approximately 255 bp and were named PCR+ strains. Pathogenicity tests performed with one PCR+ isolate and three PCR¯ isolates showed that only PCR+ isolate was able to cause cankers in walnut seedlings. Eight weeks after inoculation of PCR+ isolates on Persian walnut seedlings, water-soaked cankers appeared around the inoculation sites. The bacterium was reisolated from the bark lesions of the inoculated seedling on EMB-agar medium. To investigate the certitude of the primer pair, one PCR+ isolate and three PCR¯ isolates from different geographic locations were analyzed by sequencing 16S rDNA. The results revealed that PCR+ isolates were Brenneria nigrifluens. On the basis of this study, it can be concluded that general phenotypic tests are not reliable enough to identify B. nigrifluens isolates and that specific detection of the pathogen with pathogenicity test or PCR is necessary.

In order to study the reaction of 15 cucumber cultivars to the root-knot nematode, Meloidogyne javanica, after nematode purification on the tomato cultivar Rutgers, with the help of morphological characters and species-specific primers the nematode species was identified. Experiment was conducted on the basis of completely randomized design in the greenhouse conditions using sterile soil. In resistance assessment of different cucumber cultivars to this nematode, the growth factors of the host plants and nematode reproduction were used with different methods. Results showed that all the greenhouse cultivars used in this experiment were susceptible to M. javanica and only two local cultivars from Isfahan (Chambar and Dastgerdi) recognized as tolerant. Also the amount of vitamin C, Calcium and Potassium minerals as well as leaf area index decreased in infected plants.

In recent years, cucumber crown and root rots have been observed in greenhouses of Yasouj. In order to isolate the pathogenic agents, tissues from infected parts were cultured and twenty five isolates were recovered. Based on the morphological characters and temperature requirements, two species, namely, Phytophthora melonis and Pythium aphanidermatum were identified. Pathogenicity tests of Ph. melonis and P. aphanidermatum on different cultivars of cucumber were done under greenhouse conditions. The inoculating of crown and root systems was conducted by inoculum on vermiculite – hemp seed extract. Eventually, indices including colonization of root & crown, and seedling death were measured. It was shown that Fadia and Bahman cultivars were the most susceptible and Mehr and Close cultivars were the least susceptible in reaction to Ph. melonis, respectively. In reaction to P. aphanidermatum, Mehr and Katrina cultivars were the most susceptible for crown colonization and also Katrina cultivar was the most susceptible for root colonization.

In the course of a research project during 2008-09, diseases of mulberry shoots in Izadshahr (Mazandaran), Astaneh Ashrafieh (Guilan) were investigated. The results showed that phloeospora leaf-spot caused by Phloeospora maculans on Morus species is a widespread disease in the above mentioned cities. Also, diatrype die-back on white mulberry, caused by Diatrype stigma, is a rather important disease in Astaneh Ashrafieh. A full account of the symptoms of the diseases and also details of their causal agents are presented here. Malberry is a new host for Cytospora cincta and Diatrype stigma in Iran. Specimens of the two diseases are preserved under IRAN 14695F and IRAN 14692F in the Herbarium of Iran (IRAN) at the junior author's address.

During the study of Powdery mildew related to Erysiphaceae family from Abardeh region in Mashhad (Khorasan-e-Razavi province), one species of Rose (Rosa persica Michx.ex Juss, Summer 2006) with symptoms of Powdery mildew was collected. The symptoms observed with the naked eye including the dense cottony shape mycelia infected the leaf surfaces as well as some parts of the stem. Mycelia were dense, amphigenous, conidia with two types, primary conidia lanceolate, and secondary conidia ellipsoid to cylindric, 15-11*48-35 µm, chasmothecia with mycelioid appendages, and asci numerous, 67-90*30-40 µm, with 2-spores, 11-20*30-39µm (Fig 1). According to A Monograph of the Erysiphales (Braun,U.1987.A Monograph of the Erysiphales) the fungus was identified as Leveillula taurica (Lev) Arnaud. Based on the literature, this is the first record of the genus Leveillula on Rosa from Iran.

Members of Cyperacea family are among the common host species of rust fungi. To our knowledge, till now seven Puccinia species have been recorded on cyperaceous plants in Iran. Of these, four species were known on Carex. In present report, other rust species are reported on Carex orbicularis subsp. kotschyana (Boiss. & Hohenacker) Kukkonen. This report is based on infected specimen collected from Central Alborz. The studied specimen had the following features: Uredinia hypophyllous, arranged in row, minute, ellipsoid or oblong, light brown, on chlorotic stripes; urediniospores subglobose, ellipsoid or obovoid, 22.5-30(-32.5) x 20-27.5 µm, walls yellowish brown, echinulate, 1.5-2.5 µm thick, germ pores 3-4 equatorial. Telia hypophyllous, oblong, on short chlorotic stripes, semi-compact blackish brown; teliospores mostly clavate rounded or truncate at apex, rarely acuminate, constricted at septum, narrow at the base, 37.5-60 x 15-22.5 µm, walls light chestnut brown, 1.5-2.5 µm thick at sides, 6-12.5 µm at the apex; pedicels persistent, about the same length as spores (Fig 1). These features were not compatible with previously reported rust species on Carex spp., from Iran (ABBASI, M. et al. 2000. Rostaniha 1: 23-41). However, the characteristics were well fitted with description of Puccinia urticata F. Kern, written by Zwetko (ZWETKO, P. 1993. Bibliotheca Mycologica 153, 222 p). This is the first report of P. urticata from Iran. Moreover, C. orbicularis subsp. kotschyana is a new host for this fungus. Material examined On Carex orbicularis subsp. kotschyana, Central Alborz, Karaj-Chalus road, 1 km N of Kandevan pass, 2380 m, 17.Aug.1993, M. Abbasi, J. Fatehi and O. Foitzik (IRAN 15212 F), II+III.

Ficus benjamina commonly known as the Weeping Fig or Benjamin's Fig, is a species of Moraceae, native to south and southeast Asia and Australia. It is a topiary tree reaching 15 metres in height in natural conditions. Ficus benjamina is planted as a potted plant and as an ornamental shrub in gardens in Mazandaran and elsewhere in the country. Stem and crown gall symptoms were observed on Ficus benjamina in the greenhouses in Mazandaran. The affected tissues were transferred to the laboratory and the galls were washed in running water and surface sterilized for a few minutes in 1% sodium hypochlorite solution. The galls were rinsed with several changes of sterile distilled water (SDW), crushed in SDW and were left for 30 minutes at room temperature. A few drops of the suspension were streaked onto plates of PDA (Potato dextrose agar). The plates were incubated at 28°C. Colonies similar to Agrobacterium (white, mucoid and convex colonies) were selected. The strains were inoculated on the stems of tomato and the strains that produced galls three weeks after inoculation were selected. The strains produced ketolactose from lactose, Arabinose, sorbose, melezitose, dulcitol, fructose, sorbitol, cellobiose, xylose, raffinose, xylitol, lactose, acetate, lactate, D-tartrate and propionate were used by all strains whereas erythritol, valine, tyrosine, L-tartrate, malonate and citrate were used by none of them tested. All strains amplified a 224 bp fragment of the VirD2 gene in PCR reactions using VirD2A/VirD2C primers. In BOX-PCR, the fingerprints of the strains from Ficus benjamina showed 60% and in Is50- PCR 75% similarity to the reference strain of Agrobacterium radiobacter ICMP (Intern. Collection of Microorg. from plants) 8586. Based on morphological, physiological and biochemical properties and the results of the PCR tests, the strains were identified as Rhizobium radiobacter. In a previous report, Rhizobium larrymoorei was identified as the incitant of stem and crown gall on Ficus benjamina in Mazandaran province (Ghasemi, et al.2006. Iran. J. Plant Path. Vol. 42. P 197), therefore, confirmation of the presence of this species needs further investigation.

Gummy spike blight diseases of cereal has only been reported on wheat up to 2010 and the causative agents identified as Rathayibacter tritici and R. iranicus (Babaeezad, V., Rahimian, H. 2002. Iran. J. Plant Pathol. 38:47-55; Rahimian, H.1993. Proc. 11th, Iran. Plant Protec. Cong., Guilan Univ., Rasht, Iran. 35. Abst.). In the years 2010 and 2011, symptoms of gummy spike blight were observed on barley (Hordeum vulgare) in Ilam province and the associated bacterium was isolated and identified as a Rathayibacter species phenotypically non assignable to any species (Soltani. M., Fuladvand. A., Rahimian, H. and Babaeezad, V. 2010. Proc. 19th. Iranian Plant Protection Cong. P 429. Tehran, Iran). In the present study, strains of the bacterium associated with gummy spike blight were isolated from infected wheat and barley plants from several areas of Iran and their phenotypic and some genotypic features were determined. All strains were gram-positive, aerobic and catalase positive, but negative in tests for indole and acetoin production. The strains hydrolyzed esculin and Tween80 but not gelatin, starch, or casein. None of them produced oxidase nor arginine dihydrolase. Results of tests for potato rot, nitrate reduction, lecithinase, phosphatase, urease, tyrosinase, ketolactose and methyl red reaction were negative. The strains utilized xylose, L-arabinose, mannitol, inulin, citrate, fumarate, acetate and pectate but did not use malonate, pyruvate, citraconate and nicotinate. They produced acid from glucose, galactose, fructose, and mannose but not from rhamnose, raffinose or salicin. Strains isolated from barley, in contrast to isolates from wheat, used amygdalin but not succinate or L-tartarate. Both R. tritici and R. iranicus are found to be the species associated with these characteristics (Zgurskaya, H. I., Evtushenko, L. I., Akimov, V. N. & Kalakoutskii, L. V. 1993. Int J Syst Bacteriol. 43, 143-149). Strains isolated from barley and R. iranicus isolates in contrast to the strains identified as R. tritici, could not grow on CNS medium and were unable to use sebacate. Comparison of the protein profiles of the isolates with those of Rathayibacter toxicus strain ICMP 9525 (International Collection of Microorganisms from Plants, Aaukland, New Zeeland), R. iranicus strain IBSBF 636, R rathayi strain IBSBF 635 and R. tritici strain IBSBF 632 (Biological Institute Culture Collection Phytopathogenic Bacteria, Compinas, Brasil,) led to differentiation of the isolates into three groups. Based on the phenotypic features and the protein profiles, four isolates of wheat were similar to R. tritici and twenty two isolates were almost identical with the R.iranicus type strain. Strains isolated from barley formed an independent group. BOX-PCR wiht BOX A1R as primer (Versalovich, J., Schneider M. de Bruijn F.J and Lupski, J. R. 1994. Methods Mol Cell Biol 5: 25-40) was used to assess the genetic heterogeneity of the strains. The method was carried out as described by Versalovich et al. (Versalovich, J., Koeuth, T. and Lupski, J. R. 1991. Nucleic acids res. 19:6823-6831). Time and temperature conditions consisted of a predenaturation step for 4 minutes (min) at 95 ̊C for 4 min, followed by 34 steps of denaturation at 94 ̊C, annealed at 50 ̊C for 1 min and extended at 72 ̊C for 1 min; a final extension step of 10 min at 72 ̊C was also included. The products were electrophoresed in 1/5% agarose gel and the patterns scored for the presence or absence of prominent DNA bands. Similarity matrix of the strains was prepared using the Jaccard coefficient and cluster analysis, and the UPGMA method was performed using NTSYS-pc. At the similarity levels of 48%, 57%, 71% and 89%, the isolates fell into two, three, six and thirteen groups, respectively. The lowest similarity level (21%) was found between the strains isolated from wheat and barley with R. toxicus. Barley was similar to R. iranicus, R. rathayi and R. tritici at the levels of 75%, 56% and 50%, respectively. A wheat isolate showed phenotypically 66% similarity to type strain of R. iranicus, whereas its similarity to the type species of R. rathayi was 71%. Overall, although there was only slight phenotypic difference among the strains in each species, genetic heterogeneity was discernible in members of both species. At the present, barley isolates with characteristics intermediate among R. iranicus, R. tritici and R. rathayi, can be regarded as strains of a potentially undescribed species. Positive taxonomic identity and placement of these strains is pending the results of more experiments on genotypic features of their representative strains.