فصلنامه بیماریهای گیاهی
سال چهل و هشتم شماره 3 (پاییز 1391)

The Basidiomycetes fungi U. maydis is the causal agent of head smut of maize and sorghum. This fungus has haploid, diploid and dikaryotic forms in its life cycle. Fusion of compatible haploid cells on leaf is required to generate dikaryon form that is capable of plant infection. Although, U. maydis can penetrate through the roots and aerial parts but the disease symptoms only appear on the aerial parts. As regards root exudates play an important role in the plant-microb intractions, in this study, the effect of GR24 (as a analogue synthesis of strigolactones) on the physiological and morphological activities of U. maydis was investigated. Using different methods to measure cellular respiration, rates of respiratory changes after adding GR24 was measured. Our observation show that one hour after this induction, cell respiration increased 11 percent but in three and five hours cells induced respiration was found to be reduced by eight and five percent, respectively. This reduction can be due to detoxification effects of fungi in counter with exogenous molecule. Gene expression levels of some genes involved in biotrophy and cell respiratory were assayed using the Real-time PCR approach. The results show that gene expression involved in cellular respiratory was increased in induced cells by GR24 in compared to control cells. Nevertheless GR24 didnt effect on the morphological change and the yeast- mycelial form transition of haploid strain of U. maydis. This molecule could affect on root penetration and plant defense reactions by increase of cell respiration but has any role to play pathogenesis.

Symptoms of stone fruits canker were observed in some part of Kerman province. Strains in which the gram reaction was negative were investigated based on physiological and biochemical characteristics. Some tested strains were positive for levan production, hydrolysis of tween 80, gelatin, casein, arbutin, catalase, fluorescent pigment production, tobacco HR and tolerant to 5% NaCl but negative for oxidase, urease, hydrolysis of starch, arginine dihydrolase, growth at 370 C, potato rot, indole production and reduction of nitrate to nitrite. These strains produced acid from glucose, fructose, cellobiose, sucrose, galactose, mannose, mannitol, xylose, sorbitol, inositol and arabinose but did not produce acid from lactose, maltose and D-tartarate. Thses strains prevented the growth of Geotrichum candidum by toxin production and PCR amplification with the syrB primers yielded the expected 720 bp fragment from all pathogenic strains. The electrophoretic profiles of the cell proteins of the strains were closely similar to reference strain, Pseudomonas syringae pv. syringae(IBSBF 451). The pathogenicity of representative strains was improved by injection of suspension of 106 cell/ml to branches tip and flowing symptoms observation and re-isolation of agent. The isolates were determinate as Pseudomonas syringae pv. syringae based on phonotypical futures and sequences of ITS. Antibacterial effect of plant extracted was studied under laboratory conditions on bacterial isolates. The results were compared with effects of 13 well known antibiotics. Results indicating that among used antibiotics, Tetracycline, Oxytetracycline and Doxycycline have significant effect on the growth of all strains but, all the strains showed resistance to Penicillin, and. This study revealed that all the extracts have antibacterial effect and among them extracts of Ferula assa-foetida and Rosmarinus officinalis have the best effect that is comparable to the effect of Tetracycline.

Eight collections of Pseudocercospora species from different localities in Northern provinces of Iran were obtained and examined during Summer-Autumn of 2010. Furthermore all of specimens belonging to the genus Pseudocercospora deposited in fungal collection of Ministry of Jihad-e-Agriculture in Iranian Research Institute of Plant Protection were re-examined. Eight species on nine host plants were identified including: Pseudocercospora atromarginalis (on Solanum nigrum), P. cruenta (on Vigna sinensis), P. griseola (on Phaseolus vulgaris), P. heteromalla (on Rubus sp.), P. kaki (on Diospyros lotus, D. kaki), P. punicae (on Punica granatum), P. salicina (on Salix alba), P. vitis (on Vitis sylvestris). Among these, P. heteromalla and P. salicina are new records for mycobiota of Iran. The name of Phaeoisariopsis griseola changed into Pseudocercospora griseola.Other species were already been reported from Iran.

From 2008 to 2009 grapevine, rose and sugar beet samples with gall symptoms on crowns and roots were collected from different areas of Fars and Kohgiluye and Boyerahmad provinces. The extracts from galls were cultured on NA, DIM Agar, 1A and RS media. After purification the isolates have been characterized using standard bacteriology tests, the Agrobacterium isolates were identified as A. vitis (from grapevine) and A. tumefaciens (from rose and sugar beet). Pathogenicity tests were done on bryophyllum, tomato and sunflower. Most isolates produced gall on at least one of these plants but some of the isolates did not. The isolates were able to amplify 224 and 730 bp DNA fragments with primers A/C´ and VCF/VCR in PCR test, respectively. The isolates showed high diversity in rep-PCR and the isolates of Fars and Kohgiluye and Boyerahmad were divided into two main groups. Group I was divided into two subgroups which included A. vitis isolates subgroup (isolates from grapevine) and A. tumefaciens isolates subgroup (isolated from other hosts). Group II included a small number of A. tumefaciens isolates. Maximum similarity (88%) was observed in A. vitis species. Clustering based on genotypic characteristics somewhat confirmed clustering based on phenotypic characteristics. The rep-PCR and phenotypic characteristics could differentiate A. vitis and A. tumefaciens species.

Bermuda grass southern mosaic virus (BgSMV) and Maize dwarf mosaic virus (MDMV) despite a 90 nucleotide difference in coat protein (CP) gene are very similar in serological and molecular properties. In the present work, cross protection between BgSMV and MDMV was studied. The experiment consisted of five treatments in five replications (each replication consisting of one pot with 10 plants). The treatments consisted of mechanical inoculation of sorghum with each virus followed, after symptom appearance, by challenge inoculation with the other virus, simultaneous inoculation of both viruses, and single-virus inoculations. Two weeks after inoculations, plant samples were taken from treated plants and subjected to RT-PCR using mRNA Capture Kit (Roche) and specific primer pairs MD3F/MD1R and BgSMF90/ BgSMR90b. The results showed that prior infection of plants by either MDMV or BgSMV protects against super infection by the other virus. Based on these results and previous biological and molecular information, BgSMV and MDMV may be regarded as closely related viruses or the strains of the same virus despite their biological and molecular differences. Cross protection was also studied between each of these viruses and Iranian Johnson grass mosaic virus (IJMV) as control, and no cross protection was observed between IJMV and either MDMV or BgSMV.

Almond witche's broom (AWB) is an economically important disease associated with Candidatus Phytoplasma phoenicium, a phytoplasma belonging to the pigeon pea witche's broom group (16SrIX). Induction of different symptoms under natural conditions and after graft inoculation of bitter almond seedlings suggests genetic diversity of Keman 1, Kerman 2, Moshkan, Neyreez 1, Neyreez 2 and Sanandaj isolates of AWB in Iran. Restriction fragment length polymorphism, percent sequence homology and phylogenetic analysis were used for further assessment of genetic diversity among these isolates. RFLP analysis of P1/P7 PCR products (1.8 kbp) using AluI,haeIII, HhaI, HinfI, HpaII, Msel, TaqI and TasI revealed that Moshkan and Kerman 2 isolates are distinguishable from other isolates because of the different dijestion patterns produced by Cfo1 1 and Hinf1 enzymes, respectively. On this basis Moshkan isolate fell into group 1, Moshkan isolate into group 2 and Kerman1, Neyreez1, Neyreez2 and Sanandaj isolates into group 3. Percent homology of SR sequences differentiated AWB isolates. Moshkan isolate being distinctly different from the othes. Phylogenetic tree constructed with 16S rRNA gene and SR sequences also indicated genetic diversity of AWB isolates.Results of the present study shows that the 600 base pairs of rRNA gene operon following 1200 bp of the 5' start end of rRNA operon is the preffered fragment for studing genetic diversity of AWB phytoplasma. In the present stuy AWB disease is reported for the first time from Kerman and Sanandaj almond growing areas of Iran.

Medicinal plants such as plantain, Plantago major, due to having various medicinal compounds are of high importance in pharmaceutical industry. In the present research, while assessing the pathogenicity of root-knot nematode, Meloidogyne javanica on plantain medicinal plant, the effects of various nematode initial inoculums on plant growth parameters as well as nematode reproduction were investigated. Then seed mucilage content, thousand-seed weight, the amount of Quercitin and Ursolic acid compounds as well as Sodium, Potassium, Magnesium and Calcium elements of infected Plantago major 90 days after inoculation recorded. Plantain found to be sensitive to the nematode and in the infected plants, seed mucilage content, thousand-seed weight and the amount of Ursolic acid compound decreased but amount of root Sodium and Calcium elements and shoot Quercitin increased. While increasing the nematode initial inoculums, shoot fresh weight and root length and fresh weight decreased but root fresh weight increased. The highest reproduction factor was observed in plants inoculated with the lowest level of nematode population tested (1000J2/2kg soil).

Grapevine fanleaf virus (GFLV) is one of the most destructive grapevine viruses worldwide. The best strategy to control the virus is to cultivate virus free plant material in non-infested soils. Therefore, detection of the virus plays a key role in control of GFLV. To establish a reliable and sensitive technique for detection of GFLV in the stock grapevines or in preparation of virus free grapevines, samples from different vineyards of Iran were collected in 2009 and their infection by GFLV was assessed by DAS-ELISA. Fourteen ELISA positive samples were subjected to RT-PCR using GFLV published primers and the primers designed in this study. Most of the primers available in literature failed to detect GFLV in all 14 tested samples, whereas the primers designed in this study detected the virus in all mentioned samples. The efficacy of the designed primers in this study was confirmed by detection of the virus in further 35 ELISA positive samples collected from other provinces in 2010. Moreover, GFLV infectivity of grapevines for the first time was confirmed by green grafting. The results revealed that the combination of the green-grafting with RT-PCR method, using the introduced primers in this study, offers a reliable method for detection of GFLV isolates.

Interaction between arbuscular mycorrhizal fungi Glomus mosseae and Glomus intraradices and root knot nematode Meloidogyne javanica was studied in tomato under greenhouse condition. The experiment was conducted as factorial and completely randomized design with 6 treatment and 9 replications. Plant growth indices (shoot height, root length, shoot and root wet and dry weight) as well as nematode developmental parameters (gall numbers and egg mass on each plant, egg numbers in each egg mass, J2 numbers in soil and reproduction factor Rf=Pf/Pi) used for fungal effects on nematode and, fungal developmental parameters (spore numbers and root colonization percent) were used for nematode effects on mycorrhizal fungi. Results showed mycorrhizal fungi improve growth of different plant parts and decreasing nematode damages. No significant differences were observed between two mycorrhizal fungi. Despite decreasing in fungal indices (spore numbers in soil and root colonization rate) affected by nematode, there are not significant differences among indices with or without nematode inoculation.

Zucchini yellow mosaic virus (ZYMV (was isolated from squash samples with symptoms of stunting, leaf blistering and distortion of leaf and fruits, collected in cucurbit fields in Kaftarak area (Shiraz) and verified by ELISA test, designated Fars isolate (ZYMV-F). The virus was propagated in squash plants under greenhouse conditions and purified from infected tissues. The antiserum obtained from purified virus was used in serological tests. Complete sequence of the virus genome was determined using mRNA capture kit and PCR with specific primers followed by cloning and sequencing. Following the sequence walking technique a contig of 9591 bp (JN183062) representing the whole genome of the virus was generated. Phylogenetic analysis based on the nucleotide and amino acid sequence of the whole genome and individual genes showed that ZYMV-F is closest to Israel and Slovakia isolates. Analysis of partial sequence of CP gene showed that ZYMV isolates in Iran are very similar, with Fars and Karaj isolates clustering in the same clade.

Ceratorhiza hydrophila is frequently isolated from rice sheath spot in paddy rice of Guilan province. In this study, morphology, pathogenicity and its phylogenetic affinities based on rDNA sequence data were studied. Results revealed that the fungus can infect rice plants and cause small yellow to pale brown spots on the leaf sheaths one week after the inoculation. On PDA colonies were white at the beginning and were eventually changed to brown, with visible growing rings. Sclerotia were usually round, regular, very small, measured 275-737.5 µm, and were formed on the surface of Petri dishes four days after the cultivation. They were initially white, and then changed to buff or blackish brown. Mycelial cells were 3.75-7.5 × 37.5-192.5 µm. To study phylogenetic position and molecular identification, the 530 bp of rDNA ITS region was sequenced. Results showed that C. hydrophila is closely related to Rhizoctonia species rather than other Sclerotium taxa. These results are consistent with the morphology of colony and sclerotia of this fungus. This is the first report of the pathogenecity of this fungus on rice from Iran.

To determine the prevalence of Potato virus Y (PVY) in tobacco fields of Golestan province, a total of 182 samples showing virus type symptoms PVY infection in collected samples was assessed using a polyclonal antiserum in DAS-ELISA test. Of all collected samples 90 were infected by PVY. Viral RNA was extracted using RNX-Plus kit. The extracted RNA was reverse transcribed using oligo dT primer to create the respective cDNA, wich was amplified by PCR using a PVY-specific primer pair designed on CP region. After verification on 1% agarose gel in electrophoresis, the expected band of ~1000 bp size was amplified. PCR products were directly sequenced and the sequences of about 850 nucleotides were aligned with 30 sequences of the GenBank. Multiple alignment and phylogenetic analyses were performed by ClustalX and BLAST programs in BioEdit software and phylogenetic dendrogram was produced using maximum parsimony method. The results showed that all PVY sequences can be placed into 4 groups. Aliabad and Fazelabad isolates were grouped with isolates from Italy, Spain and Boushehr, while Minoodasht isolate was grouped with isolates from the Netherlands, United Kingdom and Taiwan.

Alfalfa witches’ broom (AWB) is a serious disease of alfalfa in Bushehr province. For characterization of Bushehr AWB phytoplasma, alfalfa plants with typical symptoms of AWB were selected in different regions of Busher province including Bondarooz(1 isolate),Sarkoreh (2 isolates), Poshtpar(1 isolate) and Tange- Eram (1 isolate), transferred to greenhouse and used as the source of the disease agent for transmission and molecular studies. Agent of AWB was transmitted from each isolate to periwinkle via dodder inoculation. Agents of all isolates caused little leaf, internode shortening, yellowing, virescence, phyllody and witches’ broom symptoms in periwinkle plants. DNA fragments of approximately 1800 bp of ribosomal operon were amplified with primer pair P1/P7 by direct PCR from total DNA samples extracted from naturally alfalfa infected plants and periwinkle plants inoculated with agents of AWB isolates. P1/P7 primedPCR products (1800 bp) were analyzed by digestion with AluI, HpaII, Cfol, HaeIII and RsaI enzymes. RFLP patterns analyzed with these enzymes were similar to each other and those of AWB phytoplama from Fars (Mobarak Abad and Juyom 1) and yazd, 16SrII group Related phytoplasmas but distinguishable from a Fars isolate of AWB(Juyom2) by a CfoI site. Phylogenetic analyses of full length 16S rRNA gene sequence also clustered agent of Bondarooz AWB phytoplasma with phytoplasmas of 16SrII group. Based on the results of the present study, a phytoplasma relatede to peanut witches’ broom (16srII) is associated with AWB disease in Bushehr province. This is the first report of AWB in Bushehr province

In this research, the effect of (spinach, capsicum, beet, turnip and radish) extracts the preventive and curative control of cucumber powdery mildew in greenhouse tests was investigated for. The acetonic, methanolic or water extracts of leaf tissues of each plant at concentrations one and 5% (w/v) was sprayed on the plants at either one day before, or 1 day after the plant inoculation with spore suspension of pathogen. Disease severity was evaluated based on the number of disease spots per leaf ten days after inoculation. The effectiveness of this extracts was also compared with Penconazole fungicide and Reynoutria sachalinensis (Rs) extract, two commercial compounds for the control of disease. Results showed that all tested extracts reduced the disease severity on the treated leaves, but in varying degrees. In comparison with Rs, the acetonic extracts of pepper and beet, and the methanolic extracts of spinach and turnip at the concentration of 5% showed relative effectiveness in prevention of disease. On the other hand, the methanolic extract of turnip at the concentration of 5% showed the highest curative effect and its curative efficacy was the same as penconazole. Also, the most effective time for application of extracts of spinach and Rs was 24 hours before pathogen inoculation, the application of extracts of pepper and turnip at 24 hours after pathogen inoculation, was more effective for the control of disease. Thus, application time and concentration and kind of solvent influence the efficacy of the extracts.

Citrus is one of the most important fruit crops in Iran. Among the citriculture areas of Iran, Mazandaran province has the highest acreage (about 100,000 ha), production and number of varieties nationwide. Unauthorized importation of citrus species and cultivars and propagation by growers and nurserymen have led to perpetuation and spread of virus, viroid and other graft-transmissible pathogens of citrus in Mazandaran. Nonetheless, the putative agents of several of such graft-transmissible diseases including citrus ring pattern(CRP) have remained undetermined and the use of certified disease-free propagative stocks has not gained widespread attention. With the onset of the aphid transmission of Citrus tristeza virus (CTV) in the area, trend has shifted toward the use of CTV-tolerant rootstocks, mainly trifoliate orange (Poncirus trifoliata) and its hybrids, citranges (P. trifoliata × Citrus sinensis) and citrumelos (P. trifoliata × C. paradisi) with concurrent use of disease free (mostly CTV-free) budwoods and budlings. Some nurserymen and growers are becoming keen about the impact of several viroid species which have, herebefore, been regarded as harmless to orange and mandarins produced on sour orange(C. aurantium) rootstock but would adversely affect vigor and yield of trees on trifoliate orange and its hybrids. Preliminary attempts to detect viruses causing citrus psorosis and CRP diseases in symptomatic orchard trees and in plants graft-inoculated with the infected sources in the greenhouse, have remained unsuccessful and no amplicon specific for Citrus psorosis virus has been obtained in PCR trials thus far. In the present study, bark patches from twigs of Satsuma mandarin(C. unshiu) trees showing psorosis young leaf symptoms and those displaying CRP symptoms on mature leaves were budded onto one-year-old seedlings of Etrog citron(C. medica, Arizona 861) and sour orange and 2-year-old Washington navel sweet orange on sour orange rootstock. The budded plants were kept in the greenhouse at 22-30oC, and observed periodically for the appearance of symptoms. Leaf flecking appeared on all citrus plants inoculated with buds from the psorosis-infected trees within one to three months. Midvein browning and leaf epinasty developed on Etrog citron seedlings budded with bark strips from several of the CRP-infected trees. Sweet orange budlings started to show some ring and line patterns in three to four years after inoculation with two CRP sources. Sour orange seedlings remained symptomless up to the termination of the experiment(year 6). Ribonucleic acid(RNA) was extracted from both symptomatic sweet orange and Etrog citron plants with TRIzol reagent(Invitrogen, Paisley, UK) and subjected to Reverse transcription-polymerase chain reaction(RT-PCR) using primers specific for CPV and citrus viroids in single PCR. The PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. Bands representing those of citrus viroids including citrus bent leaf, hop stunt, citrus dwarfing, citrus bark cracking and citrus viroid VI were present in the Etrog citron and sweet orange plants inoculated with several sources of CRP and CP-like symptomatic Satsuma mandarin trees. Citrus exocortis viroid was not detectable in any sample tested. This is the first report on the simultaneous presence of multiple citrus viroids in citrus trees showing symptoms of psorosis and ring pattern diseases in Iran.

The mycorrhizal fungi are the most important micro-organism among the soil micro flora which via genetical, physiological and ecological changes lead to more yield in symbiotic plants. The Piriformospora indica Sav. Verma, Aj. Varma, Rexer, G. Kost & P. Franken 1998, is one of well known endomycorrhizal fungus with different beneficial effects on diverse monocots (Poaceae) and dicots (Brassicaceae and Chenopodiaceae) plants. P. indica belongs to Sebacinales, Hymenomycete and Basidiomycota phylum. It has been reported that P. indica is capable to symbioses with different plants. Several studies showed that P. indica increases root and stem length in plants via induction of growth promotion hormones, more absorption of nutrients such as phosphorus and increase tolerance to salinity and drought stresses. As well P. indica causes induction of systemic acquired resistance against different diseases on roots and shoots. In this study P. indica (kind gift from Prof. Kogel, Head of Institute of Phytopathology and Applied Zoology, University of Giessen, Germany) culture was grown on complex medium (CM) at 27 oC for one month. Disinfected rice (Local Cv.Tarom) seeds with 1% active chlorine were germinated on water soaked filter paper in Petri plates. Four days old seedling immersed in 106 ml–1 of P. indica chlamydospores suspension for 12 hours and shacked with 40 rpm. Infected and mock seedlings transferred in plates containing aqueous uchida medium for planting in soil. To detect of P. indica roots samples were stained with ink following by Vierheilig et al. method with minor changes. Molecular detection of P. indica in plants carried out using Tef specific primer pairs (accession no. AJ249911). Result of this study showed that treated plants with P. indica, contain round to pear shaped chlamydospore formed in infected roots. The rate of tillering increased up to 50% in P. indica infected plants. As well as, the number of fertilized spikes and root length increased noticeably in P. indica treated rice plants. Studying of induced diseases resistance in p. indica treated rice plants is under progress. This is the first report of symbiosis in Iranian local rice cultivar and P. indica.