فصلنامه بیماریهای گیاهی
سال چهل و نهم شماره 1 (بهار 1392)

Angular leaf spots of sleepy mallow Malvaviscus penduliflorus have been observed in some areas of Mazandaran and Golestan provinces. The spots were angular، brown and necrotic. A levan positive and oxidase negative pseudomonad was isolated from the symptomatic leaves. The biochemical and physiological characteristics and profile of fatty acid methyl esters of the causal bacterium was very similar to Pseudomonas syringae pv. syringae. Pathogenicity of selected strains was confirmed by inoculation of sleepy mallow with bacterial suspension of several strains. PCR amplification using the syrB specific primer pairs yielded the expected 720 bp fragment from all strains. In rep-PCR، the fingerprints of the strains from sleepy mallow showed 28% similarity to those of the standard strains of P. s. pv. syringae. However، on the bases of difference in host specifity، some genotypic properties، sequencing of some highly conserved genes (rpoD and gyrB)، the strains isolated from angular leaf spot on sleepy mallow just were similar to Pss but were genetically distinct from other groups and may represent a new pathovar of P. syringae belonged to another genemospecies.

Barley yellow dwarf viruses (BYDVs), Cereal yellow dwarf virus (CYDV) and Wheat dwarf virus (WDV) are the causes of dwarfing and yellowing in small grain cereal crops in Iran. WDV consists of at least two strains each adapted to wheat (WDV-W) or barley (WDV-B). In this research, the distribution and genetic variation of WDV-B and WDV-W isolates in Iran were investigated. Infected barley and wheat plants showing dwarfing and yellowing symptoms were collected from cereal fields of Chahar Mahal and Bakhtiari, Fars, and Yazd provinces during 2009-2010 and from other provinces in 2004-226 growing seasons. These samples were subjected to DNA extraction, PCR, and sequencing, using specific WDV primers at species and strain levels. Results showed that WDV was detected in 155 of 270 samples. WDV-W and WDV-B accounted for 55% and 45% of WDV positive samples, respectively. While WDV-B was only isolated from naturally infected barley plants, isolates of WDV-W was isolated from both wheat and barley. The results of this study indicated that in addition to BYDVs, WDV is a major component of yellows complex in cereal fields in Iran. WDV was transmitted from wheat and barley infected plants to healthy plants using leafhoppers collected from cereal fields and reared on plants in the greenhouse. Morphological characteristics especially those of male genitalia indicated that Psammotettix alienus is the WDV vector in Iran.

During the observations over nurseries and almond orchards of Kermanshah Province in 2010-2011, saplings and fertile trees suspected to infection by Pythium and Phytophthora and the soil around their crown and foots were sampled. After culturing these samples on common and semi-selective media, some isolates of Pythium (sensu lato) and Phytophthora were obtained. These isolates were identified based on their morphological and several physiological characteristics. Pathogenesity tests were performed on excised shoots and seedlings In vitro and In vivo respectively. In this research, 12 isolates, belong to 4 species including Phytophthora cactorum, Pythium aphanidermatum, Ovatisporangium helicoides and Pythium group-G were identified. The isolates were mostly from the soil around the roots and foots of saplings. In pathogenesity test on excised shoots, highly significant differences were found among the treatments based on the rate of disease extension. In this experiment the most disease extension was induced by P. cactorum followed by O. helicoides. There was no significant differences among P. aphanidermatum and P. group-G when compared to the control. In pathogenesity test under greenhouse condition, P. cactorum isolates caused seedling death after 7 days; but the symptoms of seedling death appeared after 40 days, when almond seedlings were exposed to P. aphanidermatum and O. helicoides. P. group-G did not make any pathogenesity on almond seedlings in both experiments. This is the first report of identification and pathogenesity of O. helicoides in Iran.

Rice aggregate sheath spot disease is caused by Rhizoctonia oryzae-sativae (Sawada) and occurs more or less at all paddy fields in Guilan province. In this study some aspects of biology of Rhizoctonia oryzae-sativae and optimal growth temperatures were examined. Rice straw colonized with mycelia and sclerotia of Rhizoctonia oryzae-sativae were incubated in different field conditions such as natural field soil surface, 5 cm deep under the soil, flooding condition, on soil surface at the borders of the field. The effects of variable conditions on viability of fungus were studied. Moreover, the effects of different temperatures (5, 0, -5, -10, -15, -20 and -72) on germination and viability of sclerotia were assessed during one month. Optimum growth rate was found to take place at 32 ºC. Results showed that R. oryzae-sativae overwinters as mycelia on the rice straw on the soil surface in the field, soil surface at field margins and as sclerotia in the soil. According to the results 30.4% of sclerotia were able to germinate after one month storage at minus 72 ºC.

Alternaria blight disease, caused by Alternaria spp., damages severely to oil-producing species of Brassica spp. all over the world and reduces the quality and quantity of the oil. To determine the relationships between incidence (I) and severity (S) of Brassica blight disease, and making a model for predicting S based on I values, this research was performed on two genotypes of mustard (j-98-102/51-5 and Bard-1), two genotypes of turnip (Rainbow and Candle), three genotypes of canola (Hayola401, Shiralee and RGS003) and genotype Select 4 (fourth generation of rapeseed-mustard cross) in agricultural research station of Gonbad, Iran as a completely randomized block design in three replications. Severity of disease in the specified time intervals was measured until the appearance of withdrawal symptoms. Results showed that the Allometric model (natural logarithm transformation of I and S) with about 66 % of R2 had the best fitting for the collected data and therefore it was the best model to describe I-S relationships in Alternaria blight of Brassica spp. Based on the regression slope of Ln transformed values of I and S, the genotypes were classified in 3 groups. The first group includes Bard-1 genotype with high slope (1.48). Candle, Select4 and RGS003 were placed in the second group with moderate slope (1.25) and the third group was included genotypes Rainbow, Shiralee and J-98 with the least slope (1.15).

Bermuda grass southern mosaic virus (BgSMV) is a widespread potyvirus inducing mosaic on Bermuda grass in warm regions of southern Iran. In this study, various geographical isolates of the virus were compared phylogenetically. Bermuda grass samples showing mosaic symptom were collected from Jiroft, Bushehr, Borazjan, Ramhormoz, Andimeshk, Shushtar, Behbahan and Darab. Viral RNA was extracted and reverse transcription polymerase chain reaction (RT-PCR) was conducted using specific primers for amplification of the 3′ region (CP- UTR) of the viral genome. PCR- products were cloned and sequenced. The sequences of CP- UTR region of BgSMV isolates were compared with each other and with available sequences of cereal potyviruses in the GenBank. Phylogenetic analyses showed that all sequences were grouped into six clades of BgSMV, Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Johnson grass mosaic virus (JGMV), Sorghum mosaic virus (SrMV), Iranian Johnson grass mosaic virus (IJMV) and Zea mosaic virus (ZeMV). IJMV and ZeMV were grouped in the same clade. Among potyviruses, BgSMV was close to MDMV. However it had an additional 90- nucleotide stretch in frame (30 amino acids) in the 5′ region of the coat protein compared with MDMV. This difference was consistent in all BgSMV isolates. The mean of the nucleotide similarity in CP- UTR region among BgSMV isolates was 98.1% which indicates low level of genetic diversity in intra- population of BgSMV. Despite the high nucleotide sequence similarity between BgSMV and MDMV, differences between the two viruses in host range and serology, and presence of additional 90 nt in the 5' region of CP gene, make BgSMV a distinct potyvirus species close to MDMV.

Leaf spot disease is one of the most destructive foliar diseases of sunflower. The use of resistant genotypes is potentially one of the most economical means for the disease control. A greenhouse assay was used to evaluate resistance of six sunflower cultivars, namely Dorsefid, Ghalami, Pestei, Master-op, Azargol, and Euroflor against three isolates of A. alternata. The isolates showed significant differences in pathogenicity. The reactions of cultivars to fungal isolates were different; however, none of the cultivars showed high levels of resistance. This is the first study on the evaluation of partial resistance of some of the sunflower cultivars to the Alternaria leaf spot disease in Iran.

Turnip curly top virus (TCTV) is a unique geminivirus which has been recently reported from turnip growing farms of Fars province. Despite differences in the gene organization of this virus compared to other geminiviruses, most of its biological properties including vector and natural hosts have remained uncharacterized. In the present work the ability of two leafhopper species, i.e., Circulifer haematoceps and Orosius albicinctus to transmit TCTV was studied. The results showed that TCTV is efficiently transmitted from infected plants to healthy turnip seedlings by C. haematoceps under greenhouse conditions. Infection of turnip plants was verified by symptoms appearance and PCR. Typical TCTV symptoms including inward rolling of the leaf margins and swelling of veins on the lower leaf surfaces of inoculated plants was observed 10-20 days post inoculation. The presence of virus in symptomatic plants was confirmed by PCR using specific TCTV primer pairs. In order to identify more natural hosts of the TCTV, plant samples from different crops and weed species were collected in severely TCTV infected farms and tested by PCR using two specific primer pairs. PCR results showed the presence of TCTV infection in radish samples showing curly top and chlorosis symptoms. Furthermore, TCTV infection of symptomless weed species including herb sophia, bugloss, black nightshade and flower of an-hour was confirmed by PCR test. To compare the sequence of radish isolate of TCTV with available TCTV sequences in GenBank, full-length genome of the radish isolate was amplified by rolling circle mechanism using phi DNA polymerase, cloned and sequenced. Sequence comparison showed 93-99% homology between radish and TCTV isolates. Results of this study indicated that in contrast to most curtoviruses, TCTV has a narrow natural host range.

Cauliflower mosaic virus (CaMV) causes yield losses in plants of Brassicaceae family including canola (Brassica napus). In order to detect CaMV in canola, a total of 684 leaf samples showing various virus-like symptoms were collected in several provinces of Iran including Tehran, Ghazvin, Chaharmahal-Bakhtiari, Mazandaran, Ardabil, West Azarbayejan and Fars provinces. Using a polyclonal antiserum and DAS-ELISA method, samples were tested for the presence of CaMV. ELISA positive isolates from 7 provinces, were used for further studies. Biological diversity of CaMV isolates was evaluated by their ability to infect turnip, kohlrabi and datura. All isolates under study infected turnip, producing local lesions of different sizes and timing, whereas, no symptom was obsereved on kohlrabi and datura. Specific primers amplified an 840 bp fragment of ORF V. Phylogenetic analysis grouped all Iranian isolates and two GenBank isolates (Cabb-D/H, M10376, Xinjiang, AF140604) in one cluster, while all other CaMV isolates from GenBank formed a separate cluster.

Cercospora leaf spot, caused by Cercospora beticola, is one of the most important foliar diseases of sugar beet. Due to the problems associated with traditional methods in the identification of Cercospora species, in the present study two sets of previously designed Cercospora beticola-specific primers based on calmodulin and actin genes were used for the molecular identification of C. beticola isolates. For this purpose, two standard isolates of C. beticola together with the other isolates recovered from sugar beet plants with leaf spot disease symptoms in present study were subjected to PCR amplification. Efficacy of primer sets were tested on the Cercospora isolates recovered from weed plants with leaf spot symptoms, in present study. Our results showed that the primer set based on actin gene amplified a 959 bp PCR product from the all of the isolates tested in this study. This primer set failed to distinguish C. beticola from the other Cercospora spp. isolated from weeds as well as other fungal groups. The results of multiplex PCR assay using primer sets based on calmodulin gene yielded 234 bp PCR product from all of the Cercospora spp. tested in this study and only a 176 bp PCR product from C. beticola isolates; such that successfully discriminated C. beticola from the other Cercospora isolates. The calmodulin genes primer sets can be used for the screening of Cercospora beticola from the other Cercospora spp. and for the detection and the determination of the host range of Cercospora beticola as well.

The following new findings about Iranian smut fungi are presented: 1-Ustilago hordei × U. nuda hybrids are reported on Hordeum vulgare from Yazd province. Our study showed that main causal agent of covered smut in the province is above hybrids. 2- Urocystis tianschanica is newly reported from Iran on Critesion violaceum. 3- Tranzscheliella iranica is also reported from Yazd province, Deh Bala. Before our report, this species was known only from the type locality. 4- Revision of herbarium specimens of infected Loliolum subulatum revealed that smut species on these specimens belongs to Tilletia lolioli not T. bromi. All above mentioned taxa are documented by microphotographs and Persian description.

Sesame damping-off caused by Fusarium oxysporum f.sp. sesami is one of the most important diseases of Sesamum indicum L. in the world. In this work,the activity of peroxidase enzyme was assayed as a resistance mechanism in Asfij local germplasm (Bahabad, Yazd province) as resistant and Kahnoj local germplasm (Kerman province) as susceptible germplasm. Fifteen isolates of causal agent were isolated from sesame farms in Yazd province and one isolate with the highest pathogenicity potential was selected and used to determine hostrange. Among ten species of several plant families that were artificially inoculated with this pathogen, only sesame showed disease symptoms. Evaluation of peroxidase activity in tolerant and susceptible germplasms in 2, 4, 6, 8, 10 and 12 days after inoculation with Fusarium oxysporum f. sp. sesami showed that peroxidase enzyme activity increases in resistant germplasm with highest level in 4 days after inoculation. The activity of peroxidase was decreased 4 days after inoculation. In susceptible germplasm, enzyme activity was increased slightly in low quantity. In conclusion the results show that peroxidase activity play probable role in induction of plant resistance against outbreak of plant pathogens.

Grafted and rooted cuttings of grapevines were inoculated with six pathogenic strains of Rhizobium vitis. Evaluation of infection severity was done based on the number, size and weight of galls on vines, four months after inoculation and in experiment duration of six years. Results showed that vinifera varieties were very sensitive to crown gall. Weight, number and size of galls induced on grafted vines on H4 and H6 was less than those on other vines. Scions grafted on rootstocks H4 and H6 had a 21.5% and 6.8% incidence compared to 55% for self-rooted vines. During six years 90% of self-rooted vines died, while 18% and 5% of the grafted vines on H4 and H6 hybrids were dead, respectively. The highest yield was found in the Red Sahebi grafted vines on H6 rootstock (2.98 kg/ vine) and the lowest was in self-rooted vines (1.25 kg/vine) and on others (2.25 kg/ vine), respectively.

Biocontrol activity of Metschnikowia pulcherrima alone and in combination with silicon against Penicillium expansum and its machanisms of antagonisms was evaluated. Percentage of inhibition mycelial growth of pathogen in dual culture, volatile test and non-volatile test were evaluated. To evaluate the direct effect of silicon on mycelial growth of P. expansum in vitro, it was added at different concentrations to PDA medium. The effect of silicon on populations of yeast in NYDB was determined after 24 and 48h. Silicon at 0.6%(wt/vol) or above completely inhibited mycelia growth of pathogen and decreased yeast populations. For determining the effect of M. pulcherrima alone and in combination with silicon in controlling disease during storage, fruits were wounded using a sterile nail. Each wound was treated with 20µl of M. pulcherrima suspension, solution of silicon in different concentrations, antagonist suspension amended with silicon at different concentrations, and sterile distilled water as the control. After 24h, 20µl of conidia suspension of the pathogen was applied, and fruits were transferred to storage at 4 and 20˚C. At 4˚C and 20˚C after 45 and 15 days, respectively, lesion areas were measured and the population of yeast in the wound was determined by serial dilution. A combination of silicon with yeast reduced the decay of apple better than silicon and yeast alone. Peroxsidase activity and total phenolic content were significantly enhanced by yeast treatments.