فصلنامه بیماریهای گیاهی
سال پنجاهم شماره 1 (بهار 1393)

Ochratoxin A (OTA) is a mycotoxin which contaminates different plant products, including cereals, coffee, and dried grape. Aspergillus and penicillium species were identified as the main producer of OTA. Ochratoxin A is a potent nephrotoxin. The presence of OTA in raisin is an important barrier to raisin export. The maximum tolerable level of OTA in raisin is 10 µg /kg. During 2010- 2012 a research was carried out on the monitoring of ochratoxin A and ochratoxin A - producing fungi in raisin of Khorasan, Azarbaijan, and Qazvin provinces. The samples were collected from different processing stages of raisin, storages and factories. To study fungal contamination two methods of plating surface disinfected raisin and washing of raisin surface (suspension) on artificial media were used. Aspergillus section Nigri species were isolated and the growth of Aspergillus section Nigri species which were assumed to produce ochratoxin A were recorded after incubation period. To confirm morphological identification of A. carbonarius isolates, DNA of three A. carbonarius isolates were extracted and their ITS regions of ribomal DNA and β- tubulin genes were amplified by PCR and sequenced. Ochratoxin A contaminations of the raisin samples were estimated using HPLC- IAC method. To study ochratoxin A producing ability of Aspergillus section Nigri species isolated from raisin, 14 isolates selected as representative were cultured on PDB medium and OTA produced by each isolate was evaluated using HPLC- IAC method. All raisin samples were contaminated by Aspergillus section Nigri species. The mean of the assumed ochratoxin A producing -fungi contamination of raisin samples was 56. 31 (8-100%) but varied depending on the processing method, raisin variety and location. The highest contamination was found in injured not directly edible raisin. Twenty percent of the isolates belonged to A. carbonarus species and the rest to A. nigra. Ochratoxin A was found in five samples out of 44 (11%) raisin samples at the contamination levels of 0. 4 to 100 ng/g. 71% of tested A. carbonarius isolates produced ochratoxin between 0. 78- 108. 8 ppb. DNA sequencing data were generated for a part of 18S-ITS1-5. 8S-ITS2 and 28S region of the rDNA, and β- tubulin gene region. These sequences were compared to other sequences of A. carbonarus in GenBank and showed 100 % and 99% similarity, respectively. These data confirmed the morphological examinations. Sequences were submitted to GenBank under the accession numbers KF434631, KF434632, and KF434633 for ITS, and KF434634، KF434635 for β- tubulin gene. Since the highest contamination was found in injured raisins, they can be easily removed to reduce contamination by fungi and ochratoxin.

Phytophthora drechsleri and P. cryptogeaare phylogenetically closely related oomyceteous plant pathogens. In order to evaluate genetic variation among these species, isolates of P. drechsleri and P. cryptogeafrom various hosts and localities. Also some isolates of another close taxon, P. erythroseptica,were characterized by the random amplified microsatellites (RAMS) technique using three degenerated anchored primer: VHV(GT)7G, DDB(CCA)­5 and DHB(CGA)5. Two different banding patterns were observed among isolates consisting of 26 RAMS markers among P. cryptogea and P. erythroseptica isolates and 18 among P. drechsleri isolates. UPGMA-analysis of RAMS markers showed that P. cryptogea has considerable molecular variability and illustrates intraspecific variations. There are at least three distinct genetic groups within P. cryptogea populations worldwide with average similarity of 54-60% between groups. P. erythroseptica isolates produced almost the same RAMS polymorphic markers as P. cryptogea and interspersed as a divergent lineage of P. cryptogea in the P. cryptogea UPGMA dendrogram. It seems that P. erythroseptica isolates are much similar to two of the P. cryptogea groups than the third group to the others. There was no evidence for biogeographically structured lineages which could be as a consequence of wide host range and transglobal transposition of lineages associated with worldwide movement of agricultural and horticultural plants. Data showed that P. drechsleriis a more homogenous species but there are some lineages that seem to reflect the geographic distribution. The RAMS analysis did not reveal any trends for host specificity between different lineages of these two species.

Mycorrhiza colonization can induce qualitative and quantitative changes in the microbial community. The mechanisms which control interactions between bacteria and AM fungi in plant roots can be synergistic, competitive or antagonistic. In this study, biological and molecular studies indicated a competitive and antagonistic interactions between G. intraradices, Pseudomonas and Bacillus. In inoculated plants by beneficial microbial agents, plant immunity system sat in priming position and resistance pathways activated systemically and more effective in rice plants by pathogen infection. Treatments including, B. subtilis and B. subtilis+P. fluorescens, exhibited disease severity (DS) score less than 1.5, were considered resistant to infection by M. oryzae. Like MIR, rhizobacteria mediated ISR is mainly effective against necrotrophic pathogens and is based on priming of JA-regulated responses. This is the first study to evaluate the interaction between AMF and bacteria in rice plant, therefore rice as a monocotyledonous plant, could be a suitable host for studying the mechanisms and interactions between AM fungi and other microbial agents.

Tomato mosaic virus (ToMV) with wide host range is one the most important viruses in tomato plants. A survey was conducted to analyse incidence, host range and genetic diversity of the virus during 2006 until 2008. Symptomatic samples were collected from tomato, pepper, Kidney bean and weeds in Yazd, Kerman, Hamedan and Hormozgane provinces and checked by double antibody sandwich ELISA (DAS-ELISA). Of 434 leaf samples from tomato, pepper, Kidney bean and weed species, 27 samples were ELISA positive. Among the weed species tested Common lLambsquarters (Chenopodium amaranticolour) and Black night shade (Solanum nigrum) were found to be infected with ToMV. Based on the geographical location and host range, 10 ToMV isolates were selected for molecular and biological characterization. The full-length coat protein (CP) gene from 10 isolates was PCR amplified, cloned and sequenced. Phylogenetic analysis showed that these isolates fell into three groups I, II and III. Two weeds- and one pepper isolates were clustered in group I and the rest of isolates in group III. The lowest percent nucleotide sequence identity (84.4%) was between Iranian isolate Jir.Che.4 in group I and other four isolates collected from Kerman and Hamadan provinces in group III. Inoculation of 10 Iranian ToMV isolates on test plants induced different symptoms on tobacco. This is the first report of Kidney bean as the natural host of the virus in Iran. Study of genetic diversity (π) showed a remarkable variation in the ToMV isolates in the Iran and Brazil. This value was about twice for Iranian ToMV isolates in compared to the reported isolates in the world.

The effect of several rhizobacteria isolates on activity of root-knot nematode, Meloidogyne incognita, was studied under laboratory and greenhouse conditions. Initially, 52 rhizobacteria isolates from the rhizosphere of cucumber, eggplant and tomato plants and their inhibitory effects on egg hatching and mortality of the second stage juveniles (J2s) were investigated in vitro. Then, nine most effective isolates were selected and identified by biochemical and morphological tests. Using filtered culture of five isolates of Pseudomonas fluorescens and one isolate of P. putida, Bacillus subtilis, B. brevis, and Serratia sp., caused 35-48% reduction in egg hatching and 32.2-48.8% increase in the mortality of J2s. The inhibitory effects of the isolates on nematode activity and growth indices of tomato cv. Early Urbana were evaluated under greenhouse conditions. Treatment of the sterilized and non-sterilized field soil and mixture of sand and field soil (3:1 ratio) with suspension of 108CFU ml-1 bacteria and four eggs and J2s of nematode (per g soil) significantly reduced nematode reproduction factor and increased the growth indices of tomato plants. The results showed that nematode indices were reduced more in non-sterilized soil, compared to sterilized soil. The isolates of P. putida, P. fluorescens PF19 and Bacillus subtilis were the most effective isolates in reduction of nematode population.

The effect of lime witches’broomphytoplasmainfectinon the content ofpolyphenols, flavonoidsand two other important compounds dealing with the biosynthesis ofphenolics (i.e. carbohydrates and phenylalaninammonialyase) in lime leaves was investigated. Sampling from healthy and infected plants weretaken. at12 and 16 weeks after inoculation.Despite many reports indicating the increase of phenoliccompounds under stress conditions, the results obtained from the qualitative and quantitative study of these compounds usingspectrophotometryand gas-liquid chromatography showed a significant decrease ofsoluble carbohydrates, phenylalaninammonialyae, total phenolicandflavonoidcompoundsin infected compared to that in healthy plants. The content ofphenoliccompounds decreased in both sampling times.

Citrus nematode (Tylenchulus semipenetrans) is a serious pest of citrus trees in north and south of Iran. During 2010-2012, the effect of soil solarization on canopy soil of individual infected sweet orange trees evaluated in a randomized complete block experiment, in Darab region, Fars province. Trees were treated at the beginning of the nematode activity in spring for two periods, 45 and 60 days and at the onset of maximum temperature in June 21 for a period of 60 days. Non-solarized plots were used as control. Recorded data showed that the maximum mean of monthly temperature reached 39. 7- 41. 8 °C, under plastic covering, at soil depth of 15cm. The results indicated that the percentage of the nematode survival reached to zero or near zero in all treatments. There were no increases in the citrus nematode populations three months and one year after solarization (P≤0. 05). The population densities of some plant ectoparasitic nematodes belong to other families and also rhabditids were less affected by the treatments. Due to severe reduction of citrus nematode population density, the vegetative, as well as the reproductive factors of the treated trees were enhanced in the consequent year (P≤0. 05). After soil solarization, the mean yields in different treatments were increased 71. 6-77. 9 and 70. 2-77. 8% in comparison to the initial measurement and control٫ respectively.

Nine monogerm lines and their 36 F1 hybrids were evaluated for their resistance to Cercospora leaf spot in a triple lattice design at Safiabad Agricultural Research Center, Dezful, Iran during 2008-2009 growing season. The plants of the experimental field were scored three times at approximately 30-day intervals. Scoring was accomplished by using 1-9 scale (KWS). The disease severity was determined by two methods, plot score and score of ten plants per plot, which in the second method, the average of ten plants score was considered as a plot score. Results obtained from each scoring method were independently analyzed. Results showed that RR607 genotype had the highest level of resistance to CLS. Hybrids derived from RR607 genotype: RR607×7112-36 and RR607×452 were more resistant to CLS than other hybrids. Based on yield traits in the presence of the disease 7173×474, 436×436-104, 436-104×SBFIROZ, 436-104×7173 and 436-104×452 hybrids were scored best in performance and resistance to CLS.

Species of the Cucurbitaceae family especially melons (Cucumis melo L.) are important sources of human food in the world. Members of C. melo are highly sensitive to viral diseases. Zucchini yellow mosaic virus (ZYMV) is one of the most serious agents in production of these crops in Iran. In the present study, reaction of 12 hybrids and 32 Iranian landraces of melons to ZYMV is examined using DAS-ELISA method. Results of ANOVA showed significant difference at 1% level among studied melon genotypes for ZYMV contents of plants. Results confirm higher level of resistance in hybrids in contrast to landraces and demonstrate genetic control of this trait. Charantaio T showed the highest sensitivity and ELISA value. In contrast, PI414723 was the least sensitive. PI414723 can be used as resistance source to ZYMV in breeding programs.

During the surveys of greengroceries in Urmia during the summer and autumn of 2013, banana fruits with rot symptoms were frequently observed. At first, fruit skin color was changed to brown, then the browning was progressed into fruit flesh and after a short period, all the fruit became brown and rotted. At the surfaces of the rotted fruits, the fungal white mycelium was grown normally and then, small black spots representing fungal acervuli were formed (Fig. A in Farsi section). Fungal isolation was done in two Culture the infected fruit tissues and picking up the conidia directly from acervuli. On the basis of morphological characteristics of the purified fungi, Pestalotiopsis theae with the following characters was identified: Fungal colony on PDA medium at 25ºC and photoperiod of 12 h is white. Colony is fast growing and fills the 80 mm Petri plates after 5 days. After this time, small black pustules (acervuli) are formed at the surface of the colony, at first scattered then aggregated, which contain aggregations of black slimy conidial masses (Fig. B in Farsi section). Conidia are fusiform, staright or slightly curved, 5 celled, with constriction at the septa, 20-25 ×5.5-7 µm, basal and apical cells are hyaline while three median cells are brown to dark brown. At the basal cells of the conidia, there is a hyaline central appendage, 3-8 µm long, but, at apical cells, there are 2-4 appendages, 15-40 µm long. The tips of the appendages are clearly swollen (Fig. C in Farsi section) (Chang et al. 1997; Suwannarach et al. 2013). Pathogenicity tests of the isolates were done on healthy banana fruits in laboratory. For this, fungal inoculation was done both with placing a suspension of 1×106 conidia/ml on the surface of a wounded fruit skin and with mycelial plugs from the edges of the actively growing fungal colonies on the surface of fruits with or without wounding. In controls, water or agar plugs without fungus mycelium was used. Although Pestalotiopsis theae was previously reported from Camellia sinensis (L.) Kuntze in Iran (Ershad 2009), but to our knowledge, this is the first report of Pestalotiopsis theae associated with banana fruit rot.

During April 2013, Samples of broad bean (Vicia faba L.) plants showing root rot and wilt symptoms were collected from different farms located in Tarom region of Zanjan province. Laboratory observation indicated sporodochium of the genus Fusarium on infected roots. Single spore isolation of the fungus was carried out from sporodochia on infected host tissues. Based on morphological characteristics including; growth features on PDA and CLA media, size and shape of macroconidia and microconidia and other microscopic characteristics, the fungus was identified as Fusarium avenaceum (Fries) Saccardo (Leslie and Summerell, 2006;GerlachandNirenberg, 1982). Growth rate was relatively rapid on PDA. Surface of the colony on PDA was white in color and reddish orange from the bottom of Petri dishes. Abundant pale orange sporodochia formed on PDA and CLA. Macroconidia were long and slender, straight to slightly curved with 3 to 5 septa, apical cell long and tapering, basal cell with dorsal curvature and foot shaped, 42 – 55 (50.3) × 2.5-5 (3.3) µm. Macroconidiaformed in vitro on CLA were longer and narrower than those produced in natural habitat. Microconidia and mesoconidia were fusiform, 1-3 septate, produced singly on monophialides and polyphialides and variable in size. The pathogenicity test of the fungus was carried out on cultivar Barakat in pots containing 1/1/1 (v/v/v) ratio of field soil, sand and compost under greenhouse conditions using wheat inoculum according to Burgess et al. (1994). After 12 days, the inoculated plants showed root rot and wilt symptoms similar to those occurred in the field. F. avenaceum is one of the species causing damage to broad bean in the world. This is the first report of isolation and pathogenicity confirmation of F. avenaceum from broad bean in Iran.

Polymyxa betae was first described as the parasite of sugarbeet root by Keskin in 1964. However, it is its role in the survival and transmission of Beet necrotic yellow vein virus which makes P. betae economically important. Weed hosts may play a more important role than sugar beet and spinach in the spread and persistence of P. betae. The reported weed hosts of P. betae include Amaranthus retrosflexus, Atriplex patula, Chenopodium album, Portulaca oleracea, P. grandiflora, Papaver argemone, Silene alba, S. vulgaris, S. noctiflora and Stellaria graminea. In the present work, we examined weeds of sugar beet fields in Lorestan and Fars provinces for the presence of P. betae. Sampling of roots was carried out in 2012. The weeds sampled included Amaranthus blitoides, A. retrosflexus, Asperugo procumbens, Chenopodium album, Cirsium arvense, Conringia orientalis, Convolvulus arvensis, Heliotropium europaeum, Hibiscus trionum, Malva sylvestris, Melilotus officinalis, Portulaca oleracea, Raphanus raphanistrum, Setaria viridis, Sonchus oleraceus, Spergula arvensis, Tragopogon graminifolius and Xanthium strumarium. DNA was extracted from the roots by Guanidin method (MacKenzie et al., 1997) and subjected to amplification by conventional PCR using P.betae specific primers. P. betae was detected in A. retroflexus, C. album, P. oleracea, R. raphanistrum and S. arvensis. R. raphanistrum and S. arvensis as the hosts of P. betae are new world records. These weeds may contribute to the survival of P. betae and BNYVV while rotating sugar beet with other crops.