فصلنامه بیماریهای گیاهی
سال چهل و هفتم شماره 2 (تابستان 1390)

Contamination of pistachio fruits by aflatoxin-producing isolates of Aspergillus flavus has caused critical problems in pistachio production, consumption and exportation in Iran. In order to study aflatoxin production rate in A. flavus isolates collected from pistachio fruits, 46 isolates of the fungus were tested using ELISA method. 80.44 percent of isolates produced aflatoxin and 19.56 percent of isolates were non-aflatoxigenic. Moreover, to study the effect of hyphal anastomosis on the reduction of aflatoxin production by heterokaryon isolates, toxigenic and non-toxigenic mutants R5 and Mb2 were crossed, respectively. Among the 30 heterokaryon isolates, only ten percent of isolates were not able to produce aflatoxin. Results showed that hyphal anastomosis can affect reduction of aflatoxin production in heterokaryon isolates. Also, the results of DNA fingerprinting patterns, resulting from rep-PCR, showed that all parental isolates (R5 and Mb2) had similar patterns except for one fragment with the size of 566 bps. Also, fingerprinting patterns for heterokaryons isolates (R5Mb21 to R5Mb215) were similar to those of toxigenic parents (R5).

Iranian wheat stripe virus (IWSV) is a tentative member of the genus Tenuivirus. In this study, several properties of the virus were studied. To determine the polarity of the IWSV RNA 1 segment and compare it to corresponding segment of other tenuiviruses, about 650 bp fragments of the 3´ end of this segment was amplified, cloned and sequenced. Sequence comparison showed that similar to other tenuiviruses, the IWSV RNA 1 is of negative polarity and more similar to tenuiviruses causing white leaf in American continent. Previously, only wheat had been reported as the natural host of IWSV. In this study, we identified rice as an economical natural host of the virus in rice growing areas in north of Shiraz. The coat protein (CP) and non-structural protein (NS4) genes of IWSV rice isolate were amplified, sequenced and compared with the corresponding genes of the wheat isolate. The nucleotide and amino acid sequence identities of the CP and NS4 genes between two isolates were 99.6 and 99-100%, respectively. The serological relationships between IWSV and five tenuiviruses were determined by agar gel diffusion (AGD), dot-immunobonding assay (DIBA) and/or indirect enzyme-linked immunosorbent assay (ELISA) using antisera against CP or NS4 proteins. These results showed that among five tenuiviruses, IWSV is related only to Rice hoja blanca virus (RHBV). Serological and molecular studies again confirmed similarities between IWSV and tenuiviruses causing white leaf in American continent. IWSV was successfully transmitted by embryo injection of wheat seeds presoaked in water for 30 mins at 25°C with an efficiency of 5.2%.

In the study of nematode collection of Nematology Laboratory of Tarbiat Modares University, seven species of the family Dolicodoridae belonging to the genera Merlinius, Tylenchorhynchus and Quinisulcius were identified viz, M. bavaricus, M. brevidens, M. plerorbus, T. divittatus, T. goffarti, T. latus and Q. capitatus. Among these species, M. bavaricus, M. plerorbus and T. divitatus are new records for nematodes fauna of Iran. M. bavaricus is distinguished from other species by cephalic skeleton not refractive and fine striae on head and body, thin stylet with rounded knobs, bilobed spermatheca and tail length unstriated terminus. The female characters of Merlinius plerorbus include constricted head, stylet 15-16 µm long, 39-50 µm long tail with 24-32 annules, a smoothly rounded terminus and lobed spermatheca. T. divittatus is distinguished from related species by having lateral fields marked by only three incisures, a well set-off lip region bearing six to seven annuls, stylet 17-20 µm long, presence of a post-anal extension of the intestine and phasmids located behind middle of tail.

Pratylenchus neglectus and P. thornei are the common root-lesion nematodes in irrigated wheat and corn fields in Marvdasht region, Iran. Quiescence of these nematodes in response to water stress (anhydrobiosis) and their return to active stage in wet condition is their important strategy to survive in dried soil. Anhydrobiotic states and infectivity of these two root lesion-nematodes were studied under laboratory, greenhouse, microplats and field conditions. The laboratory results indicated that the highest percentage of quiescent P. thornei became active after the infested dried soil was wetted and kept at 25 °C for 72 hrs. In the greenhouse, 86.3% of P. neglectus were alive in 4 cm air-dried soil layer after 4 months. In microplats, without rainfall the populations of two species remained constant for eight months; however, in plots that received natural rainfalls, the populations of both species were dramatically decreased. Under field conditions, all stages of nematodes survived in anhydrobiotic state. In general, anhydrobiosis is important in relation to population maintenance and survival of root lesion nematodes during winter and summer in Marvdasht region.

Bacterial vascular necrosis and root rot of sugarbeet caused by Pectobacterium betavasculorum is one of the important causal agents of sugarbeet root rot in Fars province. The disease has become widespread in recent years in the region. In order to determine host range of this pathogen in cucurbitaceae and solanaceae, two representative virulent isolates were used. Isolates were inoculated into stem, petiole, root or fruit of plants. Plants were kept at 28+ 2oC in a growth room or a glasshouse. Control plants were treated with sterile distilled water and kept in similar conditions and checked daily for symptoms development. Disease symptoms in the form of black streaking lesions and rot around inoculation site developed during 2-10 days in leaf, stem, root, fruit and tuber of cucumber, beans, melon, tomato, squesh, maize, potato, eggplant, carrot, turnip, garlic, onion, garden beet and date palm fruit. Disease symptoms were less severe on maize than other plants, however, inoculation induced water soaking and rot in the crown area and finally killed maize young seedling after a week. Restricted rot developed on garlic and onion. The P. betavasculorum was re-isolated from inoculated plants. Based on the research, melon, cucumber, squash, maize, bean, and eggplant are introduced as potential new hosts of P.betavasculorum. The results of distribution studies in various regions in Fars province showed that the disease was widespread in Marvdasht, Kavar, Fasa, Zarghan,and Shiraz vicinity but it was not found in Eghlid.

Citrus blast is one of the important diseases of citrus in north of Iran. The disease causes considerable damage during and immediately after the cold seasons. The causal agents have epiphytic survival on diverse weeds during spring and summer. During 2009-2010, samples were collected from several candidate weed plants including: Poa annua, Phalaris canariensis, Sorghum halepense, Cynodon dactylon, Setaria viridis, Acalypha sp., Aegilops ovate, Lolium sp. and Amaranthus retroflexus. A rod- shaped, gram negative, aerobic, fluorescent bacterium was isolated from all samples. The bacterium possessed several polar flagella. Strains were negative in oxidase and arginine dihydrolase but positive in catalase and urease tests and tolerated 5% NaCl. They were variable in production of levan from sucrose and rotting potato tuber slices. All strains hydrolyzed gelatin,casein and Tween 80. Nitrate was not reduced to nitrite, and production of reducing substances from sucrose was variable. All strains produced a hypersensitive reaction in geranium (Pelargonium X hortorum Baily). Glucose, fructose, xylose, mannitol, raffinose, glycerol, alanin, leucin, asparagine, tryptophane, fumarate, pyruvate, D- and L- tartrate,citrate and malate were utilized as carbon sources by all strains; none could utilize arabinose, dulcitol, ethanol, propanol, tyrosine, methionine, and oxalate. They were variable in utilization of sucrose, esculin and pectate. Pathogenicity of most of strains on citrus was confirmed 5-7 days after inoculation.Based on The protein profile strains were devided into four groups. The first group consisting of isolates from Poa annua, Phalaris canariensis, Sorghum halepense, Setaria viridis and Lolium sp. were between Pseudomonas viridiflava (Pv.) and P. syringae pv. syringae (Pss.). The second group consisted of Acalypha sp. which was different from Pv. and Pss. In the third group, the isolate from Amaranthus retroflexus was similar to Pss. The isolates of the fourth group including those from Aegilops ovate and Cynodon dactylon resembled Pv. Polymerase chain reaction (PCR) assays with gyr B and rpo D primers, the specific fragments of 850 bp and 1200 bp long respectively, were amplified from most strains. Multiple sequence alignment was performed using ClustalW software, with sequences of this region of all Pseudomonas species in GenBank (NCBI). The phenotypic tests and PCR assays results showed that the strains belonged to Pseudomonas. The strains isolated from Aegilops ovate and Cynodon dactylon belonged to Pv. and the strains from Amaranthus retroflexus belonged to Pss. Isolates collected from Poa annua, Phalaris canariensis, Sorghum halepense, Setaria viridis and Lolium sp. had some characteristics half way between Pseudomonas viridiflava (Pv.) and P. syringae pv. syringae (Pss.)

Gummy bark disease of sweet orange (Citrus sinensis) trees has been reported from a few citrus growing regions of the world. The disease symptoms consist of mild to severe stem pitting and brown gummy flecks in the bark of sweet orange trees on sour orange (C. aurantium) rootstock, being conspicuous several centimeter above the bud union on the trunk. The pits were usually impregnated with brown gummy deposits. In surveys conducted in 2005, several sweet orange trees, cv. Moro Blood, with symptoms of gummy bark were encountered in Mahdasht groves in Sari. One year old sweet orange budlings on Troyer citrange (Poncirus trifoliate × C. sinensis) rootstock and sour orange seedlings were graft-inoculated with buds from two symptomatic sweet orange trees. The budded plants were cut back to force growth of new flushes one month after budding. Gum impregnation of the bark of sweet orange on Troyer citrange rootstock was observed three to five years after graft-inoculation. RNA was extracted from graft-inoculated symptomatic sweet orange and symptomless sour orange plants and healthy seedlings of both species, as controls and a sweet orange budling previously proved to harbour several citrus viroids (Alavi et al., Iranian J. plant path. 41: 65-66) using TRIZOL (Invitrogen, Carlsbade. CA). RT-PCR was conducted using specific primers for citrus viroids and for the major coat protein gene of citrus tristeza virus. Electrophoresis of PCR products revealed presence of hop stunt viroid, citrus bent leaf viroid, citrus viroids III and IV and citrus exocortis viroid in the samples. This is the first report on the infection of the gummy bark diseased oranges with five different citrus viroids. The role of these viroids, if any, in gummy bark disease of sweet orange needs to be determined.

Alder (Alnus subcordata subsp. Subcordata) is one of the most important tree species in northern forests of Iran and is economically considered as the fourth commercial tree. A leaf spot disease of alder caused by Xanthomonas sp. has been reported from several forest regions of Mazandaran province (Rahimian et al. 1383. 16th Iran. Plant Protec. Cong., 439). According to a recent genomic study, the bacterial agent of this disease has the highest similarity to Xanthomonas arboricola (Rahimian et al. 1387. 18th Iran. Plant Protec. Cong., 428). Though limited studies have been performed about the homology of isolates and the distribution of the disease (Rahimian et al. 1383. 16th Iran, Plant Protec. Cong., 439), detailed information is not available about the genetic diversity of isolates from different regions. This study was conducted to evaluate the genetic similarity or diversity among the isolates obtained from different regions of Mazandaran and Golestan provinces. Phenotypic characteristics of isolates were very similar and they were different only in utilization of carbon sources including L- Serin, Leucine Dextrin, Quinate, Tyrosine. The population genetic diversity analysis of the bacterial pathogen of alder angular leaf spot was performed using BOX-PCR and REP-PCR. Genomic DNA was extracted from isolates using alkaline lysis, and PCR was performed as recommended by Versalovich et al., but by reducing the deneturing time (Versalovich et al. 1991. Nucleic Acids Res, 24: 6823 -6831). PCR products were electrophoresed in 2 percent agarose gel and the gel was stained with ethidium bromide. Similarity, matrix of the isolates was obtained using Jaccard's coefficient and cluster analysis by employing the UPGAMA method and NTSYS software. Cluster analysis by BOXAIR primer showed that the isolates were divided into 24 groups at 80 percent level. However, the isolates of alder showed the same similarity with standard isolate Xanthomonas arboricola pv. coryli. At 63 percent level, alder isolates were divided into 15 groups, but with the same amount of similarity they were clustered with Xanthomonas arboricola pv. pruni. At 40 percent level, alder isolates were divided into seven groups and showed similar homology with reference isolate of Xanthomonas arboricola pv. juglandis. According to these findings, it can be concluded that isolates inciting alder angular leaf spot have high variability, while BOX-PCR was unable to differentiate X. arboricola pathovars. Results obtained with REP1R and REP2I primers showed that at 78 percent level, isolates were divided into 14 groups. In contrast, the isolates showed the same level of similarity with X. arboricola pv. juglandis and X. arboricola pv. coryli pathovars. At 57 percent similarity level, the isolates were divided into seven groups and were clustered with X. arboricola pv. pruni with the same amount of similarity. The data show that alder isolates have high variation and REP-PCR has a better efficiency for grouping causal agent of angular leaf spot of alder isolates.