مجله دانش گیاه پزشکی ایران
سال چهلم شماره 1 (بهار و تابستان 1388)

A 'worst case' laboratory method was employed for evaluating total effect of four pesticides (imidacloprid, dichlorvos, pymetrozine and abamectin) on the nymphs mortality and adults reproduction in Orius albidipennis Reuter (Hemiptera: Anthocoridae). The bioassays were carried out in laboratory using Drum cell method, under controlled conditions of: 27±1?C, RH of 65±5% and 16-h photo phase. The effects of the pesticides were assessed on the N1 stage and determined by the total effect, considering the mortality rate and oviposition reduction in audlts. Toxicity effects were classified according to scale proposed by IOBC. Corrected mortality percentages (Ma) were 75.55±8.59, 50.22±6.34, 32.88±4.81, and 28.28±2.37 for imidacloprid, dichlorvos, abamectin and pymetrozine treated populations, respectively. For the pesticides, mean no of eggs/female (R) were 15.56 ±7.61, 38.44±15.25, 6.89±5.56, 10.22±5.71. The pesticide's effect on ovipositon (Er) was recorded 0.54±0.24, 1.33±0.49, 0.33±0.29, 0.35±0.16, and while the total effect (E) being 86.76±5.75, 33.82±4.20, 77.92±9.67, 74.94±11.77%, respectively. According to IOBC standards, dichlorvos was classified in class II (slightly harmful), abamectin and pymetrozine, with SE being taken into account, were classified in class II to III (slightly to moderately harmful), whereas imidacloprid standing in class III (moderately harmful). Further investigations are recommended on all of the chemicals, and under semi-field conditions.

An experiment was conducted, using two year old Kinnow plants, budded on rough lemon rootstock, inoculated with different isolates of AMF fungi viz., Glomus manihotis, Gigaspora gigantean and Glomus mosseae singly as well as in mixed culture. Five primers VANS1, NS61,NS21,VAGLO,VAGIGA specific to Glomus and Gigaspora species were designed and used in a polymerase chain reaction with DNA extracted from mycorrhizal roots varying in colonization. Glomus family-specific VAGLO primer in conjunction with Glomerales-specific VANS1 primer, successfully amplified 190bp fragment from samples of G. manihotis and G. mosseae. This 190bp product was not detected with G. gigantean and in the un-inoculated, non colonized control plants. Use of primer pair VANS1-NS61 for amplification of around 1kb fragment belonging to nearly complete gene coding for the small subunit rRNA was successful. Further use of this 1kb product as template DNA after one more round of PCR (for more amplification) in conjunction with primer pair VANS1-VAGLO and VANS1-VAGIGA produced an amplicon with an expected size only for the first primer pair and not for the second primer pair. This study clearly indicates the possibility of using this technique as a tool for rapid and precise detection and identification of arbuscular mycorrhizal fungi in Kinnow roots.

Biological control of tomato root-knot nematode, Meloidogyne javanica, by the isolate BI of Trichoderma harzianum was evaluated in greenhouse and laboratory conditions in tomato cv. King Stone. Effects of fungal spore suspensions in various densities (103 to 108 spore/ml) were studied on different disease intensities (average of gall diameters, average of root weight and average of stem weight). Average number of eggs per each mass of eggs per plant and average of egg number per each egg mass were determined. The results indicated that Trichoderma at 106 spore/ml concentration significantly reduced disease intensity as compared with concentrations less than 106 spore/ml (p?0.05%). Inoculation of tomato seedlings with Trichoderma at 106 spore/ml induced peroxidase activitiy in roots and revealed its maximum activity 7 days following inoculation. Peroxidase activity in tomato seedlings inoculated with nematode+Trichoderma (as treatment) significantly increased as compared with that in the only fungus inoculated seedlings (as control) reaching its maximum activity within 5 days after inoculation.

Two hundred and thirty one leaf samples of virus infected and mosaic and as well dwarf mosaic symptoms showing maize plants from various corn fields in Tehran province were collected during the growing season 2003. By performing serological tests of DAS-ELISA, DIBA and TPIA, using SCMV, MDMV, SrMV and JGMV antisera, only SCMV was detected in samples, with all samples reacting strongly to the SCMV antiserum. SCMV was determined as the main and prevalent potyvirus on maize in Tehran province. The virus was transmitted to sweet corn and sorghum plants using 0.1 M potassium phosphate buffer (pH 7) containing %2 Polyvinyl Pyrrolidon (PVP) for mechanical inoculation. For propagation and maintenance in greenhouse, the virus was propagated on sweet corn cv. Pars 403 and on grain sorghum cv. Kimia. The host range study with selected isolates of SCMV showed that the virus isolates were not transmitted by mechanical inoculation on Avena sativa, Panicum miliaceum, Setaria italica, Pennisetum americanum, Hordeum vulgare and Triticum aestivum. Purification of isolates was done using a minipurification method. UV absorbance spectra were obtained for purified viruses a ratio of A260/280=1.2 being calculated. Electron microscopic study using ISEM and decoration method with SCMV antiserum revealed filamentous flexuous particles of SCMV. In SDS-PAGE and western blotting tests on infected samples and purified isolates, the molecular weight of the virus coat protein was approximately 37-38 KDa. A difference among the CP of various SCMV isolates however, was not detected. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was done using SCMV F3 and SCMV R3 primers. where amplified fragments were found of approximately 900 bp in size as expected. The study indicated that SCMV is the prevalent potyvirus and the main causal agent of mosaic and dwarf mosaic on maize plants in the province.

Fire blight, a bacterial disease caused by Erwinia amylovora (Burr.) Winslow et al. 1920 is considered as a serious disease on pome fruits. During recent years, this disease has caused serious damages to the fruit growing industry in Iran. Precise and reliable methods for identification and detection of the pathogen can help in its early control, prevent the disease spread and help eliminate the infected trees in orchards and particularly young trees in the nurseries. In this study, two specific polyclonal antisera were developed and used in double diffusion in agar test and Dot Immunobinding Assay (DIBA) to detect E. amylovora (Ea) cells. As many as 1×107 cells per ml from pure culture and as well from infected tissues were detected through DIBA. The sensitivity level was increased to as low as 10 cells using the bacterial enrichment step. The efficiency of serological methods was compared with some published PCR based identification methods, too, in which, a single 1kb DNA fragment (pstI) of pEA29 plasmid was amplified in Polymerase Chain Reaction (PCR) by using A and B primer pair. The sensitivity levels of this method in water and tissue extract were 2×104 vs 2×106 CFU/reaction mixture, respectively. The three mentioned methods were able to identify Ea strains representing various hosts and regions of the country and were specific for Ea, in contrast with other bacterial genera. In Nested-PCR, AJ75 and AJ76 specific primers were used to amplify a single 800- bp fragment. In each reaction mixture, this highly sensitive method could detect as few as one and as many as 100 Ea cells in water and in tissue extract, respectively.

Verticillium wilt Verticillium dahliae is now a days commonplace in olive orchards in north of Iran. Root-knot nematode, Meloidogyne javanica, as well is one of the most widely disseminated plant parasitic nematodes in olive orchards of Iran. This study was arranged to find the effects of two sets of pot beds on these pathogen's interaction. Microsclerotia of Verticillium dahliae and J2s of Meloidogyne javanica were employed as the source of inoculum for the experiment. One-year-old seedlings of olive cultivar, Zard, were transplanted to the different sets of pot beds containing 720 cm3 of sterilized sandy loam soil (sand: 72.8%, silt: 13.8%, clay: 13.4%, organic substance: 2.2%, ECE = 2.76 ds/m, pH = 7.76) vs gravel (particle diameter: 5-7mm). The experiment was conducted in a completely randomized design of eight replications: Treatments were: control, nematode alone, fungus alone, nematode and fungus (simultaneously), nematode and fungus (concomitantly, fungus two weeks prior to nematode), nematode and fungus (concomitantly, nematode two weeks prior to fungus). Pots were inoculated with 1500 J2s of nematode and/or 7200 microsclerotia according to the treatment. The experiment was terminated after a lapse of nine months. Results showed that fungus colonization in root was more in seedlings transplanted into sandy loam soil, but female population as well as knots in the root system were higher in pots filled with gravel than in the sandy loam soil. The number of plant chlorotic, necrotic and wilt leaves was more in plants grown in sandy loam soil. In any case, variance within the pots containing sandy loam soil vs gravel was not different (P?0.05). Presence of nematode prior to fungus causes reduction in colonization of the fungus in the root and stem and inversely the presence of fungus prior to nematode caused reduction in number of galls produced by the nematode (P?0.05). Severe symptoms on aerial parts of the plants were observed when both pathogens were inoculated simultaneously (in the sandy loam soil, 97%) (P?0.05).

Forty monoconidial isolates of Magnaporthe grisea were examined, for an identification of vegetative compatibility group and a characterization of genetic diversity, using rep-PCR genomic fingerprinting. The isolates were collected from weeds Digitaria sanguinalis (crabgrass), Setaria italica (foxtail millet), Echinochloa crus-galli (barnyard millet), and some other unknown ones during 2003 - 2005 and preserved in the Mycology collection of the College of Agriculture and Natural Resources, University of Tehran, Karaj. Nit mutants were obtained from fast growing sectors on Minimal Medium (MM) containing 5-6% potassium chlorate. Complementation between nit mutants of isolates was tested on MM. Three vegetative compatibility groups were determined including VCG1, VCG2 and VCG3, the VCG1 with 29 isolates forming the dominant VC group among others. Genetic diversity of M.grisea isolates was studied based on DNA fingerprinting through rep-PCR, using two primers previously designed based on ERIC and BOX regions. They generated variable length fragments ranging from 420 to 3000 bp. Phenetic analysis differentiated six distinct clonal lineages designated as A to F. Clonal lineage A with a 62.5% frequency, was the largest fingerprinting group. This study revealed that isolates obtained from foxtail millet and crabgrass were designated in the same VC groups, forming heterokaryons with each other, however these isolates separated from each other the by 42% of similarity using rep-PCR marker. The correlation between VCGs and clonal lineages demonstrated low genetic diversity in M. grisea population within weeds.

To evaluate fertility status and mating type distribution of Cryphonectria parasitica, causal agent of chestnut blight in Guilan province, four sites in two main growing regions of: Shaft (Visrud, Taleghan and Babarekab), and Rezvanshahr (Doran) were investigated. During the study, fifty four isolates of C. parasitica were examined. C. parasitica isolates were crossed with each of the two mating type tester strains M1115 (MAT-2) and M1297 (MAT-1) of C. parasitica on autoclaved pieces of Castanea sativa shoots. According to the results, one of MAT idiomorphs (MAT-1 or MAT-2) were identified in most of the isolates (72.3%). A 20.4% of isolates were not able to produce perithecia with mating type testers. Moreover, 7.4% of isolates were sexually compatible with both mating types and produced perithecia.

To determine rice yield loss resulting from blast disease (Magnaporthe grisea) at the corp's different growth stages, a field experiment was conducted at the Rice Research Institute of Iran, Rasht. Rice local cultivar Hashemi was transplanted to 3×4m plots for the experimental treatments (time of disease evaluation) Each treatment was replicated 4 times. Under natural infection conditions, percentage of diseased leaf area and panicle blast incidence on each sample were scored 40 and 50 days following transplanting, 15 and 25 days after panicle appearance and as well at harvest time. Fungicide (Tricyclazole) was applied to prevent blast development after each time of disease scoring, until harvest time. Linear regression analysis was employed to calculate percentage yield loss due to leaf and panicle blast at each appearance time. Only the equation y= -0.74 + 0.5x (R2= 0.91*) was found significant to predict yield loss (y) from panicle blast (x) at harvest from the treatment in which no fungicide had been applied at the development stages. This treatment had also been more effected than the others in its increase of unfilled grains. Differences of blast disease rate and its development were not significant at the different levels of urea fertilizer application due to the high temperatures occuring at the most susceptible stages of rice growth.

One-year-old seedlings of olive cultivars, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to different sets of pots each pot containing 2000g of sterilized sandy loam soil. The experiment was conducted in a completely randomized design of 32 treatments, each in five replications. Treatments were comprised of: control, mere nematode, mere fungus, and fungus+nematode. Pots were inoculated with (0, 2000, 3000, 4000) J2 of nematode and/or a number of 10 microsclerotia per gram of soil in accordance with the treatments. Quantitative changes in phenolic compounds were studied 1,10,20 and 30 days following inoculation. Ten days past inoculation, the phenolic compounds in different treatments (as compared with control) were increased, but it was only in Koroneiki cultivar that did the fungus+nematode (4000 J2) treatment show a significant difference against control at 5% level (p?0/05). It was in the twenty and thirty days past inoculation treatments and in other cultivars (Roghani, Zard and Manzanilla), that the phenolic compounds in fungus+nematode treatment of 4000 J2 showed a significant difference (at 5% level) as compared with control. Phenolic compound's content (as compared with mere nematode and fungus alone treatments) was increased in the host plants and for the fungus+nematode treatments (p?0/05). Maximum and minimum total phenol content in olive roots in their and leaves were respectively recorded for cv. Koroneiki, Roghani, Zard and Manzanilla.