نشریه پژوهش های کاربردی در گیاه پزشکی
سال پنجم شماره 1 (بهار 1395)

The efficacy of trifloxystrobin tebuconazole (Nativo® WG 75%) in control of apple scab disease was studied in a Randomized Completely Block Design with 5 treatments in Talegan, Zanjan and Meshkin-Shahr. The treatments included Nativo 0.02 and 0.03% as the target fungicide, trifloxystrobin (Flint® WG 500) 0.02% and kresoxim methyl (Stroby® WG 500) 0.02% as standard fungicides plus no spraying control. Sprays were done at silver tip, petal full stages and 14 days after 2nd spraying based on Chapman and Catlin. Efficacy of spraying of fungicides was evaluated 14 days after early symptoms observed on leaves in control plots using disease severity index and before harvesting time on fruits by Marsh scale. The results revealed that Nativo 0.02% and 0.03% decreased disease of leaves by the 97.57% and 97.92% in Talegan and Zanjan trials, respectively 92% and 98.6% in Meskin-Shahr trial compared to control with the same effects. Also assessments of treatments effects on fruit scab revealed that Nativo 0.02% and 0.03% decreased scab by the 96.3% and 97.5% respectively in Talegan trial and 88.5% and 94.3% in Meskin-Shahr trial compared to control with the same effects. These results showed that Nativo could be used as a suitable candidate in chemical control of apple scab as an alternative fungicide to Flint and Stroby.

Temperature is one of the important factors that can influence the predator- prey dynamics. In this research, functional responses of males and females of Orius laevigatus towards Tetranychus urticae nymphs were investigated in laboratory condition at three constant temperatures, 20, 25 and 30°C. Different densities of mixture of protonymph and deutonymph of mite (2, 4, 8, 16, 32 and 64), on cucumber leaf discs (6 cm in diameter), were separately offered to male and female predators. After 24 h, the number of consumed preys was recorded. Ten replicates of the experiment were performed. Logistic regression and nonlinear regression of SAS software were used to determine the type of functional response and estimate the parameters, respectively. Consumption rate of both male and female predators increased with increasing temperature and prey density. Functional responses of both male and female predators to the mite were Type II at 20 and 25°C and Type III at 30°C. At 20 and 25°C, attack rates were estimated to be 0.0452 and 0.0498 h-1for females; and 0.0457 and 0.0460 h-1 for males, respectively. The lowest handling time for both male and female predators was estimated at 25°C. The results revealed that the predatory bug O. laevigatus may be more effective for biological control of T. urticae at higher temperature.

In this study formulations were prepared by emulsion of different vegetable oils (sunflower corn rosemary olive sesame and walnut) of Metarhizium anisopliae (Metschnikoff) Sorokin conidia. Control formulation was also prepared and germination of fungal conidia in all formulations was assessed. Results showed that control and olive oil formulations had the highest average conidial germination while the sesame oil formulation had the lowest germination. Effect of four concentrations (108 107 106 and 105 conidia/ml) of these formulations were applied against populations of Microcerotermes diversus Silvestri (Isoptera: Termitidae) termites. Due to high mortality rate in the first three concentrations the treatment with 105 conidia/ml was used in comparisons. The highest mean percent termite mortality (91.25%) was obtained with the sesame oil formulation and the lowest (58.33%) with control formulation. The lowest LT50 (2.38 days) occurred with the sesame oil formulation while the control formulation had the greatest LT50 (15.9 days). The lowest calculated LC50 (1.7  104 conidia/ml) was also for sesame oil formulation.

The effects of elevated temperature and CO2 concentration on spot blotch disease of wheat (caused by Cochliobolus sativus) were studied in controlled conditions. Wheat plants were grown in phytotron chambers in two levels of temperatures (22 and 25°C) and CO2 concentrations (360 and 550 ppm) and artificially inoculated with spores of pathogen at 30 days old. Influence of elevated CO2 was noted in terms of increased leaf area, spot size, percentage of leaf necrotic area and decrease in spot numbers cm-2 of leaf, incubation period and latent period. Temperature rise increased spot number and necrotic area of leaves and decreased incubation and latent period but did not affect mean size of spots as well as leaf area. By increasing both CO2 and temperature levels, significant decrease in incubation and latent period and increase in percentage of leaf infection was noted. The maximum percentage of infection and the shortest duration of incubation and latent period were observed in 550 ppm CO2 and 25°C. The effect of temperature rise under temperature gradient chambers was also observed as significant increase in disease severity in all wheat genotypes and varieties studied. Therefore, CO2þand temperature rise likely to contribute definitive increase in spot blotch severity in wheat.

In this research, insecticidal efficacy of the diatomaceous earth Sayan® in combination with a plant pharmaceutical formulation MG® containing myrtle (Myrtus communis L.) essential oil was evaluated on adult Rice weevils Sitophilus oryzae. Five doses of 0.125, 0.25, 0.5, 1.0 and 1.5 g Sayan®/kg grain in combination with MG® at four concentrations of 0 and estimated LC10, LC20 and LC50 (equal to 1267.0, 1790.0 and 3451.0 µl/L air, respectively) were assessed. All experiments carried out at 26±2ºC, 65±5% R.H. and L:D 16:8. Adults’ mortality was recorded after 24, 48 and 72 hrs of exposure. To determine progeny production at F1 and F2 generations, dead and alive adults were removed from treatment containers after the 72 hrs counts and containers were left at the same conditions for 45 and 90 days more. After these periods, emerged adults were counted (as dead or alive). Using Sayan® alone did not result in notable mortality even after 72 hrs so that at the highest dose of 1.5 g/kg it was 16.66%. Different doses and concentrations applied in combination increased the mortality significantly even after 24 hrs so that after 48 and especially 72 hrs it was highly notable and increased from about 80% by using LC10-0.125 g/kg after 48 hrs up to 100% by using the LC50-1.5 g/kg. No offspring produced by using treatments containing MG® essential oil but Sayan® applied alone resulted to progeny production in F1 and F2 generations. Results of this study indicated that applying the diatomaceous earth Sayan® in combination with the myrtle essential oil significantly increased the insecticidal efficacy of Sayan® and remedied dilatory effect of it (decreased the exposure interval form about more than 14 days down to about 72 hrs). This could be considered in the case of stored-products pest control.

Fire blight, caused by the bacterium, Erwinia amylovora, is a serious disease of pome fruits in many regions of the world and Iran. Several models based on climatic and phonological data were developed to prevent fire blight during bloom, thus improving our ability to control this phase of the disease by the timely application of antibiotics sprays in orchard. In this study (i) the causal agent of fire blight were detriminated in infected orchards (ii) removal infected plant tissues (iii) air temperature, and relative humidity were recorded (iiii) spraying and application of treatments were performed to prevent the blossom blight and based on claimatic condition, phonological trees and Thomson model. Experiment was performed as Random Complete Blocks Design (RCBD) with three replications. Results showed minimum effects in streptomycin 100ppm treatment (6.22%- 8.5%) on preventing of infection, especially when applied three bloom sprays during sensitive stages of flowering at 150C-18.10C. Then oxytetracycline 200ppm and starner 300ppm were ranked next respectively (8.533%-16.33%). The effective hess of copper oxycloride 1/1000 is depending on relative humidity and rain. Results that indicated interaction among bloom period, time of flowering and temperature could reduce number of spraying compared to Thomson model. Therefore we can use this technique in orchards of Golmakan, Chenaran, Nyshabour, Mashhad and other geographic areas where pome fruits are grown and have similar climatic condition.

Purification of expressed proteins is done to pursue different goals such as preparation of mono- and polyclonal antibodies, structural studies of the expressed proteins, identification of the amino acids and polypeptide sequences by chromatography which are generally costly and time- consuming. In this research, after expression of the coat protein (CP) gene of Cucumber mosaic virus (CMV) in Escherichia coli, purification of the expressed protein was done from polyacrylamide gel. To this aim, recombinant plasmid containing the CP gene was transformed into E. coli, induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and followed by polyacrylamide gel electrophoresis of the extracted protein. The result showed that the yield of the recombinant protein was optimized to purification. After express the recombinant plasmid in large volume of LB, extracted protein was electrophoresed on the polyacrylamide gel. The location of the expressed protein was determined, and then the expected protein band was separated from the polyacrylamide gel before subjecting to protein purification. Next, the recombinant purified protein was injected to two rabbits to raise the antiserum. Titration of the prepared antiserum was performed by the use of an indirect ELISA. It appeared that the method applied in this study to purify the expressed protein was more efficient than other purification methods such as chromatography. Also, results of DIBA and rabbit immunization showed that the purified protein could be used as the antigen to prepare the antiserum and antibody for detection of the CMV isolates.

One of the most pathogenic viruses in Solanaceous plants is Potato X virus (PVX) which causes systemic mosaic spots and economic loss in Nicotiana tabacum. Use of the tobacco resistance varieties is the best method in management of this disease. In this study, to establish the tobacco plants resistant to PVX, an RNAi construct was designed for a 400 bp fragment of PVX’s P25 gene which is a suppressor of RNA Silencing in plant. The P25 fragment was amplified with specific primers by PCR and was cloned in two steps (in sense and antisense directions) into the pFGC5941 plasmid. Then, the RNAi construct was transformed from recombinant plasmid into the tobacco explants (Samsun) by means of Agrobacterium cells. The transgenic plants were identified by selection on Basta containing media and propagated, followed by molecular and phenotypic analyses. PCR test using plant’s DNA confirmed the presence of the transgene in transgenic plants. The next generation of the transgenic plants was inoculated with PVX in greenhouse. ELISA using anti-PVX-CP antibody showed that three transgenic lines are resistant to PVX. Molecular confirmation of three tobacco resistant lines was performed by RT-PCR using plant RNA and CP-specific primers and PVX did not detected in transgenic plants. This study resulted in production of three tobacco lines resistant to PVX. Study of their resistance in the field condition is in progress.

Iranian Johnson grass mosaic virus (IJMV) is one of the most important cereal viruses and can cause viral symptoms such as stripe mosaic and red necrotic stripe in corn and sorghum. Recently, these symptoms widely observed in corn and sorghum fields of Golestan province. With regard to native origin and possibility of the virus survival in wild plant hosts and importance of the cereal cultivation in Golestan province, it is necessary to study its incidence, especially in important regions of corn growing farms. In this study, in order to serological and molecular detection and studying the distribution of IJMV in Golestan province, a total of 350 samples were collected from maize, sorghum and some weeds from poaceae family showing mosaic and red necrotic stripe symptoms in 4 stages. Infected samples were checked by ELISA test. A part of coat protein gene of IJMV was amplified in RT-PCR test using general primer pair of potyviruses. The result showed that through 350 samples, 164 (47 per-cent) showed positive reaction and 186 samples showed negative reaction in ELISA test. Maximum infection was in maize samples. The RT-PCR test led to amplification of expected fragment of 327 bp. The purpose of this study is determining of the distribution of IJMV and its extent of infection in different areas of Golestan province. According to the result of this study it has shown that IJMV has a high incidence in Golestan province.

This study was aimed to characterize the Monilinia species associated with brown rot disease of stone fruit trees in West Azarbaijan province, Iran. For this purpose, during the 2013-2014 growing seasons, samples were collected from stone fruit trees showing blossom and twig blight, rotten and mummified fruits symptoms. Monilinia isolates were identified based on cultural characteristics and morphological features of conidia on PDA, MEA and CHA culture media. A total number of 89 Monilinia isolates were recovered from symptomatic tissues. Based on the comparison of cultural and morphological characteristics of the isolates obtained in this study with those of reference isolates for M. laxa, M. fructicola and M. fructigena, all of the isolates were identified as M. laxa. The identity of our isolates was further confirmed using sequence data of ITS-rDNA region for the representative isolate. A phylogeny inferred based on the sequence data from ITS region, clustered our isolate in M. laxa clade together with other sequences retrieved from GenBank for this species. All of the attempts made for the induction of sexual stage under laboratory conditions were unsuccessful.

Take-all, caused by Gaeumanomyces graminis var. tritici is an important disease of wheat in the Fars province (south Iran). Five isolates of the pathogen were isolated from infected wheat plants of this province, and their virulence tested on Alvand cultivar. Biological control method of disease investigated with dual culture test of native isolates of Pseudomonas fluorescens, Trichoderma harzianum and T. virens in vitro, at first. Then impact of treatments of seed and soil with three top isolates of bacterium and two of T. harzianum were tested on disease severity caused by the most virulent isolate of the pathogen, under greenhouse condition. All microorganisms significantly reduced hyphal growth of pathogen isolates, in dual culture test. P. fluorescens isolates in two treatment methods, and isolates of T. harzianum in seed treatment were most effective in reducing disease severity, in greenhouse test, therefore it is possible to use them for biological control of the disease. Ability of Fars province native isolates of these bacterium and fungus in biological control of wheat take-all disease is reporting for the first time.

During evolution, various compounds against herbivors were developed in plant species. Disturbance in digestive system by plant enzyme inhibitors is one of the effective methods in integrated pest managment programs. In this study, the effects of total Goebelia alopecuroides and Vicia ervilia seed extracts and their fractions were assessed against proteolytic activity of cotton bollworm. Total extracts of G. alopecuroides was effectively inhibited digestive proteinase of cotton bollworm compared with that of V. ervilia. The inhibitory effect of G. alopecuroides on Bovine trypsin and chymotrypsin as well as insects’ trypsin and chymotrypsin was higher than that of V. ervilia. Total seed extracts of V. ervilia significantly decreased digestive chymotrypsin activity. Ammonium sulfate precipitation of seed extract also showed significant inhibitory effect among the fractions. The fifth fraction of G. alopecuroides showed the highest inhibititory efect agianst digestive proteolytic activity of cotton bollworm and bovine trypsin. The first fraction of V. ervilia significantly inhibited total proteolytic activity.The first fraction of both plants seed extracts inhibited digestive trypsin of cotton bollworm compared to the other fractions. The third fraction of V. ervilia and the forth fraction of G. alopecuroides showed higher inhibitory effect on digestive chymotrypsin of cotton bollworm comperd with the other fractions. Comparing crude extracts effects of both plant species on laboratory and wild populations of cotton bollworm showed that wild ones was susseptible compared with the laboratory population.

Potato virus Y (PVY) is one of the most damaging viruses of solanaceae family which has been reported from potato and tobacco growing areas in Golestan province; however, this virus has not been studied in pepper fields yet. Due to importance of pepper plants as one of the important hosts of PVY and also cultivation of solanaceous crops in close proximity to one another, the occurrence of PVY in pepper growing farms in Golestan province has been studied. A total of 80 pepper leaf samples showing symptomatic mosaic, mottle, dwarfing and deformation were collected. The PVY infection of the samples was investigated using PVY polyclonal antibody in TAS-ELIASA test. Among 80 pepper samples, 10 samples from each two regions of Daland and Gorgan had the infection. Two positive samples were inoculated on Nicotiana tabacum cv. samsun by mechanical method. Inoculated plants showed symptoms of necrosis and chlorosis in two weeks after infection, and infection of symptomatic plants was confirm by using monoclonal antiserum specific against PVYN, PVYO and PVYC in ELISA test. Results of this study indicated that Daland sample was infected by PVYC, whereas Gorgan sample was infected by PVYN and PVYO strains. Results of RT-PCR test using both general and specific primers covering a part of coding region of CP confirmed infection of samples to PVY. This is the first report of PVY infection of pepper plants in Golestan province.

Gray mold, caused by Botrytis cinerea, is one of the most important diseases of grape worldwide. Although, it has recently become a major problem in West Azarbaijan province causing significant yield losses, no fungicide has been registered to control the disease. In currentstudy, susceptibility of 103B. cinerea isolates collected from vineyards in different regions of the province was assessed to three fungicides including benomyl, iprodione and fenhexamid using discriminatory doses of fungicides in in vitro conditions. Results indicated that isolates resistant to benomyl and iprodione were widely distributed across the surveyed regions at different frequencies, whereas the isolates resistant to benomyl were not detected in the samples collected from Salmas. Isolates resistant to fenhexamid were detected at low frequency in six sampling locations. The results also showed that the isolates with multiple resistances to two fungicides were widely distributed in the studied regions. It is likely that application of benomyl and iprodione or fungicides with similar mode of action to control fungal diseases in grape other than gray mold, have resulted in selection of B. cinereastrains resistant to these fungicidesas non-target pathogen leading to increase in their frequencies. The results revealed that the use of benomyl and iprodione or fungicides with similar mode of action would be ineffective to control the disease, while fenhexamid seems to be an alternative and effective fungicide to be registered and used to control the disease.

The effect of different concentrations of Metarhizium anisopliae strain EUT115 along with control was investigated on various life stages of green peach aphid Myzus persicae, and different canola cultivars, Brassica napus. Thirty aphids of each stage were treated with the fungus and then transferred onto Petri dishes containing leaf-discs placed on 2% water-agar medium. The aphid mortality was recorded daily for 14 days after the beginning of the test. According to the results, after 7 days, the highest mortality was obtained on the RGS003 (5.47 ± 0.44) and the lowest mortality was occurred on the Zarfam genotype (4.983 ± 0.45) and the significant difference was observed between the two genotypes. The highest mortality among the different life stages happened for the adults and was calculated 7.64 ± 0.65 and 8.82 ± 0.50 aphids after 7 and 14 days, respectively. Among the various fungal concentrations, 107 conidia/ml of the fungus caused the highest mortality after 7 (8.38 ± 0.24 aphids) and 14 days (9.60 ± 0.12 aphids), respectively. The results showed that there was interaction between the fungal concentration and the aphid life stage. It is suggested that to control enormous infestation of the green peach aphid in canola fields, the RGS003 genotype and 107 conidia/ml of the fungus can be used. The mean mortality of the aphid was increased by increasing the time from spraying the pathogenic fungus. It shows the importance of fungus-sporulating cadavers of the aphid as a reservoir in surrounding environment.

According to high postharvest losses of peaches and restriction of the use of chemical fungicides, it is necessary to find proper methods to control postharvest pathogens. For this purpose, natural antifungal and antimicrobial compounds like antagonists can be used to increase shelf life of fruits. Rhizopus rot that caused by Rhizopus stolonifer is one of the most important decays of peach fruit losses. In this research we evaluated the antifungal activity of two isolates of antagonist Aureobasidium pullulans (L1 and L8) against R. stolonifer at different concentrations of yeast suspension (108, 107, 106 CFU ml-1) and antagonist forms (culture filtrate (CF), washed cell suspension (WCS), autoclaved cell suspension (AC)) to control the Rhizopus rot on peach and discovering the possible modes of action on peaches at 20ºC. Results showed that just washed cell suspension could control significantly incidence of peach rot. Between applied concentrations of antagonist, the highest concentration could control incidence of Rhizopus rot on peaches.

Potato, Solanum tuberosum L. is an important vegetable crop throughout the world. This crop is attacked by various pests such as Colorado potato beetle, Leptinotarsa decemlineata (Say). Since many plant compounds have been found with insecticidal and deterrent effects against many insect, in this research the effects of essential oils of three medicinal plants including: Satureja hortensis L., Artemisia dracunculus L. and Thymus vulgaris L. were investigated on nutritional indices of Colorado potato beetle. This study was conducted in a completely randomized experiment with three treatments (three essential oils) and each treatment with four concentrations (4-10 ppm for 2nd and 3rd larvae and 10-16 ppm for 4th and adult) and each concentration with 15 replications in a growth chamber set at 25±1ºC, 65±5 % RH in 16:8 (L:D) h conditions. Essential oils of all three medicinal plants in this research showed insecticidal and nutritional deterrence effects on 2nd, 3rd, 4th and adult of Colorado potato beetle but in all concentrations, essential oil of S. hortensis decreased relative growth rate, relative consumption rate and efficacy of conversion of ingested food of larval stages and adult of Colorado potato beetle significantly in comparison to the other oils and affected feeding deterrence index in all stages. Therefore, if a suitable formulation method of the essential oil of S. hortensis could be found and the persistence of the formulated material is increased under the field conditions, it could be used for applications in integrated management of the Colorado potato beetle.