فصلنامه گیاه پزشکی
سال چهل و پنجم شماره 2 (تابستان 1401)

Angoumois grain moth, Sitotroga cerealella, is one of the most critical storage pests with a wide range from the hosts in Iran and worldwide. Excessive consumption of chemical insecticides leads to developing the resistance to chemical insecticides, environmental pollution, and the emergence of new pests. Therefore, we have decided to reduce the risk of the environment in pest’s control via plant essential oils.

In the current study, to examine the environment- friendly pesticides, effects of three plant essential oils from the families Mentha longifolia L., Thymus daenensis Celak (Lamiaceae), and Artemisia aucheri Boiss (Asteraceae) compared to dichlorvos (DDVP®,as a positive control) an organophosphate insecticide were studied. Antioxidant enzymes, including: superoxide dismutase (SOD), catalase (CAT), and lipid peroxidation marker on the fourth instar larvae of S. cerealella after 24 hours were examined. The essential oils were prepared using the water distillation method. Essential oils were used in five concentrations and three replications. The concentration was selected based on the primary experiment for each essential oil. Insects were exposed to fumigant toxicity of the essential oils and insecticide in laboratory conditions (25±1 °C, 70±5% in 12 hr light/12 hr dark).

The results showed that LC50 for the essential oils of M. longifolia, T. daenensis, and A. aucheri were recorded 0.04, 0.68 and 6.47 μL/Lair, respectively. The highest mortality rate increased with high concentrations (0.09, 3 and 9 μL/Lair) of M. longifolia, T. daenensis and A. aucheri essential oil were 84, 80.67, and 77.33 %, respectively after 24h. Enzymatic activity increased with essential oil high concentrations. Data indicated a significant increase in the activity of catalase, superoxide dismutase enzymes and the concentration of malondialdehyde as peroxidation lipid indicator in treated larvae with essential oils of M. longifolia, T. daenensis and A. aucheri compared with the untreated (negative control), and DDVP® (positive control) on treated 4th instar larvae of S. cerealella after 24h. 

 Conclusion

It seems that different essential oils of M. longifolia, T. daenensis and A. aucheri with the effect on the enzymatic activities of antioxidants, including catalase, superoxide dismutase enzymes and malondialdehyde as lipid peroxidation indicator of the fourth instar larvae of S. cerealella via the disruption of physiological processes and survival. Regarding the existence of different aromatic plants in Iran, we can use the species with higher toxicity that would be more economical to produce the botanical insecticides and use in the integrated pest management programs for this pest control.

The Greenbug, Schizaphis graminum Rondani (Hemiptera: Aphididae), is one of the most important pests in grain, especially wheat, and is an important cause for transferring a number of pathogenic viruses in plants. The purpose of this research was to evaluate the lethal and sublethal effects of Pymetrozine insecticide on life history parameters and α-esterase and β-esterase enzymes activity of S. graminum. Detoxifying enzymes play a vital role in the detoxification of chemical compounds in many living organisms. These enzymes increase the resistance of insects to chemical pesticides by the catalysis of these compounds in their body.

The S. graminum was reared on the wheat host in a growth chamber set at 25 ± 2 °C, 60 ± 5 % RH, plus 16: 8 (L: D) hour photoperiods. Daily observations and records were made for the development period and survival rate. The crude data were analyzed based on age-stage and two-sex life table analysis. TWO SEX-MSChart was used to evaluate the raw data based on the age-stage and two-sex life table. In addition, alpha-naphthyl (α-NA) and beta-naphthyl (β-NA) acetates were employed as substrates for the α-esterase and β-esterase activity, respectively. Esterase enzymes of tested treatments were measured using a microplate reader.

Exposing adults to bioassays indicated that the lethal concentration (LC50) of insecticide treatment in S. graminum adults was 84.68 mg a.i./L. The present study demonstrated that Pymetrozine insecticide showed more acute toxicity on adult stages of S. graminum. Moreover, exposure of adults to sublethal concentrations (LC30) negatively affected the development and reproductive characteristics and demographic factors of the Greenbug. In specific, results for sublethal experiments showed a significant increase in the development time of the pest on the insecticide treatment compared with the control. On the contrary, the lowest fecundity in pests was observed on Pymetrozine insecticide. In addition, the tested insecticide significantly reduced the population growth parameters of S. graminum, which can be identified using detoxifying enzymes as biochemical markers of the pest. Estimation values for the intrinsic rate of increase (r) in the control and insecticide treatment were 0.394 and 0.280 per day, respectively. Furthermore, the activity of alpha and beta esterase was not significantly different between control and insecticide treatment.

The results revealed that Pymetrozine insecticide exerts high lethal and sublethal effects on S. graminum, and thus, it is recommended to be applied in an integrated pest management program (IPM). Semifield and field studies are needed to obtain more applicable results using insecticides.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is one of the key pests that can damage a wide range of crops in farms and greenhouses. Using chemical pesticides is one of the main methods for its management. Laboratory bioassays were conducted to evaluate the effects of sub-lethal concentrations (LC5, LC10 and LC20) of Biomite (a formulation based on Citronellol) on biological characters and life table parameters of T. urticae under laboratory conditions (25±2°C, 60±5% RH, and L:D16:8 hours). Data were analyzed based on age-stage, two-sex life table analysis. Our results indicated that when adults of T. urticae were treated with LC5, LC10 and LC20 of Biomite, the oviposition period and total lifetime were significantly reduced compared with the control. The highest and lowest values of the fecundity (61.1 and 41.56 eggs/female) and longevity (13.01and 9.97 days) were obtained in control and LC20, respectively. The individuals treated with LC10 (R0 = 38.1) and LC20 (R0 = 32.92) showed a significantly reduced net reproductive rate, compared to individuals that treated with LC5 (R0 = 47.01) and to the control (R0 = 48.88 individuals/female/generation). The intrinsic rate of increase (r) and finite rate of increase (λ), were not significantly reduced in different treatments compared to the control. The mean generation time (T) decreased significantly at upper concentration (LC20=15.58 days), in comparison to LC5 (16.66 days). Due to the obtained results of current study and considering the detrimental effects of Biomite on some biological parameters, it could be incorporated in IPM programs of T. urticae.

Diamond back moth, Plutella xylostella L. (Lepidoptera,Plutellidae), is the cosmopolite insect pest of cruciferous plants causing significant injury to the plants of this family. Although many integrated approaches have been proposed and developed for DBM management, the most common method of controlling this pest is chemical control. Organophosphates (OPs) are mainly used to control the agricultural pests in Iran, especially DBM. This study was performed to determine and compare some toxicological and biochemical properties of detoxification enzymes (ESTs and GST) and cholinesterase between two resistant (Esf-R) and susceptible (Ard-S) field populations of P. xylostella.

The phosalone-susceptible (Ard-S) population was collected from Ardabil, Ardabil province, Iran and the phosalone –resistant (Esf-R) population from Esfehan, Esfehan province, Iran. The toxicity of insecticides was measured using a standard leaf-dip bioassay. To determine the role of metabolic degradation as a mechanism for phosalone resistance in DBM, PBO (piperonyl butoxide), TPP (triphenyl phosphate) and DEM (diethyl maleate) were bioassayed for synergistic activity with phosalone (Kodwo and Tanaka, 2005). EST and GST assays were determined based on the method of Van Asperen (1962) and Habig et al. (1974) with minor modifications. AChE activity and its kinetic parameters were measured with two artificial substrates, ATC and BTC, along with the modified method of Ellman et al. (1961). Statistical analyses were evaluated by LeOra software (1978) through ANOVA followed by Tukey test.

According to the bioassay results, the (Esf-R) population showed a significantly high resistance to phosalone compared with Ard-S population (17-fold). Diethyl maleate (DEM) and triphenyl phosphate (TPP), as glutathione S-transferase (GST) and esterase inhibitors, increased phosalon toxicity on both resistant and susceptible populations, but the synergistic ratio in the resistant population was higher than that of susceptible one. This confirms the greater role of esterase and GST enzymes in phosalone resistance. Metabolic resistance mechanisms to phosalone were surveyed by biochemical assays. The results indicated that specific activities (SA) of GST, α-esterase and ß-esterase were 2.1-, 2- and 1.7-fold higher in the resistant populations than those of susceptible population, suggesting higher expression of GST and esterase enzymes in resistant population. Furthermore, target site insensitivity was surveyed by biochemical assay. Kinetic parameters of acetylcholinesterase (AChE) on the hydrolysis of acetylthiocholine iodide (ATC) showed no change in the affinity of AChE of resistant population to this substrate and the phosalone resistance mechanism was not related to altered AChE active site.

The results distinctly indicated that metabolic detoxification mechanisms such as GST, esterases created phosalone resistance in the Esf-R and AChE structure were not involved in resistance. According to the result, use of synergists can be helpful for suppressing the phosalone resistance.

The bacterial rot of pepper, onion, and potato is one of the most crucial diseases in the East and West Azerbaijan provinces of Iran. Therefore, accurate identification of the causes of these diseases and the study of their genetic diversity can be beneficial in preventing their occurrence. In the present study, in addition to studying and identifying their morphological and molecular characteristics, the genetic diversity of bacterial species from selected hosts in the provinces of East and West Azerbaijan is studied to obtain comprehensive information.

Infected potatoes, onions, and peppers with soft rot symptoms were collected from different areas in the East and West Azarbaijan provinces of Iran in the timeframe between 2019 and 2020. Infected samples were cultured on the NA medium. Moreover, purified bacterial colonies were streaked on the Kings’B medium. All isolates that produced fluorescent pigments were identified using biochemical, nutritional, and physiological tests, including LOPAT tests (levan production from sucrose (L), the presence of oxidase (O), the ability to cause rot on potato tubers (P), the presence of arginine dihydrolase (A), and the ability to induce hypersensitivity reaction (HR) on tobacco leaves (T)), nitrate reduction, and the utilization of various carbon sources. All isolates that produced fluorescent pigments were identified as Pseudomonas using phenotypic properties. To assess the genetic diversity within the strains, BOX and ERIC-PCR were analyzed. The UPGMA method was employed to evaluate the similarity matrix and clustering between the strains. For genetic identification, 16S rRNA, gyrB, and ropD genes of strains were amplified and sequenced. 

A total of 59 isolates were obtained from infected onion, pepper, and potato plants. The bacterial isolates revealed cream colonies with a low convex height, jagged edge, and rugged surface on the nutrient agar medium. All strains produced fluorescent pigments on the King’s B medium and demonstrated Gram-negative and positive reactions to the catalase. In pathogenicity tests, all strains were pathogenic on healthy plants. Moreover, clustering ERIC and BOX-PCR results with UPGMA and Jaccard’s similarity coefficients showed that the strains of potato, pepper, and onion were clustered into five and four main groups, respectively. The gyrB, rpoD, and 16S rRNA nucleotide sequences showed high similarity between the strains and Pseudomonas cichorii.

The results showed that Iranian P. marginalis strains are very diverse and are related to the isolation site and the host plant. This high diversity can be due to the long history of cultivating these plants in the provinces of Iran and the high genetic diversity in the bacterial population. Based on previous studies published on Iranian P. marginalis, this is the first report of isolation and identification of P. marginalis, the agent that causes soft rot in pepper, onion, and potato in East and West Azarbaijan provinces.

The larvae of the Mediterranean flour moth, Ephestia kuehniella (Zeller), are widely used, as an alternative host, to rear some biocontrol agents such as Habrobracon hebetor (Say) for biological control programs. To enhance the quality of mass production, it is necessary to optimize the rearing of natural enemies on alternative hosts. The aim of this study was to investigate the possibility of diapause induction in the Iranian population of E. kuehniella larvae for optimizing the mass production of biocontrol agents such as H. hebetor.

To study the diapause induction in E. kuehniella larvae, the insect eggs (0.04 g) were released on 200 g of wheat flour and kept under optimal rearing conditions (25 ± 1°C, 65 ± 5% relative humidity, and 16:8 (L:D) h). After the emergence of each larval instar, they were divided into two groups. The first group included larvae that had already been reared in each instar (first to fifth instars) at 15 ± 1°C and total darkness for one week and the second group under the same conditions for two weeks. Then, the larvae of both groups were transferred to the optimal rearing conditions. After the emergence of fifth (final) instar, the percentage of larvae entered the diapause where the duration of diapause was evaluated. The control group consisted of larvae that were reared in the optimal rearing conditions.

The results revealed that 8 to 45% of the fifth instar larvae from the first group and 100% of the fifth instar larvae from the second group entered the diapause. The percentage of diapausing larvae from the first group was the highest when they were reared in the fourth or fifth instars at 15°C and total darkness. The duration of diapause in the larvae from the second group (19.41 to 20.56 days) was significantly longer than those from the first group (15.20 to 16.92 days).

The present study is the first attempt showing diapause induction in Iranian population of E. kuehniella. By increasing the availability of host larvae by inducing diapause, the efficiency of mass production of biocontrol agents such as H. hebetor can be enhanced.

Wheat (Triticum aestivum L.) is one of the most important crops worldwide, responsible for a large portion of the human diet. In light of the increasing population, increased demand of 20 million tons by 2025 has been predicted by the Iranian Ministry of Agriculture. However, Fusarium graminearum and Xanthomonas translucens are among the most destructive diseases that lead to high yield loss. The present study aimed to isolate and evaluate the antagonistic plant growth-promoting endophytic bacteria in Iranian wheat cultivars.

In this study, endophytic bacteria were isolated from different tissues (e.g., seeds, roots, and shoots) of 14 wheat cultivars: Arg, Bahar, Baharan, Behrang, Chamran, Chamran 2, Dena, Falat, Heidari, Parsi, Pishgam, Sayonz, Sirvan, and Sivand. Bacterial strains were cultured in different media (i.e., NA, LBA, and TSA 10%). Then, their antifungal and antibacterial activity against F. graminearum and X. translucens were analyzed by dual culture assays. Moreover, the effect of isolates on seed germination was evaluated in vitro. In addition, extracellular enzyme production and plant growth-promoting properties were assessed. Furthermore, the effect of endophytes on plant growth was evaluated in greenhouse trials, and finally, the selected isolates were identified by 16S rRNA gene sequencing using two sets of universal primers (rD1, fD1, and 27F, 1492R).

In this study, a total of 464 endophytic bacteria were isolated from wheat surface-sterilized tissues. Forty-four isolates successfully suppressed both Fusarium and Xanthomonas in vitro. The highest percentage of the fungal growth inhibition was evaluated at 86.66% (CB2), and the highest clear halo around isolates, which indicated the antibacterial activity, was 49.33% (PaR28, PiR5). Among the isolated bacteria, twelve were capable of increasing seed germination in vitro by at least 10% more than untreated seeds and could increase shoot and root length, which were selected for further characterization. The clear halos, which indicated enzyme production, were calculated as follows: protease 0.36 to 2.53, cellulase 1.03 to 1.75, pectinase 1.06 to 2.65, and amylase 0.43 to 2.1 cm. The diameter of siderophore production on CAS agar medium varied from 0.21 to 1.46 cm. Moreover, IAA was between 7.05 and 14.14 µg/mL, ammonia production varied from 0.29 to 34.49 mg/mL, and phosphate solubilization was between 6.09 and 14.95 µg/mL. Greenhouse experiments indicated that the biomass was significantly increased in plants treated with endophytes. In addition, DNA sequencing using the 16S rRNA gene showed that the isolate CB2 had 99.06% homology with B. subtilis, B. velezensis, and B. amyloliquefaciens. Plus, the isolates PaR28, SeR8, BaR15, and BR20 indicated a homology of more than 99% with P. jamilae, P. polymyxa, and P. peoriae. The sequence of the mentioned isolates has been deposited in the GenBank under accession numbers MT258405 (CB2), MT258403 (BaR15), MT258407 (SeR8), MT258404 (BR20), and MT258406 (PaR28).

The present study is the first report on the endophytic population in mentioned wheat cultivars and an indication of their significance in the biocontrol of X. translucens. As isolates identified in this study have several desirable characteristics, a better-informed knowledge of them can be expected to have promising applications as biofertilizers and biopesticides.

Many aphids attack cucumbers in the greenhouse and reduce their quality and quantity. Various varieties of greenhouse aphid species, particularly Aphis gossypii Glover, are responsible for considerable damage to cucurbits and vegetables, including cucumbers, tomatoes, watermelons, melons, etc. In addition, these insects can transmit many plant viruses from infected plants to healthy ones.

This study was conducted in the cucumber greenhouses of the three provinces of Sistan and Baluchestan, western Azerbaijan, and southern Kerman by a randomized complete block design with five treatments and four replicates. The efficiency of Emamectin benzoate 4.8% + Acetamiprid 6.4% EC at a ratio of 1.5/1000 and 2 /100 was compared with Flonicamid (Teppeki 50 WG) at a ratio of 0.25 /100, Primicarb (DF 50%) at a ratio of 0.7 /1000, Dichlorvos (EC 50%) at a ratio of 0.5 /1000, and the control (water spraying). The sampling was done one day before and 3, 7, and 14 days after treatment. The Henderson-Tilton formula was used to calculate the efficiency of the pesticides. And the means were compared by Tukey's test and SAS software. In the second part of this study, to measure the pesticide residues, 11 samples (one sample from each both different doses were collected three times: 3, 6, 8, 13, and 15 days after spraying, while one sample was taken as a control with no treatment) were collected in West Azerbaijan Province and transferred to the Pesticide Residues Reference Laboratory in the Pesticide Research Department. The samples were crushed, homogenized, and prepared. The organic phase obtained in the process was used for the cleanup stage and then centrifuged. Furthermore, residual pesticide content was identified and quantified by optimizing the operating conditions of the LC-MS/MS instrument. Finally, a comparison of the calculated levels with the national MRLs was made to determine the level of the toxin residues present in the product.

The results showed the following on the third day after spraying:The concentration of Emamctin benzoate + Acetamiprid treatment in Sistan and Baluchestan province was 2/1000 (77. 30 ± 4.6%). The concentration of Imamctin Benzoate + Acetamiprid insecticides in the Western Azerbaijan province was 2/1000 and 1.5/1000, and the concentration of Flonicamid was 95.02 ± 2.35, 94.42 ± 1.07, and 91.68 ± 3.65% respectively. Subsequently, Emamectin benzoate + Acetamiprid in the concentration of 2/1000 with an efficiency of 100% performed better than the others. The examination of the pesticide residues in the insecticide-treated cucumbers showed that the residues of Emamectin benzoate in all the samples sent after harvest were so low that LC-MS/MS was not able to detect it both quantitatively and qualitatively. Although the Acetamipride residue was 0.058 and 0.063 mg/kg respectively in both treatments on the third day after spraying, the level was below the permissible limit. Moreover, it was not detectable from the sixth day after spraying.

Essential oils are a variety of compounds, and their monoterpenoids, as the main constituents may impair insects' physiological and behavioral functions and could thus be applied in pest management. In addition to causing damage to stored products, the Mediterranean flour moth, Ephestia kuehniella Zeller, 1879 (Lep.: Pyralidae), is actually an easy model to grow in the laboratory and is one of the suitable insects to determine toxicity and in vivo interactions of xenobiotics with physiological systems of insects. In the present study, in order to better understand the mechanism of action of trans-anethole as one of the most important secondary metabolites of the Apiaceae family, the oral toxicity of this compound was evaluated on the activities of digestive and detoxifying enzymes, and metabolites involving in intermediate metabolism of the fourth instar of E. kuehniella.

Fourth instar larvae of E. kuehniella were randomly selected and separately exposed to 1 g of the artificial diet containing 1, 2, 4, 8, and 16 μL/g of trans-anethole while the control larvae fed on the diet containing acetone. The bioassay test was performed in 3 replicates with 10 larvae per replication. Mortality was determined after 24 h and LC50 value was calculated by POLO-plus software. Ephestia kuehniella larvae were exposed to the LC50 value of trans-anethole to determine its effects on the enzymatic and non-enzymatic components. After 24 of exposure, the larvae hemolymph, midgut, and fat bodies were extracted. Hemolymph samples were immediately centrifuged at 15000 × g at 4°C for 20 min, while samples of midgut and fat bodies were initially homogenized by a glass pestle and then centrifuged under the same conditions. The hemolymph samples were used to assay the detoxifying enzymes and enzymes of intermediary metabolism, while the midgut samples were used to assess the digestive enzymes. Fat body samples and head capsules were also used for energy reserves.

The LC50 value of trans-anethole against the larvae was 7.03 μL g-1. Significant decrease in the digestive enzyme activities (α-amylase, α and β-glucosidase, lipase) and specific proteases (trypsin, chymotrypsin, elastase, amino and carboxy peptidase) were observed. In contrast, the activity of the detoxifying enzymes (esterases, and glutathione S- transferase) increased in the treated insects. The activities of amino transferases (alanine, aspartate, and gamma-glutamyl) significantly increased in the treated larvae by trans-anethole. Significant decreases in lactate dehydrogenase and phosphatase (acid and alkaline) were observed after treatment. Moreover, the amount of storage macromolecules (total protein, glycogen, and triglyceride) in the treated insects was significantly decreased compared to the control.

Our research revealed that trans-anethole is a toxic compound to E. kuehniella by reducing the survival and digestive efficiency of the larvae. Moreover, trans-anethole significantly enhanced the detoxifying enzymes activities. When the larvae were exposed to LC50 of trans-anethole, the digestive activity was reduced due to the cytotoxic effects of trans-anethole on epithelial cells of the larval alimentary canal. Our results demonstrated that trans-anethole might have a promising potential to develop as a safe compound to suppress E. kuehniella population. For the practical use of trans-anethole to control E. kuehniella, it is required to determine the accurate mode of action, and using appropriate formulations to increase its efficacy in long-term applications.

Bacterial leaf streak of the cereal is one of the most important diseases of wheat and barley which was reported from Iran in 1989. The severity of this disease has increased since 2016 in some of the irrigated wheat fields of Iran. Using the resistant cultivars is the best way to manage this disease. In the past, the response of some elite lines and cultivars to this disease was studied and determined. As a result, it's critical to investigate how new promising lines respond to this illness. Under this context, the reaction of a variety of elite wheat lines and cultivars to this disease was examined in greenhouse settings in a recent research. In this study, the reaction of a number of wheat lines to this disease was evaluated in field conditions in three regions with different climates.

Seventy-one advanced lines received from the Seed and Plant Improvement Institute, were planted in the experimental farms of research stations in three provinces, including Hamadan, Lorestan, and Jiroft.  Each line was planted in two one-meter rows in a randomized complete block design in three repetitions. They were then sprayed with bacterial suspension (regional isolates) at a concentration of 108 cfu / ml by spraying at three times (during tillering, after stem elongation and at the beginning of heading/flowering). The percentage of leaf surface infection was recorded and evaluated fifteen days after the last inoculation. Data were analyzed using SAS software, and the means were compared using Duncan test at a 1% probability level.

The response of various lines to this bacterium was different in three regions. But, in general, the studied lines due to the reaction to this disease were evaluated susceptible (such as S-92-19, ICSBWEYT-17-3, N-91-17, ICSBWEYT-17-2, ICSBWEYT-17-9 and ICSBWEYT-17-7), Semi-sensitive (such as ICSBWEYT-17-11, ICSBWEYT-17-10, ICSBWEYT-17-16, ICSBWEYT-17-1 and ICSBWEYT-17-24) and tolerant (such as: C-91-4, N-91 -8, N-92-9 and CD-94-9).

The grouping of the lines' susceptibility to this disease in six specific groups has high overall coordination and fit with each other, in three areas. However, the infection severity of wheat lines with this disease significantly varied in different regions. Therefore, the severity of wheat line infection in Boroujerd was higher than in the other two areas. On the other hand, some lines in various regions had different reactions to the disease. For example, MS-92-18 line had relatively good tolerance in Boroujerd region, but not in Hamadan and Jiroft regions. Climatic and geographical conditions can affect the behavior of wheat genotypes to the disease and change their response in different climates. Many other researchers have proved this. The results of this study showed that although most of the studied wheat lines are susceptible to this disease, but there are also lines among them, that have good relative resistance which can be used as resistance sources in wheat breeding programs.