فصلنامه گیاه پزشکی
سال چهل و هفتم شماره 3 (پاییز 1403)

The brown snail is a major pest affecting citrus orchards in Iran. Using chemical pesticides can contaminate the air, water, and nearby crops. Due to the emergence of pest resistance to chemical toxins and their harmful and destructive effects on human health and the environment, one of the methods to control snails is to evaluate the activity and inhibition of their digestive enzymes. α- amylase and α- glucosidase enzymes are key enzymes in the digestive system, and their inhibition is crucial in biotechnology. Using natural sources as inhibitors for these two enzymes is a new goal due to fewer side effects than chemical inhibitors. The purpose of this study was to use the essential oils of thyme (Thymus vulgaris L.), lavender (Lavandula angustifolia Mill.), and marjoram (Origanum vulgare L.) plants from the mint family to inhibit these two enzymes of the digestive gland of the brown snail.

In this study, α -amylase and α -glucosidase enzymes were extracted from brown citrus snails. The digestive glands were isolated from snails in starvation conditions. The enzyme extract was prepared after homogenization and centrifugation methods at 4 °C. Kinetic parameters and activity of α-amylase and α-glucosidase measured in the enzyme extract were measured. The total amount of proteins in the crude extract was measured by the Bradford method. The essential oil was prepared using a Clevenger apparatus. The activities and kinetic parameters of α-amylase and α-glucosidase enzymes in crude extract were measured in the presence of different concentrations of the essential oils of T. vulgaris, L. angustifolia and O. vulgare. α-amylase enzyme activity was measured according to Bernfeld's method at a fixed concentration of the starch solution, essential oils (0.1-6 mg/ml) and 250 µl of the enzyme extract. The total volume was 2.5 ml in each experiment. α-glucosidase enzyme activity was determined based on the method of Siegentaler and Low with a slight change. In each experiment, different amounts of the essential oils (0.25-5 mg/ml) were incubated with the enzyme extract (15 µl) and para-nitrophenyl alpha-di-glucopyranoside (20 µl). The total volume was 200 µl.

The amount of protein in the crude enzyme extract was 0.2 mg/ml. The α-amylase activity was obtained at 0.278 U/ml, and the specific activity was 1.39 U/mg. The Km and Vmax values for α-amylase were measured as 0.13 mg/ml and 0.062 mg/ml/min, respectively. The α-glucosidase activity was obtained at 0.4 U/ml, and the specific activity was 2 U/mg. The IC50 values for the α-amylase in the presence of essential oils of T. vulgaris, and L. angustifolia. and O. vulgare were obtained at 2.462, 0.2816, and 0.114 mg/ml, and for α-glucosidase was obtained at 0.741, 2.036, and 1.668/ml, respectively. The GC-MS analysis found that the main constituents of the essential oils of T. vulgaris, L. angustifolia and O. vulgare were Carvacrol, D-Camphor, and Terpinene-4-ol, respectively. The essential oil of O. vulgare acted as an α-amylase inhibitor, and the thyme essential oil acted as an α-glucosidase inhibitor. Our experiments showed that the essential oil of L. angustifolia competitively inhibited the α-amylase. On the other hand, O. vulgare essential oil can act as an effective competitive inhibitor for the α-glucosidase. Therefore, compounds of D-Camphor and Terpineol, which are the main components of L. angustifolia and O. vulgare essential oils, are effective competitive inhibitors for α-amylase and α-glucosidase of the snail.

Citrus snail is one of the most significant pests of agricultural products, which damages all kinds of citrus cultivars in gardens and nurseries by feeding on leaves, stems, fruits, and tree bark. Iran's rich pharmaceutical plant flora contains diverse compounds, including volatile ones like essential oils, which can be explored for their medicinal, insecticidal, fungicidal, and other properties. According to the literature, the snails have more than 20 types of carbohydrases. α-amylase and glucosidase are key enzymes in carbohydrate digestion and metabolism in insects that hydrolyze starch and glycogen. Our results showed that essential oils of T. vulgaris, L. angustifolia and O. vulgare or their active components can be proposed as environmentally friendly, low-cost, and accessible sources for the production of effective inhibitors against α-amylase and α-glucosidase of brown citrus snail.

The Agonoscena pistaciae Burckhardt & Lauterer (Hem.: Psyllidae), commonly known as the pistachio psyllid, is a major pest that infests pistachio trees. As the population of this pest increases, this leads to a significant decline in both the quality and the yield of the pistachio harvest. Over the past few years, various types of sulfur have been commonly used to control pests. The common green lacewing, Chrysoperla carnea (Stephens) is a cosmopolitan polyphagous predator, attacking various pests, including eggs and nymphs of the common pistachio psylla. This study aimed to evaluate the effectiveness of three concentrations (1500, 2500, and 4000 ppm) of a commercially available sulfur formulation (WP80%) on C. carnea, which is one of the most important natural enemies in pistachio orchards.

The green lacewing eggs and larvae, collected from pistachio orchards in Yazd, were treated with sulfur using both the dipping method for two days-old eggs and the residual dry-on-glass plate method for larvae. The study was conducted under laboratory conditions at 26 ± 2 ˚C, 65 ± 5% relative humidity (RH), and a photoperiod of 16:8 h (L: D). Several parameters were measured such as mortality rate, developmental time of the immature stages of the green lacewing, adult longevity, and fecundity. The fertility life table data were analyzed using the TWO-SEX MS Chart 2024 software. A one-way analysis of variance (ANOVA) with a post-hoc Tukey test was conducted to statistically assess the mean percentage of formation across different biological stages and the pre-adult mortality rate data, using SPSS version 27.00. Normality was tested using a non-parametric test, Kolmogorov-Smirnov test.

The results revealed that the mortality rates of the green lacewing's immature stages in the treatments (4000, 2500, and 1500 ppm) and the control group were 69.2 ± 0.8, 58.9 ± 1.1, 43.7 ± 2.4, and 14.4% ± 1.1, respectively. A significant difference was observed between the control group and the treatments at the 5% significance level. All concentrations resulted in a significant decrease in oviposition period, total fecundity, longevity, and total life span compared with control. Maximum and minimum mean lifespans of C. carnea individuals were observed in the untreated group and a concentration of 4000 ppm of sulfur. Additionally, the net reproduction rate (R₀) was 6.1 ± 1.4, 12.7 ± 2.7, 29.2 ± 5.7, and 61.2 ± 7.3 offspring per individual, while the gross reproduction rate (GRR) was 23.6 ± 3.9, 36.4 ± 5.9, 59.03 ± 9.8, and 81.1 ± 8.4 offspring per individual, in the treatments (4000 ppm, 2500 ppm, and 1500 ppm) and the control group, respectively. The mean generation time (T) was 43.9 ± 0.6, 44.2 ± 0.4, 47.23 ± 1.1, and 46.6 ± 0.8 days, and the finite rate of population increase (λ) was 1.05 ± 0.01, 1.07 ± 0.01, 1.097 ± 0.006, and 1.12 ± 0.04 day-1 across the treatments and the control group, respectively. The highest values were observed in the control group, whereas the lowest ones were recorded in the 4000-ppm sulfur treatment. A significant difference was noted between the control group and the treatments at the 5% significance level.

The adaptability of pesticides with biological agents is one of the major concerns of IPM practitioners. Hence, it is essential to have necessary information about the action mode of pesticides on non-target insects. Furthermore, finding efficient biological control agents is considered the first step in developing biological control programs. Therefore, the effects of a commercially available sulfur formulation (WP80%) on C. carnea were discussed to design integrated management programs.  The results of this laboratory study demonstrated that sulfur application negatively affects the efficiency of C. carnea, significantly reducing its population. If these findings are confirmed under field conditions, adjustments in the method and timing of sulfur application in integrated pest management (IPM) programs will be necessary to avoid disrupting the sensitive stages of this predator.

The control of fungal and bacterial plant diseases mainly relies on the application of mineral and chemical fungicides/bactericides, which are considered to be environmental pollutants and toxic to humans. Moreover, the prolonged use of bactericides and antibiotics has led to the development of resistance among pathogens, as well as an increase in environmental and health threats. Therefore, the development of non-toxic and non-polluting treatments to control plant diseases has been the focus of extensive research in agriculture. Synthetic antimicrobial peptides have recently received extensive attention as the potential alternatives to other conventional methods in terms of their strong broad-spectrum antimicrobial activity. In vitro assays using various chemically synthesized peptides showed that the broad-spectrum peptide thanatin derived from the spined soldier bug (Podisus maculiventris (Say)) had the greatest potential for eliminating aflatoxigenic fungi. However, the antibacterial effect of thanatin against bacterial plant pathogens was less studied so far. The thanatin encoding sequence was codon optimized for expression in Pichia pastoris. This coding sequence cloned into P. pastoris expression vector pPICZαA and used for synthetizing the recombinant thanatin. Then, antibacterial activities of the constructed peptide were studied under in vitro and in vivo condition. The result showed that, all construction, cloning and expression processes were successfully performed in yeast. The results of the MIC and MBC tests showed that the growth rate of the majority of bacterial plant pathogens including gram-negative and gram-positive bacteria was inhibited by recombinant thanatin under in vitro conditions.MIC values for different bacterial isolates ranged from 0.016 to 2.048, while the MBC values for the same bacterial isolates were determined to be between 0.064 and 4.096 μg/mL. These amounts were significantly lower than the MIC and MBC values obtained by applying copper oxychloride. The application of 4.09 μg/mL of recombinant thanatin against rice leaf blight splendidly controlled this bacterial disease under greenhouse conditions. However, this promising antimicrobial peptide requires further investigation for the development of novel molecules for the control of plant pathogens.

As a necrotrophic fungus, Ascochyta rabiei possesses various compounds that bypass host plant defense mechanisms. Understanding the genes responsible for its pathogenicity on chickpea plants is crucial for developing resistant cultivars. This study investigates the induction of programmed cell death by A. rabiei and its potential involvement during interaction with the chickpea plant. By tracking homologues of effector proteins from other necrotrophic pathogens, a candidate effector protein named GSh200 was identified in A. rabiei genomic data. The protein sequence of GSh200 was annotated using web-based tools and submitted to Gene Bank with the following accession number OR567501. Additionally, a homologue of the GAPDH gene (ArGAPDH) was identified using a homology-based search in the genome of A. rabiei and used as a reference gene. Specific primers were designed to confirm the presence of A. rabiei isolates in chickpea plants after fungal infection. Sequence analysis and alignment of the GSh200 gene in different A. rabiei pathotypes revealed identical sequences, indicating a potentially conserved role in pathogenicity. Further investigation is needed to elucidate the functional role of the GSh200 protein in inducing programmed cell death in chickpea plants.

Yellow or stripe rust caused by the fungal pathogen Puccinia striiformis f. sp. tritici is one of wheat's most prevalent diseases. It poses a significant threat to wheat production worldwide, including in Iran. Diseases are considered one of the most significant biological challenges that can reduce wheat performance by up to 20% globally. Given the total wheat production, this incurs an economic impact of several hundred million dollars. Annual losses caused by rust diseases in wheat are estimated to reach up to 50 million tons worldwide. Integrated management using agronomic and chemical control methods and genetic resistance (developing resistant cultivars) is considered the most effective strategy for controlling this disease. Identifying sources of resistance at various genetic levels within wheat germplasm is crucial in national wheat breeding programs for establishing effective and heritable genetic resistance against this disease.

Two distinct races with high and low virulence were obtained from the yellow rust pathogen collection of the Pathology Unit at the Seed and Plant Improvement Institute for phenotypic screening of resistance in wheat cultivars and promising lines at the seedling stage (under greenhouse conditions). The elimination of urediniospores from other rust species was achieved through several rounds of purification on the susceptible Bolani cultivar using the rub-in method. To identify effective and stable resistance sources against yellow rust pathogen races, the resistance reaction of 41 commercial cultivars and 45 promising wheat lines (from four climatic regions of the country), along with the sensitive cultivar Bolani (as a control), was evaluated at the seedling stage against two yellow rust races from the pathogen collection of the Pathology Unit at the Seed and Plant Improvement Institute in Karaj, with high and low aggressiveness (174E191A+, Yr27 and 6E134A+, Yr27), and at the adult plant stage in the research station of the Ardabil (Moghan) Agricultural and Natural Resources Research and Education Center.

In the 2023 and 2024 growing seasons, the climatic conditions in the Moghan Plain were highly conducive to the emergence and spread of yellow rust disease. The weather was characterized by cool temperatures ranging from 8 to 17 degrees Celsius and high relative humidity from late March to mid-May. During the 2024 growing season, the prevalence of yellow rust disease was notably more severe in various regions of the country, particularly in the northern areas. The resistance genes Yr1, Yr5, Yr10, Yr15, Yr24, YrSU, and YrSP were identified as effective resistance genes against the yellow rust races. The results indicated that approximately 56% of the commercial wheat cultivars exhibited acceptable resistance reactions, while about 44% lacked effective resistance (showing intermediate to susceptible reactions). Furthermore, around 87% of the promising wheat lines displayed acceptable resistance reactions, whereas about 13% lacked effective resistance. The yearly variation in results could be attributed to differences in the abundance and virulence of dominant pathogen populations in the region, and environmental conditions affecting the two years.

The findings of this study indicate that heritable genetic resistances are present in most of the commercial cultivars currently cultivated extensively and in the lines nominated for introduction as new commercial wheat cultivars. In the present study, the number of wheat cultivars and promising lines exhibiting resistance to yellow rust races was greater than those showing susceptibility and intermediate susceptibility. This indicates that effective resistance genes have been incorporated into the new wheat lines within breeding programs. New wheat lines must possess high yield potential and desirable agronomic traits and demonstrate acceptable levels of resistance to the most significant wheat diseases, particularly rusts, to be introduced as commercial cultivars. If they do not possess such resistance, they should be discarded, as environmental conditions during the growing season can favor rust pathogen activity, especially yellow rust, leading to significant yield losses. The identified resistant cultivars and lines from this research can be utilized in breeding programs to develop wheat cultivars resistant to yellow rust.

Fusarium is a significant soil fungus found globally. The fungus responsible for the disease can survive as chlamydospores in infected seeds and plant debris for more than five years in the soil. When conditions are right, it can devastate entire crops. This fungus releases spores that grow when they come into contact with chemicals from the chickpea roots. The fungal threads invade the plant's roots and vascular system, which blocks the plant's ability to absorb water. As a result, the chickpea plant can die from lack of moisture. In Iran, the disease is common in many areas where chickpeas are grown, resulting in a 17% decrease in chickpea yields. In Lorestan Province, it causes root rot and wilting in chickpeas, severely impacting their production. Under suitable conditions, this disease can entirely devastate crops. To effectively manage this issue, it is crucial to identify the various Fusarium species, understand their detrimental effects, and evaluate which plants they can infect. Also, studying these species' genetic relationships can facilitate the development of improved disease control strategies.

This study focused on identifying Fusarium species that cause root rot in chickpeas in Lorestan Province. Researchers visited chickpea farms, collecting 155 samples from plants exhibiting yellowing, wilting, and root rot symptoms. The samples underwent isolation, purification, and identification in the laboratory based on their morphological traits. Pathogenicity tests were conducted on a susceptible chickpea cultivar named Arman to evaluate the pathogenic potential of the identified Fusarium species. This study focused on analyzing specific genetic markers to identify particular isolates. Researchers amplified two gene regions: the internal transcribed spacer (ITS) of ribosomal DNA and the ef1-α gene, utilizing materials from SinaGen Company. The amplified DNA fragments were sequenced by Biomegic Company in Tehran. The sequences were edited and analyzed using BioEdit 7.1 software and registered in the NCBI GenBank database. MEGA 7.0 software was employed for phylogenetic analysis to construct a phylogenetic tree, comparing the isolates with others in the gene bank. A bootstrap analysis with 1,000 repetitions was conducted to confirm the stability of the phylogenetic tree branches. The study evaluated specific isolates' harmful effects on the above-ground parts and roots of intentionally infected plants.

The phylogenetic tree presented in this study, constructed from specific gene sequences, aligns with the results of morphological analyses. The research identified Fusarium oxysporum and Fusarium solani as the most prevalent isolates. F. falciforme affects plants by causing yellowing and wilting, starting from the lower leaflets and spreading upwards. The roots also show signs of damage, with visible root rot symptoms after emerging from the soil. Additionally, there are instances of discoloration in the vascular bundles when looking at sections of the stem, both longitudinally and cross-sectionally. These findings correspond with previous studies in the field. Furthermore, the study uncovered that Fusarium falciforme is a weak pathogen for chickpea roots. Importantly, this research represents the first documented evidence of the pathogenic effects of Fusarium falciforme on chickpeas.

The phylogenetic tree presented in this study, constructed from specific gene sequences, aligns with the findings of morphological analyses. The research identified Fusarium oxysporum and Fusarium solani as the most prevalent isolates. The disease symptoms linked to most isolates from the F. oxysporum complex in this study resemble those caused by F. redolens. DNA sequencing revealed that the F. oxysporum isolates are closely related to F. redolens, with a 96% genetic similarity. All isolates of F. redolens showed symptoms similar to those of F. oxysporum f. sp. ciceris in pathogenicity tests but without any color change in the vascular tissue. Since black root rot is a localized issue, it is crucial to differentiate it from systemic diseases like wilting for effective plant pathology understanding. A combination of molecular analysis and thorough morphological examination is essential to identify these isolates correctly. These results are consistent with previous studies on the subject. Furthermore, the investigation revealed that Fusarium falciforme exhibits weak pathogenicity towards chickpea roots. Importantly, this research represents the first documentation of the pathogenic effects of Fusarium falciforme on chickpeas.