فصلنامه گیاه پزشکی
سال چهل و ششم شماره 2 (تابستان 1402)

Fusarium dry rot is one of potatoes' most significant postharvest fungal diseases, reducing crop yield and seed tuber quality. Utilizing certified seed tubers treated with chemical fungicides is the simplest and most cost-effective method for preventing dry rot disease damage to potatoes. Monitoring for diseases transmitted by tubers enhances the quality of seed tubers produced and reduces the risk of disease establishment and soil contamination. This study aimed to identify the causal fungal agents of potato seed tubers dry rot, determine the susceptibility of seed samples from potato seed farms to Fusarium dry rot agents, as well as to evaluate some of the effective factors in disease severity, particularly the levels of extracellular enzyme activity and fusaric acid production on the disease index and dry rot volume of potato tubers.

To identify Fusarium species responsible for dry rot disease in the seed tubers of Agria, Banba, Jelly, Challenger, Ramus, Sagitta, Sante, Sifra, Fabula, and Colomba cultivars of potato grown in Ardabil (Ardabil), Chaharmahal & Bakhtiari (Borujen), Razavi Khorasan (Fariman and Torbat-e Heydarieh), Zanjan (Khodabandeh), Fars (Abadeh and Eqlid), Lorestan (Khorramabad), Markazi (Arak) and Hamedan (Razan, Kabudarahang, and Bahar) provinces were sampled according to the standard guidelines of the National Seedling Health and Propagation Materials Laboratory. The pathogenicity test was carried out on artificially inoculated potato tubers tubers cv. Agria for determining the volume of dry rot caused by Fusarium isolates. Furthermore, spectrophotometry and high-performance liquid chromatography (HPLC) methods were used to evaluate in vitro activities of amylase and cellulase, as well as fusaric acid production.

The results indicated that approximately 18% of the seed tubers samples collected from different fields were infected with Fusarium dry rot disease in the 1 to 3% range. A total of 26 isolates were identified based on morphological and molecular characteristics belonging to F. solani sensu lato (13 isolates, 11%), F. oxysporum (8 isolates, 7%), and F. sambucinum (5 isolates, 5%). Analyzing the activity of extracellular enzymes revealed that all recovered Fusarium isolates were capable of producing amylase and cellulase enzymes but at varying levels. The maximum amount of extracellular enzymes produced by Fusarium spp. isolates ranged from 286 to 675 μg mL-1 for cellulase and from 103.5 to 317 μg mL-1 for amylase. According to the results, cellulase peaked sooner than amylase, but both enzymes contributed to the development of dry rot in potato tubers. The results revealed that approximately 38% of the isolates produced fusaric acid in the 13-32 μg kg-1 range. Moreover, the disease index level and the volume of dry rot caused by different Fusarium species isolates on potato tubers varied.

It appears that the amount of dry rot caused by Fusarium spp. isolates on potato tubers are affected by the production of fusaric acid and the activity levels of cellulose and amylase. This research provides new insights into the health status of potato seed tubers, the predominant Fusarium species causing dry rot disease, and the effect of the level of extracellular enzymes and fusaric acid secreted by fungal isolates on the disease progress in potato tubers, which can be applied to the revision of the national standard for seed tuber health and effective disease management during storage. The dry rot disease and its detrimental impact on tubers significantly threaten the official seed tubers certification system, production, and storage procedures. The current study is the first report on identifying the Fusarium dry rot disease agents from potato seed tubers in Iran and investigating the relationship between the disease severity of isolates and their extracellular enzyme activity, and fusaric acid production.

The wheat aphid, Schizaphis graminum, is deemed one of the most significant grain pests, causing substantial damage to the country's wheat farms. Consequently, various biological, agronomic, and chemical methods are used to control wheat aphids, where chemical spraying is the most common method. The increasing use of chemical insecticides has resulted in several issues, including the emergence of insect resistance to pesticides, adverse effects on the environment, beneficial organisms, and humans, and the development of insecticide-resistant pests. In this regard, insect pathogens like nematodes may be preferable to chemical insecticides. This study aimed to evaluate the lethal and sublethal effects of entomopathogenic nematode Steinernema carpocapsae on the life history parameters and phenoloxidase enzyme activity of S. graminum by L-DOPA (L-dihydroxyphenylalanine) substrate. This enzyme activity is essential to the insect's innate immune response.

S. graminum was cultivated on a wheat host in a growth chamber maintained at 25 ± 2 °C, 60 ± 5% relative humidity (RH), and 16:8 light/day (L:D) hour photoperiods. Daily observations and recordings were used to track the development period and survival rate. In addition, for breeding the wheat aphid S. graminum, populations from the plant protection department's laboratory were utilized and transferred to wheat plants in a growth chamber.
Aphids infested the plants once they had reached their optimal size. Age-stage and two-sex life table analyses were used to analyze the raw data. TWOSEX-MSChart was used to evaluate the raw data based on the age stage and two-sex life table. In addition, blood cell phenol oxidase and hemolymph testing were performed. Samples of the enzyme were preincubated with phosphate buffer at 30 °C for 30 min, then 50 µl of 10 mM L dihydroxyphenylalanine (L-DOPA) substrate was added and incubated at 25 and 30 °C for 5 min. The phenol oxidase activity was measured using a 492 nm enzyme-linked immunosorbent assay (ELISA) reader device.

The bioassays indicated that the lethal concentration (LC50) of nematode treatment against the adults of S. graminum was 136 larvae/insect. The current study revealed that S. carpocapsae had a higher adult mortality rate than S. graminum. Results of the sublethal effects experiment showed that the pest's fecundity in S. carpocapsae treatment (19.07 offspring/female) was lower than the control (39.16 offspring/female). Furthermore, the studied nematode significantly reduced the population growth parameters of S. graminum. The intrinsic rate of increase for treated and control nematodes was 0.300 and 0.421, respectively, per day. Based on the results, the nematode increased the phenol oxidase enzyme's activity at 25 and 30 °C relative to the control.

Considering that chemical insecticides are accompanied by problems such as resistance and residual toxins in agricultural products, it is preferable to use entomopathogenic nematodes, which have a higher immunity to environment and natural enemies. The lethal concentration (LC50) study of the examined nematode showed that the nematode S. carpocapsae exhibited good disease-causing power on the adult female aphids of S. graminum.
The investigation into the effect of experimental nematode sublethal concentration on the growth parameters of the wheat aphid population revealed that in the nematode S. carpocapsae, the characteristics of the aphid population, particularly the intrinsic rate of population increase parameter value, decreased significantly. Sublethal concentrations of the studied nematode can slow or even stop the population growth of wheat aphids and significantly impact aphid fertility, survival, and life expectancy.
In this study, the phenol oxidase enzyme's activity significantly differed between treated and untreated aphids. It appears that the insect uses phenol oxidase enzymes to counteract the nematode's effect, and this enzyme plays a role in the wheat aphid's body when dealing with S. carpocapsae. The overall results revealed that S. carpocapsae exhibited high lethal and sublethal effects on S. graminum. After additional field studies, it can be recommended for use in biological control and an integrated pest management (IPM) program for this pest.

Rice (Oryza sativa L.) is one of the most important cereal grains and serves as a staple food for nearly half of the world’s population. Trogoderma granarium Everts (Coleoptera: Dermestidae), commonly known as the Khapra beetle, is a significant pest that infests stored grains and other stored products in various countries worldwide, including Iran. The pest's polyphagous nature and high ability to survive for extended periods without food necessitate using chemical fumigants to control T. granarium in storage facilities. However, the serious adverse effects of these insecticides on humans and the environment render it imperative to explore alternative pest control approaches. One of the best safe options is using resistant cultivars in integrated pest management programs. Insect-plant interactions and the resistance of various cultivars to herbivorous insects can be studied by insects’ life history and population parameters.

The resistance and susceptibility of six commercial rice cultivars (Tisa, Khazar, Shafagh, Sadri, Nemat, and Hashemi) to T. granarium were determined by analyzing the life history and population growth parameters of the pest. The seeds of tested rice cultivars, which have a large cultivation area in Iran, were obtained from the Agricultural and Natural Resources Research Center (Gilan province, Iran). The initial colony of T. granarium was obtained from the laboratory of Department of Plant Protection, University of Mohaghegh Ardabili (Ardabil, Iran), and was reared in groups on wheat seeds (as the main host). The adult insects obtained from the primary colony were transferred to the seeds of tested rice cultivars (20 pairs on each cultivar), and were reared as a group for two generations. Then, the eggs (1-day-old) laid on each rice cultivar were used for the experiments. The beetle rearing and all experiments were carried out in a growth chamber set at 33 ± 1°C, 65 ± 5% RH, and 24 h darkness. Subsequently, the raw data obtained from the life history of T. granarium on various rice cultivars were analyzed with TWOSEX-MSChart software using an age-stage, two-sex life table model.

The study results indicated that immature survival rate on cultivar Sadri was higher than on the other tested cultivars. The incubation period was longer on cultivar Shafagh than on cultivars Sadri and Tisa. The shortest larval, pupal and the entire immature periods were observed on cultivars Sadri and Tisa, and longest on cultivar Khazar. The beetles raised on cultivar Khazar took approximately eight days longer to complete their development than those on cultivar Sadri. The fecundity of T. granarium (number of eggs laid by each female) on cultivars Sadri and Tisa was significantly higher than on cultivar Khazar. The longest oviposition period was observed on cultivars Sadri and Tisa, and the shortest on cultivar Khazar. The longevity of female and male adults was longest on cultivars Sadri and Tisa, and shortest on cultivar Khazar. The intrinsic rate of increase (r) was highest on cultivar Sadri and lowest on cultivars Hashemi, Shafagh, and Khazar. Additionally, the finite rate of increase (λ) was higher on cultivars Sadri and Tisa than on the other tested cultivars. The longest mean time generation was observed on cultivar Khazar and the shortest on cultivars Sadri and Tisa.

One of the reasons for the rapid development and high survival of T. granarium on cultivar Sadri may be the high protein content of its seeds, which has provided sufficient energy for the immature stages' rapid growth. However, the amount of protein and starch in cultivar Khazar is lower than in other cultivars, which may be a factor in this cultivar's resistance to T. granarium. Our results indicate that Sadri and Tisa are the most susceptible cultivars to potential population increase of the pest because the highest values of r and λ are on these two cultivars. However, owing to the highest values of r and λ on cultivars Hashemi, Shafagh, and Khazar, they are the most resistant cultivars to T. granarium. Consequently, resistant cultivars can be used in the genetic engineering of plants to minimize the damage caused by this key pest.

Saffron (Crocus sativus) petals are used as an organic agent in agricultural industries. In the present study, the insecticidal activity and repellent effect of the saffron plant petal extract was investigated on the confused flour beetles (Tribolium confusum Jacquelin du Val.). Storage pests are one of the important issues of storing products from harvest to consumption contributing to 10-30% of damages to the products. The flour bug T. confusum is a pest found all over the world, causing great damage to various crops such as flour, beans, etc. every year. These pests have a high reproduction rate and it is necessary to protect the stored products from their contamination. In recent years, many studies have been conducted on the insecticidal and repellent effects of plant essential oils, and some of these compounds have been introduced as suitable candidates for pest management, especially in warehouses. Research on the biological properties of plant essential oils can lead to the identification of new biological insecticides compatible with organic agriculture. A large number of plants (17,500 species) and their secondary metabolites have physiological and behavioral effects on many pests, especially storage pests, such as repellent effects, respiratory effects, and inhibition of nutrition and spawning. In some cases, plant insecticides function as an interesting substitute for industrial insecticides in pest management as a safer option for humans and the environment. In terms of agricultural pest management, plant insecticides have been adapted as the best option to preserve organic food products in developed countries.

Maceration method was used for extraction, and methanol was applied as a solvent. After 72 hours, the contents of each container were filtered twice using filter paper and the resulting extract was stored in the refrigerator. The experiments were carried out at 27±1°C, 65±5% relative humidity, and in the dark. The T. confusum was obtained from the Entomology Laboratory of the Department of Plant Medicine, Zakaria Razi Laboratory, Islamic Azad University, Science and Research Unit. For the fumigant toxicity, the adults were exposed to 100, 500, 1000, 10000, 50000 and 100000 ppm of methanol extract of saffron and mortality rate was recorded 4, 5, 6 and 7 days after exposure. While, for the repellency test, the adults were exposed to the concentrations of 4.2, 5.27, 6.34 and 7.41% and the repellency was checked 4, 5, 6 and 7 days after exposure.

Saffron flower petals extract caused fumigant toxicity in T. confusum adults. According to the analysis of statistics, the mortality rate increased with increasing concentration and exposure time. In addition, the mean repellency of saffron petal extract against adults when exposed to the concentrations of 4.20, 5.27, 6.34, and 7.41% was 13.33, 18.33, 23.33, and 26.66 % respectively. The chemical analysis of the extract by GC MS device showed that the main volatile compounds in the saffron petal extract were C8H16 (Cyclopentane Propyl npr), C8H18 (Hexane 3, 3 dimethyl), C8H18 (Octane Octane) and C16H34 (Hexadecane).

Saffron petal extract causes fumigant toxicity in T. confusum adults, and mortality increases with increasing concentration and duration of exposure to the extract. Since repellents can be considered as one of the new methods in controlling stored-products insect pests, the repellency of saffron petal extract was evaluated in T. confusum adults. The repellency of saffron petal extract on T. confusum adults increased with increased time of exposure. The chemical compounds of saffron petals confirm the presence of terpenoids, ethyl acetate, methanol, and aqueous extract of saffron petals. The presence of these chemical compounds proves the antibacterial properties of saffron petal extract.

One of the most important forest trees in the south of Kerman, which plays an important role in the region's ecosystem and beekeeping industry and is infected by wood-eating beetles, is Prosopis cineraria. These trees are an important habitat for various animals and refreshing the hot air of the south. In the sampling conducted during 1401-1400 from the mesquite forests of southern Kerman (Ghaleganj, Faryab, Anbarabad), the wood-eating beetle Xylopertha reflexicauda (Bostrichidae) was collected for the first time from Prosopis cineraria trees and is identified. The species of wood-eating beetle was identified by reliable scientific sources and was finally confirmed by Mr. Dr. Len Yu Liu. This species was first identified and described by Lesne in 1937. Due to the lack of water and recent droughts and the weakness of Iranian mesquite trees, the larvae of this beetle are active inside the trunk and bark of mesquite trees and feed on the wood and bark of tree trunks and cause great damage to Prosopis cineraria trees.

Ants are a major part of the ecosystem as they assist in the decomposition process and improve soil quality. In this study the species richness and biodiversity of these arthropods was assessed in the habitats with varying degrees of human impact in Shiraz, Fars Province, southern Iran. To this end, six habitats including three city parks with the least amount of human destruction (Be’sat, Janat, Babakoohi), two habitats with high amount of manipulation in order to agriculture and urbanization (a field and a destroyed garden) and a natural park without any human manipulation as control, were chosen and sampled bimonthly during 2015 and 2106. A total of 6270 ant workers belonging to 30 species, 12 genera, and three subfamilies were recorded. The most abundant subfamily was Formicinae (16 species) followed by Myrmicinae (12 species), and Dolichoderinae (two species). The biodiversity indices showed a greater diversity in both natural park and urban landscape: Be’sat and Janat parks, with the lowest amount of human effects, respectively as well as Babakoohi and the lowest in the field and destroyed garden. Although a significant difference was observed with the habitats (F</em>= 4.255, P</em>=0.004) and the months (F</em>= 4.327, P</em>=0.002) of sampling, there was not a significant difference between Babakoohi, natural park, Janat and Be’sat parks (P</em>>0.05) nor between field and destroyed garden (P</em>>0.05).

Entomogenous fungi include a wide range of species that are ecologically very diverse. Many of them are pathogenic to insects and mostly show host specificity. Isolation and identification of native entomogenous fungi, especially pathogenic species, and their use as a source of biological control agents is an important safe, environmentally-friendly pest control approach. This survey isolated and characterized entomogenous fungi that naturally occur in soil. Post-harvest infestations cause considerable losses in cereal production. Stored-product insect control is mainly accomplished by chemical means; however, pesticides can affect non-target species, such as plants, animals, and humans. Pesticides increase pest resistance. Another possible negative effect of pesticides is their ability to bioaccumulate and biological magnification. Therefore, alternative agents are needed for managing stored-product insect pests. Biological control of stored-product insects with entomopathogenic fungi is a successful, sustainable alternative to chemical insecticides. Mediterranean flour moth, Ephestia kuehniella, is one of the most important insect pests infesting stored grain of cereals worldwide. This study isolated and determined some entomopathogenic fungi's efficacy in flour moth biocontrol, E. kuehniella. Materials and Methods Different strains of entomopathogenic fungi were isolated from the soils of various districts of Azarbaijan province in the Northwest of Iran. Their virulence against E. kuehniella larvae was evaluated. Isolates of various entomopathogenic fungi were isolated and purified using a soil trap from twenty distinct soil specimens. The immersion technique evaluated their pathogenicity against E. kuehniella in three replicates. This was done at 104, 105, 106, 107, and 108 conidia/mL concentrations. All selected fungal isolates were identified based on morphological features and mycological parameters. Results Four isolates of B. bassiana (BVA, BVB, BVE, and BVF) and two of L. lecanii (LCA and LC) from fourteen isolates were isolated. Six fungal species, namely Lecanicillium lecanii and Beauveria bassiana, were identified among the fourteen isolates collected. The bioassay assessment revealed that E. kuehniella fourth-instar larvae were susceptible to all isolates and that the larval death rate increased with rising conidial concentration. The BVA and BVB isolates of B. bassiana showed the maximum virulence, with nearly 2.5×105 and 2.8×105 (conidia/mL) LC50, while the BVF isolate of B. bassiana had the least virulence among the other isolates evaluated, with nearly 2.2×107 (conidia/mL) LC50 on E. kuehniella. The lowest LT50 value (3.72 days) using a 108 conidia/ml concentration on E. kuehniella larvae was shown on the BVA isolate. Discussion A comparison of the means and grouping of isolates showed a significant difference between isolates and the effectiveness of different concentrations. Also, the BVA isolate of B. bassiana had greater efficiency than the other isolates, and therefore, they will be more suitable for use in plant pest biological control programs. 

In the context of pathogenicity in host plants, many fungal pathogens produce and secrete a range of small molecules, including proteins and secondary metabolites. Some of these molecules are known as pathogenic factors, and they play a specific role in manipulating interactions between the pathogen and host plants. Typically, these agents are referred to as effectors. Effector proteins are pivotal in the pathogenicity process of phytopathogenic fungi and their colonization of host plants. Identifying and analyzing the functions of effector proteins are essential steps in understanding pathogenicity mechanisms, symbiosis, and plant defense strategies.Ascochyta blight, caused by the fungus Ascochyta rabiei, stands as one of the primary limiting factors in chickpea cultivation, presenting a challenge wherever this plant is grown. The availability of genomic and transcriptomic data for A. rabiei, both in vitro and during its interaction with the plant, facilitates the identification of virulence and effector genes. In this study, our primary objective was to investigate the gsh303 gene as a candidate effector in different pathotypes (I, III, and VI) of A. rabiei.

Genomic data associated with A. rabiei were obtained from the NCBI database. We utilized SignalP v4.1, SecretomP v2.0, TMHM v2.0, and EffectorP v3.0 software for the screening of candidate effector proteins, ultimately identifying the gsh303 gene as a potential effector gene. We also conducted an investigation into the presence of homologous and orthologous sequences.Subsequently, specific primers were designed for the gsh303 gene, and we tracked its presence in different pathotypes of A. rabiei. This was achieved by performing polymerase chain reaction (PCR) both in vitro and during the interaction between the plant and pathogen, encompassing both the genome and transcriptome levels.

The findings of our study indicate that the nucleotide sequence of the gsh303 gene comprises a single exon without any introns. To detect the gsh303 gene, we designed PSh303F/R specific primers based on its upstream and downstream regions. A 599-base pair fragment was successfully amplified by PCR, confirming the presence of the gene in the genomes of all three pathotypes.While the expression of the gapdh housekeeping gene was verified at the transcriptome level in the culture medium, we were unable to confirm the detection of the gsh303 gene at the transcript level. To further investigate the expression of this gene during the plant-pathogen interaction, we inoculated two chickpea cultivars, one resistant (MCC133) and one sensitive (ILC1929), with three pathotypes of A. rabiei (I, III, and VI). After 96 hours of inoculation, we collected samples from the inoculated plants for DNA and RNA extraction. Our results from tracking the gsh303 gene during the interaction conditions at both the genome and transcript levels revealed the amplification of 599 bp and 591 bp fragments, respectively.

In our current study, we employed bioinformatics methods to predict the gsh303 effector gene and identified it as a candidate effector. Furthermore, we confirmed the presence of this gene in all three pathotypes of A. rabiei under in vitro conditions. Utilizing fungal genomic DNA as a template, a specific band of 599 base pairs was successfully amplified via PCR. Examining the expression pattern of the gsh303 gene in two cultivars infected with pathotypes I, III, and VI of A. rabiei at the transcriptome level revealed consistent expression 96 hours post-inoculation across all three pathotypes.The identification of effector genes holds promise for the development of resistance genes based on genomic data, paving the way for the production of more resilient cultivars resistant to disease.

The area under cultivation of lettuce in the country is 14,000 hectares, and the amount of lettuce production in Iran is 300,000 tons. Among the important damaging factors in lettuce production is the tomato spotted wilt virus (TSWV). With its wide host range, this virus is transmitted mechanically through infected plant sap by thrips. In Iran, various insecticides such as spinosad, diazinon, imidacloprid, profenofos, etc., which are from different groups, have been registered to control thrips. New reports show that other insecticides like thiamethoxam and imidacloprid are effective on thrips, too.The use of pesticides should be performed very carefully due not only to the residue on the surface of the products, but also its penetration into the tissue of fruits, vegetables, and even grains. Excessive use of pesticides in the production of agricultural products causes pesticide residue, which is considered a risk factor for human health and the environment. High pesticide residues in all kinds of products, especially lettuce, which is consumed fresh, is of particular importance. Therefore, appropriate measures must be taken to reduce the risks of high residue levels of pesticides.

The insecticides imidacloprid and thiamethoxam were used to treat lettuce seeds to control the thrips, which is the vector of this virus. The first treatment consisted of seed treatment of lettuce with imidacloprid and the second one with thiamethoxam. Twenty transplants from each treatment were evaluated regarding thrips infection and TSWV viral disease. To extract the samples for the measurement of pesticide residues QuEChers method was used. The extracted solution was injected into the LC-MS/MS to identify and measure the pesticide residue in the lettuce samples after eight and eleven weeks of cultivated treated seeds.

The results show that thiamethoxam, with 84.5%, and imidacloprid, with 45% efficacy, can prevent infection with TSWV up to one month after transplantation. The results show that no detectable imidacloprid residue was observed in the lettuce at the two sampling periods, and only 0.33 mg/kg and 0.28 mg/kg of thiamethoxam were detectable after 8 and 11 weeks, respectively. According to the national Maximum Residue Levels (MRL) of thiamethoxam (3 mg/kg), the measured residue levels are lower than the MRL.

Therefore, according to the results obtained, the insecticides used for the lettuce seed treatment to control the thrips with the doses mentioned above are safe for the consumers of the final product as the residue of these insecticides after treatment is well below the national MRLs.

The effect of number of mating on longevity and fecundity of the predatory mite Euseius scutalis Athias-henriot was studied under laboratory conditions (at 25±1 ºC, 60 ± 5 % RH and 16: 8 L:D). Newly emerged adult females (less than 24 h) were held individually in a 9 cm arena. A male (less than 24 h) remained with a female for limited periods of time or continuously. Four treatments were designed: virgin females, single mated females, multiple mated females (female had access to a male every five days), multiple mated females (female had continuous access to a male). Mating was necessary for oviposition. Virgin females lived the longest (25 days). However, no significant differences were found among longevity of mated females (12- 15). Similarly, no significant differences were detected among fecundity of females with different numbers of mating (18- 22 eggs). Sex ratio was significantly female-biased in progeny of single mated females and multiple mated females with periodic access to male. However, no significant difference was found in sex ratio of progeny of multiple mated females with continuous access to male.