فصلنامه گیاه پزشکی
سال چهل و چهارم شماره 3 (پاییز 1400)

 The stem rust of wheat (Puccinia graminis f.sp. tritici) is one of the most important diseases of wheat in Iran causing severe yield loss in some years in the case of favourable condition for disease epidemic. In the recent years, a new race of stem rust (Ug99) and its new variants have been emerged, which are virulent on most resistance genes, such as Sr31, Sr38, etc., and have potential to spread rapidly and cause severe yield loss. The Ug99 race was first reported in 2009 from Lorestan and Hamadan provinces of Iran.Materials and

Results of a recent study conducted during 2011-2013 in Iran showed virulence on Sr36, Sr38, SrMcN and SrTmp genes and since, these genes have been used widely in genotypes resistant to Ug 99, there is a possibility of breakdown of these genes in Iran. Therefore, genetic variability of pathogen should be continuously investigated and resistance sources should be developed based on this information. For achieving this aim, 56 stem rust differential cultivars/lines obtained from the Cereal Research Center, Manitoba State, in Canada were planted in Ardabil Province, northwestern Iran under natural field conditions. The seeds of each cultivar/line were planted in two rows with 1 m of length and were irrigated every 10 days to promote disease development. Disease severity and type of infection of each cultivar/line were recorded based on the standard protocol at late flowering stage.

In the first year, the disease was developed only in Ardabil region. The SrMcN gene was the most susceptible gene, however, there was no virulence on Sr5, Sr21, Sr9e, Sr7b, Sr11, Sr6, Sr36, Sr9b, Sr30, Sr9a, Sr9d, Sr24, Sr31, Sr38, Sr8b, Sr9h, Sr12, Sr16, Sr19, Sr20, Sr22, Sr26, Sr27, Sr28, Sr32, Sr37, Sr39, Sr40, Sr41, Sr42, Sr43, Sr44, Sr45, Sr47, Sr50 ,and Sr53 genes. Other genes were moderately susceptible to virulence. In the second year, the disease was also developed.  SrMcN, Sr9h, and Sr42 genes were the most susceptible genes. However, no virulence was observed on Sr24 and Sr47 genes. The genes of Sr31 and Sr36 were resistant and susceptible, respectively. It seems that Sr36 and Sr38 genes have no potential to be used in breeding for achieving resistance against stem rust disease in Iran. However, Sr24 and Sr47 genes, which were resistant in the study area, are recommended to be incorporated in breeding wheat for obtaining resistance against stem rust disease. In addition, Sr31, Sr26, Sr27, and Sr28 genes, which were relatively resistant in most areas, were found to have potential to be combined with Sr24 and Sr47 genes in wheat breeding programs.

Sesame is one of the most valuable crops in human nutrition and is cultivated in different regions of Iran, including Khorasan Razavi province. In this plant, various pathogens cause vascular wilt and reduce crop yield. This study aimed to detect the fungal pathogens causing vascular wilt in sesame and control its diseases using the antagonistic fungus Trichoderma harzianum under in vitro and in vivo conditions.

Twenty-one suspected sesame samples were collected and transferred to the laboratory to be studied using valid morphological identification keys. Media such as PDA, WA, and CLA were used for the isolation, purification, and identification of the fungal samples isolated from the infected sesame plants, respectively. Pathogenicity test was conducted by the root inoculation of sesame plants grown under greenhouse conditions. The biocontrol activity of Trichoderma harzianum (Isolate TR5) isolated from the rhizosphere of sesame roots was evaluated against the pathogenic isolates of Fusarium oxysporum f. sp.  sesami under in vitro (dual culture) and greenhouse conditions by the root inoculation of the plants.

According to the result, 17 isolates were related to Fusarium oxysporum f. sp.  sesami. The pathogenicity test was performed on the sesame plants at the 5-8-leaf stage. All F. oxysporum f. sp.  sesami isolates were pathogenic as they revealed a significant difference at p=0.05. The highest level of pathogenicity was noticed for the isolates BA15 and BA16, both with 87.5% wilting, and the lowest pathogenicity was observed in the isolates AN7 and KA33, both with 12.5% wilting on the sesame plants. Regarding the antagonistic properties, the highest inhibition rate in dual culture with T. harzianum was observed for the BA1 isolate with 88.45% mycelial inhibition, while the lowest inhibition rate during interaction with the antagonist was exhibited in the AN2 isolate with 71.33% mycelial inhibition.Moreover, the disease severity reduction rates were different among all isolates of the pathogen tested under greenhouse conditions, and a significant difference was observed at p= 0.05. The highest antagonistic activity of T. harzianum was observed in the BA1, AN7, and K33 isolates with 100% disease severity reduction; however, the lowest activity was related to the AN2 isolate with 54. 54% reduction.

The present study indicates that the TR5 isolate belonging to T. harzianum has a high biocontrol potential for controlling vascular wilt on the sesame plants. Consistent with this finding, previous studies have also reported the potentials of Trichoderma spp. for controlling Fusarium wilt in tomatoes, cucumbers, and beans.

The strawberry spider mite, Tetranychus turkestani Ugarov and Nikolski, is an important pest of various field and glasshouse crops in Iran. This study aimed to determine the functional response of adult females of Stethorus gilvifrons Mulsant to different densities of T. turkestani on three fabaceous host plant species: cowpea (Vigna unguiculata L.), white bean (Phaseolus lunatus L.), and red bean (Phaseolus calcaratus Roxb.) at 30±2 °C, 60±10 % RH, and a photoperiod of 16L: 8D. Beetles were starved for 24 h before testing and then isolated singly for 24 h in 9-cm Petri dishes with 4, 8, 12, 24, 48, or 96 adult females of T. turkestani. Logistic regression analysis revealed a type III response on cowpeas. Constant (b) and handling time were 0.00718 and 0.3299 h, respectively. In contrast, a type II response was observed in white beans and red beans. The maximum attack (0.1219 h-1) and the minimum handling time (0.2301 h) were recorded on white beans and red beans, respectively. The maximum attack rates (T/Th) were 72.74, 60.42, and 104.30 eggs/day on cowpeas, white beans, and red beans, respectively. This laboratory study suggests that the plant features affect the capability of S. gilvifrons to respond to changes in a high T. turkestani density.

Chickpea (Cicer arietinum L.) is the third most important legume crop in the world. Ascochyta blight caused by Ascochyta rabiei (Pass.) Lab. is one of the most important threats for producing chickpea in most of the growing areas. The pathogen invades all aerial parts of the plant and causes severe yield and quality losses. The present study aims to evaluate the effect of some defense inducing volatile compounds in inhibiting A. rabiei, as well as the effect of these compounds on some chickpea growth traits at the presence of the pathogen.

First, six volatile compounds including methyl salicylate, 2,3-butanediol, methyl-jasmonate, acetoin, indole and 3-pentanol were prepared in sterile distilled water (100 μM) containing 0.2% Tween 20, and were used for laboratory and greenhouse studies. The in vitro antifungal activity of volatile compounds was tested on chickpea seed meal dextrose agar (CDA) medium. Then, a five-millimeter agar disk containing the mycelium of the pathogen was placed on the surface of CDA medium in 9 cm diameter Petri dishes. The Petri dishes were inverted and 100 μl of the emulsion of each volatile compound was placed inside the lid. The Petri dishes were sealed and kept at the same inverted position to avoid the dropping of volatile compounds over the culture medium. A greenhouse experiment with nine treatments (including six volatile compounds, chlorothalonil fungicide, healthy and diseased controls) was conducted in a completely randomized design. In addition, the emulsions of volatile compounds were sprayed on the leaf surfaces 12 days after sowing chickpea seeds (variety Bivanij). After 48 hours, the conidial suspension of the pathogen in water (2 x 104 conidia/mL) was sprayed to the surface of chickpea seedlings. Disease severity and plant growth indices including shoot fresh weight, shoot dry weight, root fresh weight and root dry weight were measured two weeks after inoculation. Statistical analyses were performed by SAS software (version 9.3). The means were compared by Duncan's test at a statistical probability level of 5%.

All volatile compounds inhibited the mycelial growth of A. rabiei on CDA with the highest (64.91%) and lowest (4.67%) inhibition obtained by 3-pentanol and methyl jasmonate, respectively. In the greenhouse test, all volatile compounds, except methyl jasmonate, reduced the incidence of blight symptoms compared to the diseased control. The highest disease reduction was obtained by chlorothalonil (86.04%) and methyl salicylate (55.81%). At the presence of the pathogen, all volatile compounds increased root fresh weight, root dry weight and shoot fresh weight, and increased shoot dry weight with the exception of 3-pentanol and methyl jasmonate. Compared to the diseased control, the effect of indole on root fresh and dry weight and shoot dry weight, as well as the effect of methyl salicylate on shoot fresh weight was more than other compounds. Methyl jasmonate had the least improving effect on growth traits compared to other volatile compounds.

The volatile compounds used in this study inhibited A. rabiei in both in vitro and greenhouse tests. They also improved chickpea growth parameters at the presence of the pathogen. The use of volatile compounds could be considered as a new and promising strategy for the integrated management of Ascochyta blight.

The bacterial mosaic induced by Clavibacter tessellarius is one of the wheat seed-borne diseases that causes mosaic and chlorotic leaf spots on infected plants. The disease has previously been reported in a few regions within Iran. There is still no information on the disease's prevalence and distribution in the major wheat-growing areas of southern Iran. The disease may have gone unnoticed in part due to its mild symptoms and resemblance to those caused by viruses and certain non-infectious wheat diseases. This study aimed to determine the occurrence and distribution of wheat bacterial mosaic in wheat fields in southern Iran.

Between 2019 and 2020, surveys in wheat fields in four southern Iranian provinces, including Fars, Khuzestan, Kerman, and Bushehr were conducted, and 192 plant samples with symptoms resembling the bacterial mosaic were collected. Bacteria were isolated and purified using semi-selective media, and Gram-positive isolates were selected using Gram staining and KOH solubility tests. The isolates were identified using a polymerase chain reaction (PCR) with Clavibacter-specific primer pair CMR16F1/CMR16R1, as well as their phenotypic and pathogenicity characteristics. Additionally, a fragment of the gyrB gene from the representative isolates was amplified by PCR using primer pair 2F/4R. The PCR products were purified and directly sequenced. The obtained sequences were aligned, trimmed, and compared to those of related strains in the GenBank using the BLAST search method. A phylogenetic tree based on the gyrB sequences was constructed using the MEGAX software employing the neighbor-joining method.

In total, 400 bacterial isolates were obtained. Based on positive PCR results with specific primers for the genus Clavibacter along with the pathogenicity test on wheat, and the phenotypic and biochemical characteristics, 61 isolates were identified as C. tessellarius. In ten representative isolates, a ~530 bp fragment of the gyrB gene was amplified by PCR. The gyrB gene partial sequences were edited and deposited in GenBank under the accession numbers MZ014474–MZ01483. A BLAST search confirmed that all ten isolates shared the highest nucleotide identity (98.8% to 99.5%) with their corresponding sequences in C. tessellarius strains in GenBank. In the phylogenetic tree, these isolates with C. tessellarius strains from GenBank were clustered in a clade distinct from Clavibacter species and subspecies of Clavibacter michiganensis.

C. tessellarius was isolated from 53 out of 192 wheat fields (27.60%), exhibiting bacterial mosaic symptoms. The Fars, Khuzestan, Kerman, and Bushehr provinces had disease incidences of 35.7% (30 infected farms out of 84 sampled farms), 30.55%, 22.7%, and 7.1%, respectively, indicating the relatively widespread occurrence of the disease in these provinces. To our knowledge, the wheat bacterial mosaic is reported for the first time in the provinces of Khuzestan, Kerman, and Bushehr, and its occurrence in other parts of the country would not be unexpected.

This study has evaluated the age-specific functional response of the females of Nephus arcuatus Kapur, as the major predator of Nipaecoccus viridis (Newstead). The varying density of N. viridis eggs (2, 4, 8, 15, 40, 65, 90, and 115) were offered to 3 to 33 days of the lifespan of the female adults under laboratory conditions (30 ± 1 °C, 65 ± 5% RH and a photoperiod of 14L: 10D h). The age of female adults affected functional responses, its parameters (handling time (Th) and attack rates (a)), and predation rate by N. arcuatus. Females exhibited functional response type III during 16th, 24th and 28th days of their life. Conversely, the functional response during the other 30 days of female life changed from functional response type III to type II. The handling time decreased significantly from the 4th to 17th day as female age increases and then increased, whereas, the attack rates did not significantly change with higher age of the predator. The maximum attack rate values were found on the 12th, 14th, 15th, 17th, 18th, 20th and 25th days of female adults. The maximum mean predation rate was 156.6 eggs on the 17th and 18th days. Accordingly, the best time to use females of this predator in an inundative biocontrol agent is during the first 25 days of females' age, when the predator is more capable to attack the prey, especially when N. viridis is in the egg stage.

The Karkheh protected area is considered as one of the protected areas of Iran, which harbors a remarkable diversity of vegetation. A population belonging to the genus Pratylenchus was recovered and identified as P. mediterraneus during a survey on the biodiversity of the plant-parasitic nematodes in this region of Khuzestan province. This study aims to characterize the population based on the morphological and morphometric characteristics and evaluate its phylogenetic affinities using LSU D2-D3 and ITS rDNA sequences. Moreover, its phylogenetic relationships were investigated with other relevant genera and species.

Several soil samples were collected from the rhizosphere of tamarisk trees in Karkheh protected area. The centrifugal-floatation and tray methods were used for extracting the nematodes from the soil samples. The collected specimens were fixed and transferred to the pure glycerin after extracting the nematodes by using the modified De Grisse method. Then, permanent microscopic slides were prepared from the processed nematodes. The species was identified by using a light microscope equipped with a drawing tube based on the morphological and morphometric characteristics, and valid keys. The molecular phylogenetic analyses were performed by using partial sequences of the D2-D3 expansion segments of  the large subunit, and internal transcribed spacer (LSU D2-D3 and ITS rDNA) regions based on the Bayesian inference under the GTR + G + I model.

A population of the genus Pratylenchus was recovered in the present study. The morphological, morphometric and molecular studies based on the D2-D3 domains of the 28S and ITS rRNA gene indicated that the recovered population belongs to P. mediterraneus. The morphological, morphometric, and molecular data of the Iranian population of the species were presented for the first time in this study. In the phylogenetic tree inferred using the 28S rRNA gene sequences, the newly generated sequence of the Iranian population of P. mediterraneus formed a clade with other sequences of the species, and some sequences assigned to P. thornei. All other sequences of P. thornei occupied a clade, in close phylogenetic relationship with the aforementioned clade. The newly generated sequence of the Iranian population formed a maximally supported clade with other sequences of the species in the phylogenetic tree inferred using the ITS rRNA gene.  

The two species P. thornei and P. mediterraneus are morphologically very similar and can mainly be separated from each other by presence/absence of male and a functional spermatheca. Their separation is further corroborated by using the partial sequences of 28S and ITS rDNA. The sequences of the two species form the separate clades, and the identification of some sequences occupying the same clade with the sequences of P. mediterraneus need further validations in lack of the morphological data.

The greenhouse whiteflies, Trialeurodes vaporariorum (Westwood), are one of the major pests of vegetable and ornamental crops in greenhouses in the world. They damage crops through direct feeding, inserting their stylet into leaf veins, and extracting nourishment from the phloem sap. As a by-product of feeding, honeydew is excreted, which is an indirect but yet another substantial source of damage. The third most harmful characteristic is the ability of adults to transmit various plant viruses. Induced resistance is a non-chemical control method that has no negative impact on the environment and human health and can be a desirable approach in plant protection and pest control. Trichoderma spp. has spread in many ecosystems and its various species such as Trichoderma harzianum Rifai have been successful as non-pathogenic symbionts in controlling plant pathogens via different mechanisms. Some of these mechanisms include enzymatic degradation, secretion of antibiotics, and increasing root absorption capacity. Recent findings suggest that T. harzianum is also a potent inducer of Induced Systemic Resistance (ISR) and can stimulate the immune system in plants against plant diseases and pests. In this study, the efficacy of T. harzianum Tr6 was investigated on host preference and egg production of T. vaporariorum.

To evaluate the effect of T. harzianum Tr6, four tests were carried out: 1) Total phenol assay using Folin–Ciocalteu reagent, 2) The effect of time on induced resistance, 3) Free choice, and 4) Non choice. These tests were conducted on tomato plants (Falat cultivar) at 25±2°C, relative humidity of 60-70%, and a photoperiod of 8 h/16 h (day/night). Also, these experiments were performed on one-day-old adults in plots with four or six leaves in shelves equipped with organza mesh.

According to the phenol assay, the highest amount of total plant phenol was observed three and four days after inoculation by Tr6 strain respectively 0.3503±0.001 and 0.3323±0.001mg/g. Therefore, for the next three tests, plants that were inoculated after three days were used as treated samples, and plants watered with distilled water were used as control ones. In these experiments, the lowest number of adults on the back of the leaves after 24 hours was observed two and four days upon inoculation respectively 2.2±  0.86 and 2.0±  0.63. On the other hand, plants inoculated by Tr6 displayed a significant decrease in adults on the back of the leaves (3.4± 0.66), egg-laying (7.4± 1.51), and honeydew droplets (1± 0.28) compared to control plants.

The most important factor in controlling the whitefly population is reducing the reproduction rate. Induced resistance can repel insects from plants and limit their nutrition, and thus, reduce their reproduction. This study implied that the inoculation of tomato by T. harzianum Tr6 strain could induce resistance to whitefly and reduce host preference and egg production, as well as honeydew secretion. Therefore, this strain can be a promising option in preventing the outbreak of this multi-generational pest.

Alternaria leaf spot disease, with the common agent of Alternaria alternata, is one of the most important seed-borne diseases of coriander. The present study aimed to identify seed-borne fungi isolated from native coriander seed populations based on morphological and molecular characteristics. In addition, the efficiency and effectiveness of seed treatment with chemical fungicides on vigor and germination indices, as well as the control of seed-borne diseases were evaluated.

In order to identify the seed-borne fungi of coriander, seed populations from the fields in South Khorasan, Alborz, Markazi, Qazvin, and Hamedan provinces of Iran were sampled according to the International Rules for Seed Testing. After isolation and purification, fungal isolates were identified and confirmed based on morphological characteristics and species-specific primers. Furthermore, the level of pathogenicity and the aggressiveness of isolates were assessed by pathogenicity test on seedlings. The effectiveness of seed treatment with Iprodione-Carbendazim (Rovral-TS®) and Mancozeb (Dithane M-45®) fungicides on germination and vigor indices, as well as the rate of disease progression were investigated.

A total of eight isolates of Alternaria alternata were identified based on morphological and molecular characteristics. The germination and vigor indices varied among seed populations and it seemed that a part of the observed differences was due to infection by seed-borne fungi. The results of the pathogenicity test showed that approximately 62.5% of the isolates were pathogenic and 37.5% were non-pathogenic. Diverse levels of pathogenicity and aggressiveness were observed for various isolates of A. alternata. Seed treatment with chemical fungicides had a significant influence on germination and vigor indices. Fungicides did not completely control the seed-borne Alternaria leaf spot disease of coriander.

The findings of the current research demonstrated that the effective inhibitory concentrations of chemical fungicides varied based on the pathogenicity of distinct isolates of A. alternata. Disinfection of seeds with Iprodione-Carbendazim fungicide caused a lower disease index of A. alternata on coriander seedlings, compared to Mancozeb. In conclusion, the cultivation of Nahavand seed population out of all the evaluated coriander populations and the disinfection of seeds with Iprodione-Carbendazim fungicide before sowing in all areas suitable for the cultivation of this product are recommended.

Cotton bollworm (Helicoverpa armigera) is a major economic pest worldwide and damaging many crops. Microbial insecticides, botanical insecticides, and insect growth regulators (IGRs) are among the compounds used as biorational insecticides in integrated pest management programs. Due to the high dependence of this pest control on insecticides application, investigating effective and safe insecticides is an important research topic.

To evaluate the effectiveness of different insecticides to control this pest, an experiment was conducted in a randomized complete block design with nine treatments in a cotton field in Golestan province. Treatments included indoxacarb, teflubenzuron+alpha cypermethrin, thiodicarb, cypermethrin, chlorfluazuron, lufenuron+emamctin benzoate, matrin and Bacillus thuringiensis. The control plot was treated with water. The plots were treated 7-10 days after the peak flight of the moths. Sampling was performed one day before and 1, 2, 4, 7, 14, 21 and 30 days after treatment. At each sampling time, 10 plants were randomly selected for each replicate, and the number of live larvae was counted.

One day after treatment, the highest efficacy was recorded for thiodicarb (63.4%) and then for teflubenzuron+alpha cypermethrin, lufenuron+emamctin benzoate and indoxacarb with 58.1, 46.7 and 44.3%, respectively. After four days, when the mean larval population in all 10plants in the control treatment was still at the pre-treatment level, the effectiveness of these insecticides was 77.6, 81.2, 89.9, 79.8, 51.6, 83.3, 47.9, and 46.4%, respectively. Two weeks after the treatments, no larvae were observed in the plots sprayed with thiodicarb, cypermethrin, and lufenuron+emamctin benzoate, and their effectiveness in reducing the pest population was 100%. At the endpoint of the experiments, the efficacy of Bt, Matrin, chlorfluazuron, and Indoxacarb were 48.5, 55.5, 58.2, and 89.8%, respectively. The other four treatments (cypermethrin, lufenuron+emamctin, teflubenzuron+alpha cypermethrin, and thiodicarb) were completely (100%) affected the larvae population.

According to the standard classification of insecticides toxicity provided by the World Health Organization (WHO), the insecticides alpha cypermethrin, emamctin benzoate, indoxacarb, thiodicarb and cypermethrin are all classified as the group with moderate toxicity, lufenuron with low toxicity and teflubenzuron and chlorfluazuron were classified in the group without toxicity. The results demonstrated that two relatively safe insecticides, teflubenzuron+alpha-cypermethrin, and lufenuron+emamctin benzoate, are suitable alternatives to common insecticides, indoxacarb, and thiodicarb. Matrin, plant-derived insecticide, Bt, microbial insecticide, and chlorfluazuron, IGR insecticide, were relatively effective. Due to the adverse effects of using common chemical insecticides, two insecticides, teflubenzuron+alpha-cypermethrin, and lufenuron+emamctin benzoate, are recommended as alternatives concerning the relative safety and simultaneous use of chlorfluazuron with matrin/Bt as entirely safe options.