فصلنامه مطالعات حفاظت گیاهان
سال سی و چهارم شماره 3 (پاییز 1399)

Ricebakanae disease caused by Fusarium fujikuroiis one of the most important diseases of rice in Iran and in the world. Studies show that the disease has spread to a wide range of paddy fields worldwide, with losses in Japan up to 20%, in India up to 15%, and Thailand's northern and central areas have been reported to be 7.3% -14.7%. Symptoms include rice foot blackness and yellowish and then wilting of infected plants. The pathogen is more likely to attack the foot rot which is a sign of the abnormal growth of contaminated plants in the farm. Infected seedlings are slender and taller than healthy plants, and highly infected plants may die before or after transplantation. The tillering is reduced, consequently the leaves die in a short time. The fungus causes the disease threats the human and animal health through the production of phytotoxins. Identification of Fusarium species is currently confusing, on the other hand, several Fusarium species have always been isolated together with rice contaminated with the disease. Therefore, it is not clear which species of this fungus are the main reason of the disease. This fungus is soil-borne and has a long life in heavy soils. It has also a global expansion and is active in most parts of the world. Rice foot rot disease in all major rice producing countries in the world is considered as a seed disease and the transmission of the disease agent from one season to another season is mainly due to contaminated seed, but soil-borne fungus can be as well. Currently, treating the seeds with fungicides is the best method to control this disease, however using chemical pesticides can lead to environmental pollution. In this situation, it is important to achieve a healthy alternate method. For this purpose, biological control is one of the ways in which today a large investment is being made around the world.

Samples of rice exhibiting the symptoms of bakanae were randomly collected from different parts of Gilan province, Iran. Pieces of organs with rot symptoms were cut and they were surface-disinfected after washing with 0.5% sodium hypochlorite solution. At the next step, they were washed with distilled water and dried on filter papers. Then, they were cultured in Petri dishes containing potato dextrose agar (PDA) culture medium and were placed in an incubator at 28°C for 3-5 days. Afterward, they were placed on a water-agar medium for purification and morphological identification. In this research, from a total of 80 samples collected from rice farms in Gilan province, 18 bacterial strains were isolated and the antagonistic ability of 8 strains of bacteria in the laboratory and greenhouse was investigated. For this purpose, in laboratory, dual culture method, volatile compounds, siderophore and antibiotic production were used. These bacterial strains were inoculated into rice under greenhouse conditions, and then the severity of the disease was determined in the tested treatments. After calculating the severity of the disease, the height of the rice bushes was measured by the ruler. To measure the fresh weight, the rice bush with the roots was removed from the soil and measured by a scale. Each bush was then separately placed for 48 hours in an oven at 80-90°C. After leaving the oven, each of the bushes was re-weighed. This weight was recorded as dry weight.

A total of 18 isolated bacterial strains, 8 bacterial strains including Bacillus subtilis, Bacillus circulans, Pseudomonas putida, Pseudomonas syringae, Pseudomonas aeruginosa, Pseudomonas fluorescens (N47), Pseudomonas fluorescens (149) and Pseudomonas fluorescens (CHA0) were identified and used for biological control studies in laboratory and greenhouses. P. putida with 39.99 % in the dual culture, B. subtilis with 31.01% in the volatile metabolites, in the method of siderophore production, P. fluorescens (N47) and B. subtilis with 52.10 % and 45.85 % respectively, and in the antibiotic production, P. putida with 59.21% had the greatest effect on inhibiting the mycelial growth of the disease causative agent. Based on the results of the analysis of variance under greenhouse conditions, there was a significant difference between treatments for severity of disease, fresh weight, dry weight and plant height at 1% probability level. Among the studied bacteria, the least severity of the disease belonged to the treatment with P. putida and B. subtilis, respectively. Regarding height, the highest height was related to treatment with P. putida and B. subtilis and in terms of fresh weight and dry weight, the highest weight was related to P. putida.

According to the results of biocontrol studies in laboratory and greenhouse conditions, P. putida and B. subtilis strains were the most effective bacteria for controlling rice foot-rot disease.Therefore, isolating and identifying these bacterial strains as much as possible can be promising for the greater effectiveness of biocontrol methods in the management of rice crown-rot disease control.

Pomegranate is one of the most popular fruit trees and one of the most important export productions in Iran. Root-knot nematodes (Meloidogyne spp.) are the most damaging plant-parasitic nematodes of pomegranate, which cause root galls and disrupt the absorption and transfer of water and food, and eventually decrease the fruit growth. Root-knot nematodes (Meloidogyne spp.) are economically important plant parasites affecting a broad range of host plants, and thus far, 100 nominal species have been described. The development of molecular methods to identify the four major Root-knot nematodes including M. incognita, M. arenaria, M.javanica and M. hapla has been the goal of numerous studies. These species are morphologically similar, making identification difficult for the non-specialist. However, distinguishing them is important for utilizing appropriate crop rotations, managing resistance effectively and plant quarantine requirements. Therefore, some molecular methods for identification of Root-knot nematodes (RKN) species have been developed. Recently, a PCR method based on DNA has been widely used for the identification of nematodes. SCAR- Primers (Sequence characterized amplified region) were developed and are used routinely on a large number of samples with high sensitivity and specificity.

In order to investigate the infection of pomegranate orchards to Meloidogyne species, 115 soil and root samples were collected from major areas of pomegranate cultivation from Khorasan Razavi, Northern Khorasan and Southern Khorasan provinces, during 2015-2016. The highest area of pomegranate cultivation is located in Torbat Heydarieh, Bardaskan, Kashmar, Khalil Abad, Bajestan and Ferdows cities. After extraction of nematodes from root and surrounding soil, species identification was performed according to morphological and morphometric characteristics of second-stage juveniles, mature females and perineal pattern of females, and also using molecular methods and specific SCAR primers. For DNA extraction, the procedure proposed by Silva et al, who extracted DNA of second stage juvenile, was applied. In addition, the Atkin methods were employed for extracting DNA of mature female. The DNA was added into the PCR reaction with specific primer (SCAR), and then loaded in gel for further analyses.

Major species of root knot nematode were M. incognita, M. javanica and M. arenaria in Khorasan provinces identified based on morphological and morphometric studies as well as SCAR primers. M. incognita showed a band about 399 bp with Inc-14 primer, M. javanica exhibited a band about 670 bp with Jav primer and M. arenaria showed a band about 420 bp with a ar primer. Infection of root knot nematode pomegranate was observed in almost all cities but the intensity of infection varied considerably. The highest percentage of infection on root-knot nematodes pomegranate orchards was observed in Bardaskan (Anabad section of Fatemieh village) with 57.5% and Bajestan (Chah Paliz village) with 32%. M. incognita has the highest distribution in pomegranate orchards. The highest percentage of infected orchards was estimated in Bardaskan (19.93%), Bajestan (12.3%), Khalil Abad (6.9%), Kashmar (4.3%), and Torbat Heidariyeh (3.5%) located in Khorasan Razavi provinces, Ferdows (5.4%) and Nehbandan (1.3%) in Southern Khorasan provinces, and Maneh (1.1%) and Jajarm (0.8%) situated in Northern Khorasan. M. incognita has the highest distribution in Khorasan Razavi and Southern Khorasan provinces, and M. javanica has the highest distribution in Northern Khorasan provinces. The differential host shows the race 2 of M. incognita in area.

Infection of root knot nematode pomegranate is growing and there is a need for accurate identification. Using molecular methods especially SCAR primers for identification of major species of root knot nematode is fast, accurate and reliable.

Root-knot nematodes are the most common and destructive plant-parasitic nematode group worldwide and adversely influence many economically important crops, ornamental plants, and fruit trees. They are obligate parasites of the roots of many plant species, including herbaceous and woody plants. Symptoms associated with Root-knot nematodes infection include root galls, shoot chlorosis, stunted growth, nutrient deficiencies, and secondary infections by other pathogens. A high level of damage can lead to serious crop loss. Due to the considerable morphological similarity between species of Meloidogyne and high intraspecies variation, it is difficult to differentiate species from each other based on the morphology of their perineal patterns alone. Molecular techniques are used for detection, species differentiation, and phylogenetic analysis of species. Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. The objective of this study was to improve molecular diagnosis of root knot nematode in vegetable crops in Fars province to enhance our diagnostic knowledge and allow species identification of root knot nematode through PCR by species-specific primers.

In 2012, in order to identify the different stages of growth of Meloidogyne javanica species, 40 soil and root samples infected with the nematode were collected from different cultivation sites of Fars province (Darab, Jannatshahr, Estahban, Nurabad, Qavar, and Shiraz). The roots of plants were flushed with water to remove soil and then washed with soap for 10 min. To obtain the pure population of each sample, a large egg bag containing more eggs was selected. The isolated egg bag was placed adjacent to Rutgers tomato transplant. After 70-60 days, the roots were removed from the pot and prepared to identify species of the nematode. After purification of nematodes using single egg method on Rutgers tomato root and diagnosis of them on the morphological basis, the DNA was extracted at different growth stage of nematode using three optimized methods of Zhang, 1998, Silva, 2000, and Liao, 2005. PCR products (5 µl) were separated on standard agarose gels. A DNA ladder (Sinagen 100bp-3000bp) was used to determine the molecular sizes of the bands. Band patterns were photographed under UV light using the GBOX document gel system. The PCR was optimized by varying the reaction components and cycling conditions. The annealing temperature was optimized separately for each primer based on the manufacturer's Tm and formula. The roots were flushed with water to remove soil and then washed with a 0.52% NaClO soap for 10 min. Individual root knots were obtained after three to five washes. Furthermore, DNA extraction was performed using four methods.

The root-knot nematodes were recovered from the vegetable crop soil samples. Species identification in this study was based on the PCR by species-specific primers. Specific primers of Fjav/Rjav, Mj-MF/Mj-MR and Mj-DF/Mj-DR could detect M. javanica with 670 bp, 517bp and 1650bp, respectively, proving their usefulness for PCR on root-knot nematodes. No band was detected using specific primer of M. incognita (MI-F/R primers) from the samples in PCR. During optimizing PCR method, it was revealed that the best annealing temperature is 54ºC for Mj-MF and Mj-MR primers, 56ºC for Mj-DF and Mj-DR primers, and 50ºC and for Fjav and Rjav primers. Adding the DNA extracted template from 1µ to 2µ for single female and single egg cist and from 2µ to 4µ for single juvenile made the best result for detection of root-knot nematode based on PCR. Changing the MgCl2 concentration from 1.5 mmol to 2 mmol in a 25µl PCR reaction made the best PCR result. The Liao optimized method was the best DNA extraction for monitoring M. javanica. Fjav/Rjav and Mj-MF/Mj-MR primers were the best primers in detecting M. javanica.

The results of this study showed a remarkable ability of DNA extraction methods, specific primers and the cases optimized in this study in identifying root knot nematode. Identifying M. javanica species based on morphological methods is time consuming and difficult, and requires high skills and also needs to be examined for a particular stage of nematode growth. In addition, identifying of this species based on biochemical methods is time consuming, affected by environmental conditions, requires relatively large amounts of living organism and a particular stage of the nematode's life cycle. Given these difficulties, DNA extraction methods, specific primers and the cases optimized in this study can be used to identify M. javanica species. However, as M. javanica and M. incognita primer pairs were not detected for a sample taken from Shiraz, more researches are needed to be carried out on this case.

Cucurbits are among the main vegetable crops that are extensively grown in commercial greenhouses, plastic tunnels, and open farms in many areas of Iran. Many begomoviruses are known to cause serious damages in economically important cucurbit crops such as watermelon and melon. Among these viruses, WmCSV has been quoted as one of the major limiting factors for cucurbit production throughout the south of Iran. Begomovirus genus is a member of Geminiviridae family that has the most species in comparison to other viral genus infecting plants. Begomoviruses are notorious pathogens infecting dicotyledonous plants globally. Thereare transmitted exclusively by the whitefly, Bemisia tabaci. Whitefly is one of the most important and prevalent pests in the warm south and southeast provinces of Iran and infects cucurbits fields in Sistan and Baluchestan province with a widespread population. Begomoviruses are single-stranded circular DNA viruses and have been observed in a wide host range of plants. There are different reports of infectious Begomoviruses species such as Watermelon chlorotic stunt virus (WmCSV) from a world that can damage the cucurbits. The purpose of this study is to find the phylogenetic relation of WmCSV isolates from Sistan and Baluchestan province, which is located southeast of Iran, together and other isolates from the world, too. Because of this province is the largest area, with about 11.4% of the total area of Iran.

To identify the WmCSV infecting cucurbits in the southeast of Iran, several surveys in different regions of Sistan and Baluchestan were selected for the study, including Saravan, Iranshahr, Nikshahr, Sarbaz, Konarak, Chabahar, Zabol, Zahak, Hamoon, Hirmand, Nimroz, and Zahedan. Sampling has been taken from different cucurbits, such as different kinds of melons, watermelon, and squash, in 2015-2017. The samples were taken from plants showing viral symptoms such as vein yellowing, stunting, chlorotic, and mottling, deformation and severe reduction of fruit size and co-infection with whiteflies. The collected samples were transformed into the laboratory and the total DNA was extracted. To identify WmCSV were used from specific primers, Gem-CP-V-5' and Gem-CP-V-3' detecting Begomovirus genus in cucurbits. PCR products have been sequenced and blasted in the GenBank NCBI database and submitted there. Then, carried out the Alignment of acid nucleotide sequences and description of the phylogenetic tree by MEGA5 software. The Identity and differences percentage of Watermelon chlorotic stunt virus nucleic acid sequence from Sistan and Baluchestan province isolates with together and other areas of Iran carried out by MegAlign software too.

All areas of sample collection of Sistan and Baluchestan province were infected with WmCSV in this study. Positive PCR watermelon samples have been infected to WmCSV, severity. The expected PCR products, 550 bp size, from 16 amplified partial CP genes from different areas of Sistan and Baluchestan province in Iran were done sequencing. Phylogenic sequencing analysis results of isolates from other countries and other regions of Iran also WmCSV isolates in this research from Sistan and Baluchestan province, classified them in two apart groups involves A and B. The A group was divided into 2 groups, that were named AI and AII. Isolates of Sistan and Baluchestan province from this research involved both of them. These findings seem to indicate Sistan and Baluchestan as of the possible origins of WmCSV in Iran. There was no relationship between geographical source and host in this province. Some isolates of this province had a near relationship to Oman and Saudi Arabia isolates.

WmCSV can be considered as a serious disease threatening watermelon production, in Sistan and Baluchestan province in the southeast of Iran as severe symptoms. On another hand, individually or in interaction with other begomoviruses, WmCSV can cause severe damage in farms infected by whiteflies. Probably, the spread of isolates belong Sistan and Baluchestan province and Oman or Saudi Arabia has been taken by whiteflies that transformed via between Iran and these countries, because of the most relationships between the WmCSV isolates of these countries. More importantly, performing collaborative studies with neighboring countries such as Pakistan and India, seems necessary. Also, further studies on Iranian isolates of WmCSV that occurred on either cultivated or wild plants are essential to have a better understanding of the viral epidemics in the country. The results of sequencings will be used to design constructs inducing RNA silencing as a resistant strategy, against WmCSV isolates that were classified in both two groups.

Plant-parasitic nematodes, especially root-knot nematodes, cause a lot of damage to most agricultural products and a lot of efforts are made to control them. Biological control is one of the most widely studied methods to reduce nematode damage. This study aimed to evaluate the effect of two bacteria including Bacillus cereusIPRI95 and B. subtilisVUPF52 on the infection of Meloidogyne javanica on three rootstocks under greenhouse conditions.

This study was performed as a factorial experiment in a completely randomized design with five replications. In this way, the soil of the pots was disinfected before planting the seedlings, and then the seedlings were planted. After the establishment of the rootstocks, a three-day liquid culture peach for each bacterial isolate was prepared with a concentration of 107 colony-forming units in milliliters (CFU / ml) and 15 ml of suspension from each bacterium to the soil around the seedlings were added in each treatment. Two days after treating the plants with bacteria in the pots, inoculation with 2000 eggs with larvae of nematodes per kilogram of soil, entered into 3 holes created around the seedlings and then covered with soil these holes. Experimental plants to pass at least three generations of nematodes, three months in suitable greenhouse conditions with a temperature of 20 to 25 degrees, relative humidity 60-80% and optical period (13 hours of light, 11 hours of darkness) maintenance and regular watering twice in the week was over. At the end of the experiment, plant growth indices include (length, fresh and dry weight of shoots, length, fresh and dry weight of roots) and nematode growth parameters including (number of galls, number of second larvae (j2), number of egg mass in plant roots and number Eggs per egg mass, as well as a reproductive factor (RF), were measured an statistically analyzed using SAS 9.4 software. A comparison of mean treatments in all cases was performed by LSD-protected test at 5%.

Nematode contamination in the absence of biocontrol agents significantly reduced the growth index at all levels compared to other treatments. So the peeling of peach bases with nematode reduces the stem length by 34% and 30% in GF677 and Shorabi local cultivars, respectively, compared to the control treatment. The results of comparing the means between treatments in GF677 showed that stem length in these plants treated with two bacterial strains increased significantly compared to the control. Root length also decreased by 14 and 50 percent in Helendri and GF677 cultivars, respectively, following infection with nematode compared to control treatment. However, following the treatment of peach roots with bacterial strains, we saw a significant increase in root length, so that the maximum root length in Helendri cultivar in treatment with B. subtillis strain and GF677 cultivar followed by treatment with B. cereus was obtained. Statistical analysis showed that the two bacterial isolates mentioned, in addition to affecting the growth factors of vegetative bases, also had a significant effect on nematode growth and development indicators and disease reduction. So that the number of eggs and egg mass decreased in all three cultivars. The mean number of galls per gram of root in GF677 treated with B. subtilis and B. cereus bacteria decreased by 48.4% and 40.68%, respectively, compared to the non-bacterial control. The results of this study also showed that the J2 population in treatments with nematodes alone compared with treatments with bacteria have a significant difference. The number of J2 larvae in the presence of B. subtilis bacteria decreased by 67.73, 52.52 and 12.6% in Helendri cultivars, GF677 and local cultivar Shorabi compared to the control. The use of biological bacteria affected all stages of nematode growth and development. However, the ability of bacteria to reduce nematode reproductive factors was significant in the nematode-sensitive cultivar (Gf). The findings of this study showed the controlling effect of biocontrol bacteria used on the propagation and damage of root-knot nematodes. So that by increasing plant growth indices, nematode damage was reduced. Although the bacteria used were effective on all three rootstocks, due to GF677 being more sensitive to root-knot nematodes than Helendri cultivars and the local cultivar Shorabi, the effect of bacterial agents on the rootstock was less than the other two rootstocks.

In Iran, potato Solanum tuberosum L. is an important crop, and its cultivation extends over more than 186,000 ha annually. The onion thrips, Thrips tabaci Lindeman, is a highly polyphagous species and a serious pest of a wide range of economically important crops. In Iran, T. tabaci causes damage on different agricultural crops including: potato, onion, cucumber and tobacco. Onion thrips feeds by piercing the surface of the tissues (leaves and leaf buds) and sucking up the exuded cellular contents. On infested leaves, the empty cells create silvery-white spots, silver damage, which cause yield loss. Use of insecticides for controlling T. tabaci has low efficacy because females lay eggs within leaf tissues, larvae hide in leaf domatia and between the inner leaves of plants, and pupae rest in the soil. Therefore, it is necessary to use alternative management approaches for control of T. tabaci such as resistant cultivars integrated with natural enemies. The interactions between natural enemies and resistant cultivars have a direct effect on the pest by effecting its developmental rate, fecundity and survival rate. They may also have an indirect effect on natural enemies by increasing generation time of the pest and, as a result, increasing exposure time of the pest to its natural enemies. In this study, we assumed that the different potato cultivars affect population density of T. tabaci and diversity of its natural enemies. Therefore, the present research was designed to evaluate the onion thrips density and species diversity of its natural enemies on four potato cultivars under field condition.

Tubers of four potato cultivars including Agria, Savalan, Arensa and Fantasia (commonly cultivated in Ardabil region) were selected for evaluation of population density of T. tabaci and diversity of its natural enemies under field condition during 2011-2012. The tubers were obtained from the Seed and Plant Improvement Institute of Karaj, Iran. Tubers of four tested cultivars of potato were planted in four experimental fields, each of 500 m2,at four different locations in the Ardabil plain. The fields were managed according to the local practice with flood irrigation and hand weeding once every ten days. No insecticides were applied in these fields. The onion thrips populations were monitored on the four cultivars of potato from late stem elongation to petal fall stages by examining four plants from each cultivar chosen randomly from each of four fields (16 plants per cultivar). The numbers of thripslarvae and adults and each of natural enemies per plant were counted using a 20X hand lens. Moreover, onion thrips and its natural enemies were collected from each of potato cultivars, transferred to the laboratory, and identified to species by morphological characteristics under a stereomicroscope or a microscope according to the valid identification keys. The Shannon diversity index (H), Shannon evanesces index (E), and Morisita-Horn index (CMH) were then calculated for each of four potato cultivars by use of EstimateS Win 8.20 software (6). Data on population densities of onion thrips and its dominant natural enemies were analyzed using one-way ANOVA and significant differences among cultivars were compared using Tukey's HSD test. 

In two years, the lowest densities of onion thrips larvae and adults were observed on Agria. In this study, 21 species of onion thrips predator were collected and identified. Amongst the predator species, Aeolothrips intermedius Bagnall and Orius niger (Wolff) had the highest relative abundance on each of four potato cultivar. The predator A. intermedius with small size and ability to penetrate into leaf domatia, faster development rate and higher fecundity is effective in control of herbivore thrips on host plants (19). Moreover, previous researches have reported that the species of Orius are efficient in control of onion thrips. The Shannon diversity index for the complex of predators was greatest on Agria. Species richness and their relative abundance are two main factors for measuring Shannon diversity index in each ecosystem. Moreover, the values of Morisita–Horn index for the complex of predators between the four cultivars of potato ranged from 0.948 to 0.989. The value of this index varies between 0 and 1. When the value of this index increases from 0 to 1, the species complex of predators between two cultivars becomes more identical. In two years, densities of two predator species A. intermedius and O. niger on Agria and Savalan were significantly higher than on Arensa and Fantasia. Furthermore, the greatest percentage of females of two predator species was observed on the thrips-infested plants of Agria. These results indicated that the preference and performance of two mentioned predators were higher on Agria and Savalan among the tested cultivars.

Based on the results of our study, it can be concluded that the cultivation of Agria could be useful in reduction of T. tabaci population and conservation of its predators in potato fields.

Ants are the most ubiquitous insects on the earth with more than 12000 species which are classified into 21 subfamilies. Until now, more than 170 ant species have recorded in Iran. Mutualistic relationship between ants and scale insects have been reported in Iran. Such relationships between ants and insects are known as trophobiosis which is due to attraction of ants to honeydew of scale insects. Honeydew is a sugar-rich sticky liquid, secreted by aphids and some scale insects as they feed on plant sap. Ants provide protection from predators and parasitoids by building shelters around scale insect colonies. Such a relationship is mostly facultative and only a few taxa of mostly tropical or subtropical scale insects have obligate mutualism with ants. The coccoids with obligate mutualism display obvious behavioral and morphological adaptations for living with ants. Ants are also able to establish antagonistic interaction with honeydew-producing hemipterans and contribute positively to biological control by the suppression of pests.

In a study during 2013-2014, colonies of the mealybug Paracoccus ficus Moghaddam, 2014 were investigated for mutualistic ants feeding on honeydew of the mealybug in fig orchards of Neyriz, Estahban and Shiraz cities of Fars province, South Iran. Samples were collected by hand, forceps and soft brush. The specimens were preserved in 75% alcohol in small glass vials and were transferred to the laboratory. The morphological keys were used for identification. Specimens were deposited in Insect and Mite Collection of Ahvaz, at Department of Plant Protection, Shahid Chamran University of Ahvaz.

Totally, 6 species belonging to 3 subfamilies of ants were identified as follows:Myrmicinae: Monomorium abeillei Andre, 1881; Tetramorium sp.; Pheidole pallidula Nylander, 1849; Crematogaster antaris Forel, 1894; Formicinae: Cataglyphis lividus Andre, 1881; Dolichoderinae: Tapinoma simrothi Krausse, 1911.
Among collected species, T. simrothi and P. pallidula were more frequent. T. simrothi has also recorded as most frequent mutualistic ants with aphids in Central Iran. Natural enemies exploit hemipterans which receive ant protection for their benefit. For example, some parasitoid wasps soliciting honeydew directly from aphids by antennation and mimicking ants during foraging in host patches. Researchers believe that preventing ants from colonies of Pseudococcidae scale insects is necessary for success in biological control of such pests. However, not all of ants have negative effects in biological control of pests. Predatory ants are less attracted to the honeydew and mostly feed on egg, larvae and adult insects. Moreover, some ants are important in pollination, soil improvement, and nutrient cycling.  

Behavioral and ecological should be carried on ants in pest management programs, and only if they have a negative influence on biological control of scale insects, then perform control measures for ants.

Sethoxydim is a post emergence graminicidethatcontrolannual and perennial grasses such as wild oat (Avena ludoviciana Durieu.) and littleseed canarygrass (Phalaris minor Retz.) by inhibitory activity on acetyl coenzyme A carboxylase enzyme and disrupt fatty acid biosynthesis. It belongs to the cyclohexanone chemical family. It was registered for weed management in broad leaf crops. Using an adjuvant (e.g. vegetable oils and surfactants) that can increase the foliar activity of post emergence herbicides by cuticle destruction and increase leaf wetting is an acceptable way to achieve this approach. Applying vegetable oils increased graminicide penetrate to leaf and post emergence herbicides performance. Some synthetic adjuvants have been shown a side effect on wildlife, similar to agrochemicals therefore using safe and reproducible adjuvants is essential. The objective of this research is to determine the best vegetable oil on biological activity of sethoxydim on wild oat and relation between compounds of vegetable oils (fatty acids) and their effects on sethoxydim performance.

The dose response experiment was conducted in Research Greenhouse of Ferdowsi University of Mashhad in 2012. The seeds of wild oat were collected from plants in the fields of the Mashhad Agricultural and Natural Resources Research Center, in Mashhad, Iran and preserved at room temperature in paper bag. The seeds were surface sterilized by immersing in sodium hypochlorite for 5 minutes. Then seeds were rinsed by distilled water for 15 minutes. To increase seed germination before the start of experiment, the seeds were dehulled and placed in Petri dishes on top of a single layer of Whatman no. 1 filter paper. Then, 10 mL of 0.2% KNO3 solution were added to each Petri dish for breaking dormancy, then the seeds were incubated for 72 h at 4–5◦C in the dark. The germinated seeds were sown in pot (1.5 L). One week after sowing plants were tinned to four plant in each pot. The pots were irrigated every days. At four leaf stage of wild oat plants the herbicide treatment were applied. The treatments included sethoxydim concentration at seven levels (0, 23.4, 46.8, 93.75, 187.5, 281.25 and 375 g ai ha-1 of sethoxydim) and vegetable oils at ten levels (without oil and with turnip, olive, soybean, corn, sunflower, canola, sesame, castor, and cotton oil). The response of wild oat biomass were analyzed by non-linear regression by R software. To determine toxicity of sethoxydim plus vegetable oils on sugar beet and onion an experiment by recommended dose of sethoxydim was conducted. Moreover, an experiment was carried out to determine the fatty acid content and quantity of each vegetable oil. To determine the chemical nature of fatty acid oils, 15 drops of each vegetable oil were added to 7 mL N-hexane plus 2 mL of potassium hydroxide in methanol (11.2% m/v). Then, four replications of the supplied compounds were shacked for 1 min and heated to 55°C for 5 min until the solution was separated into two phases. The upper phase was desiccated with sodium lauryl sulfate and filtered to analyze with gas chromatography. The fatty acid content was determined using gas chromatography Acme 6000 (Younglin, South Korea) equipped with a flame ionization detector and a CP-Sil 88 Wcot fused silica column (100 m × 0.25 mm i.d. × 0.2 μm film thickness; Chrompack, Middleburg, Netherlands). The carrier gas was ultrahigh-purity helium; we used a 1:100 split mode and a flame-ionization detector. The GC oven temperature was maintained at 140 °C for 5 min, then ramped to 240°C at the rate of 4°C/min and maintained at 240°C for 15 min. The flow rate of helium was 20 mL/min. The injector and detector temperatures were 250 and 280°C, respectively. The volume of injected sample was 1 μL. Fatty acids were identified by matching their retention times with those of their relative standards.

Results of this study showed that sethoxydim performance improved in the presence of vegetable oils whereas relative potency were higher than 1 in the presence of vegetable oils compared to sethoxydim alone. The vegetable oil could be ranked based on their relative potency value as: turnip > olive > soybean > corn > sunflower > canola > sesame > castor > cotton. The overall results showed that the efficacy of sethoxydim was improved by increasing the content of unsaturated fatty acids in vegetable oils. Turnip and cottonseed oils with 71.17 and 20.65 percentages of unsaturated fatty acids had the highest and lowest performance, respectively. Among the polyunsaturated fatty acids compounds, linoleic acid content had a key role in the efficiency. There was a negative relationship between linoleic acid content and the performance of vegetable oils. Moreover, non-significant toxicity effects on sugar beet and onion was observed.
   

Based on this work, when the vegetable oils used the performance of sethoxydim on wild oat control based on relative potency were improved. Therefore, synthetic adjuvants can be replaced by vegetable oils as adjuvants in herbicide application. Based on results of this work, composition of fatty acids in vegetable oil is a very effective factors for increasing sethoxydim performance on wild oat control. By increasing unsaturated fatty acids, the sethoxydim performance showed more performance whereas turnip oil with higher unsaturated fatty acids was showed the highest performance. Further research is needed to determine the mechanism of action of vegetable oils in increasing the effectiveness of herbicides.

In arid regions such as Iran, water hardness, as a result of existence of calcium- and magnesium-containing minerals, is a serious challenge for irrigation practice. The irrigation systems are often applied to spray herbicides. Therefore, using hard water having high cations for herbicide application is inevitable. Such water can adversely affect the activity of some herbicides particularly weak acid ones. A solution for overcoming this problem is the addition of an adjuvant to the tank. It is well established that the addition of (NH4)2SO4 (43) and C6H8O7 (45) will efficiently remove the cations of hard water from the spray solution of weak acid herbicides. However, there is no report on whether the efficacy of sulfosulfuron (a weak acid herbicide, pKa = 3.5) is affected by the cations of hard water. Furthermore, it is observed in literature and practice that the addition of herbicide to the tank is immediately done after adding the adjuvant to the tank. Thus, there is no report on whether the sequence and time interval of adding adjuvant and herbicide to hard water could be affected by the efficacy of weak acid herbicide.

The seeds of winter wild oat (Avena sterilis ssp. ludoviciana Durieu.) were primed in the same method which was already described (3). Then, eight seedlings with a 1-cm radicle and 0.5-cm coleoptile were transplanted at a 1-cm depth within each 3-liter pot filled with a 1:1:4 ratio of sand: animal manure: clay loam soil, respectively. The experiment was carried out as a dose-response study in a completely randomized design with three factorial (6×4×7) and four replications. The 1st factor was the dose of sulfosulfuron(0, 1.25, 2.5, 5, 10, and 20 g a.i. ha-1). The 2nd factor included the type of spray carrier (distilled water, hard water containing sodium chloride (NaCl), calcium chloride (CaCl2) and iron chloride (CaFe3) at 600 mg L-1). The 3rd factor was also the sequence and time interval of adding adjuvant and herbicide to the spray carrier (with and without adding 500 mg citric acid (C6H8O7) L-1 or 20 g ammonium sulfate ((NH4)2SO4) L-1 at 30 min before, simultaneous, and 30 min after adding sulfosulfuron to the spray carrier). Five weeks after spraying, the response of individual dry weight of winter wild oat to treatments was analyzed as a nonlinear regression. This was carried out by using a four parametric logistic model (36) to estimate the values of ED50 and ED90 which are the doses of sulfosulfuroncausing a 50 and 90% reduction in the dry weight as compared to the control, respectively. 

In contrast to ammonium sulfate which was ineffective, the addition of citric acid to distilled water under each sequence and time interval improved the efficacy of sulfosulfuron. Decreasing the pH can permit the weak acidic herbicides to pass through the cuticle and then cell membrane during the ionic trap process. The presence of Na+, Ca++ and Fe+++ cations in the spray carrier increased the amount of sulfosulfuron required for 90% reduction in the dry weight of winter wild oat from 9.80 to 27.60, 47.48, and 50.32 g ha-1, respectively. There was no significant difference between the intensity of the incompatibility of Ca++ and Fe+++ cations. Similar to other weak acid herbicides, sulfosulfuron can also be ionized into anionic (a negatively charged form) and cationic (H+) fragments under alkaline conditions. The anionic part of sulfosulfuron bonds with the cations in hard water. As a result, a crystalline herbicide-cation salt is formed which is not able to pass through the cuticle due to its low solubility in water. The addition of both adjuvants 30 min after the addition of sulfosulfuron to distilled water containing the cations had no effect on removing the adverse effect of cations on the efficacy of sulfosulfuron. Conversely, adding sulfosulfuron 30 min after the addition of both adjuvants to the distilled water containing the cations provided the best efficacy of sulfosulfuron. When the adjuvants are added to hard water before herbicide, the cations of hard water can receive the cation of H+ from citric acid or NH4+ from ammonium sulfate. Then, the formation and sediment of the cation-anion salt turns hard water into soft water, having no problem with the efficacy of sulfosulfuron.

The issue of correct sequence for adding adjuvant and herbicide to hard water is important. Lack of knowledge about this issue can lead to not only the lack of weed control but also the imposition of additional costs on the farmer.

Weeds are wild plant species which grow in agricultural ecosystems and compete with crops for resources such as water, nutrients, light, and space. Wild oat is reported as one of the most troublesome autumn weeds in croplands and its dominant species in Iran is winter wild oat (Avena ludoviciana Dur.). Herbicide resistance is the inherent ability of a weed biotype in survival after being exposed to a rate of herbicide which would be lethal to the wild type species. In the recent years, there have been several reports on the occurrence of herbicide resistance in Golestan province. The following research was conducted to identify the ACCase- resistant winter wild oat biotypes and generation of distribution map for the resistant biotypes in wheat fields of Ramian Township.

The present study was conducted in 2017-2018 at the greenhouse and Weed Science laboratory of Gorgan University of Agricultural Sciences and Natural Resources. Plant material included 80 putatively resistant winter wild oat biotypes which were collected from 100 wheat fields of Ramian Township. The susceptible biotype was also collected from the regions with no history of herbicide spray. Three Acetyl coenzyme A carboxylase (ACCase) inhibiting herbicides including clodinafop propargyl, fenoxaprop-P ethyl, and diclofop methyl which are common graminicides applied in wheat fields of the country were used to confirm the occurrence of resistance in winter wild oat biotypes. Three herbicides including pinoxaden, clethodim, and haloxyfop-R methyl ester were also applied on the biotypes as the possible alternative herbicides. First the winter wild oat seeds were hulled by hand. To obtain a more uniform germination, the seeds were placed in 9 cm Petri dishes topped with a filter paper and 2.5 mL of distilled water was added to the Petri dishes. Then, the Petri dishes were transferred to a refrigerator with a temperature of 4-5°C under darkness conditions. After pre-chilling, the petri dishes were incubated at the room temperature to germinate. To perform the discriminating concentration, pre- screening and concentration- response assays, 10 pre- germinated seeds from each biotype were placed on the Petri dishes as described above, then were treated with various concentrations of the mentioned herbicides. Three Petri dishes were used for each concentration, with each Petri dish serving as a replicate. Also, the Petri dishes treated with distilled water were regarded as control. The Petri dishes were kept at the room temperature for seven days and then the length of their coleoptile was measured. Four parametered log-logistic function was fitted to the data using R software (drc package). ArcGis V.10.3 was used to generate the distribution map of the herbicide- resistant biotypes.

Results of the present study indicate the confirmation of resistance to clodinafop propargyl, fenoxaprop-P ethyl, and diclofop methyl herbicides in 58 out of 80 winter wild oat biotypes gathered from wheat fields of Ramian Township. Resistance factor to clodinafop propargyl, fenoxaprop-P ethyl, and diclofop methyl herbicides were 48.69 to >851.33, 52.57 to >752.57, and 310.27 to >1443.90, respectively. For further investigation on the response of these resistant biotypes, three herbicides including pinoxaden, clethodim, and haloxyfop-R methyl ester were applied on the biotypes as a Petri dish assay. Two biotypes with the highest resistance factors (ram1, ram18) and two with the lowest resistance factors (ram15, ram4) were selected for this assay. In the haloxyfop-R methyl ester treatment, the highest and lowest resistance factors were observed in ram1 with 423.35 and ram15 with 284.50, respectively. In the other two herbicide however, resistance factor had no significant difference with the value 1.  Application of herbicides possessing different modes of action may lead to elimination of both susceptible and resistant biotypes. However, this will serve as a new selective pressure which will eventually result in intensification of resistance and furthermore, evolution of cross and multiple- resistant species. Thus, meticulous application of integrated weed management methods are of great importance to prevent or delay the evolution of resistance to herbicides.

Implementation of cultural methods for weed management and preventing the distribution of herbicides to which the weeds have readily developed resistance will lead to reduction of selective pressure. Adoption of crop and herbicide rotation principles will serve as a tool to debilitate the weed in competition with the crop, which in long term may contribute to reduced frequency of resistant alleles.