فهرست مطالب محمدرضا عالی منش

One of the damaging agents for wheat is the cereal cyst nematode (Heterodera avenae). Plant Growth Promoting Bacteria (PGPR) are a group of bacteria that have beneficial properties for plant growth, including iron chelation, siderophore production, phosphate dissolution, nitrogen fixation, as well as the production of protease and chitinase enzymes. These bacteria can also be used as biological control agents. In this study, wheat roots showing suspicious symptoms of cereal cyst nematode were collected from the Dezful county in Khuzestan province during 2022-2023.  After initial investigations, nematode identification and purification was done. According to the morphometric and morphological characteristics of the larvae and cysts, especially its cone top, H. avenae was diagnosed. the effect of seven native growth-stimulating bacterial strains on egg hatching rate and mortality of cereal cyst nematode larvae in the Petri dish was investigated in the laboratory. The effect of four selected bacterial strains with sulfur treatment and Nematex EC nematicide was investigated in the greenhouse against grain cyst nematode. The results showed that in laboratory conditions, the bacterial strains were able to reduce the egg hatching rate by 62% on average. Bacillus pumilus bacteria caused the greatest decrease in egg hatching by 73%. The percentage of mortality of larvae in the laboratory test was also shown to be 63% on average, and the treatment of B. pumilus bacteria showed the highest percentage of mortality with 70%. The results of the greenhouse investigations showed that the treatments involving sulfur-healthy plants, sulfur-nematode, and the B. pumilus and P. fluorescens bacteria were most effective in promoting healthy plant growth and controlling nematode infestation. These treatments resulted in significant improvements in plant height, as well as fresh and dry plant and root weights. Additionally, the greatest reduction in the number of cysts was observed in the B. pumilus treatment, with an average of 49 ± 1 cysts. When examining the number of eggs within the cysts, the most substantial decrease was observed in the Nematex EC and B. pumilus bacteria treatments, with 67 ± 1 and 111 ± 2 eggs, respectively. Furthermore, in the assessment of the number of live larvae in the pot, the greatest decrease in the number of larvae was observed in the Nematex treatment, with 87 ± 1..

Pectobacterium carotovorum carotovorum (Pcc) is one of the main causes of soft rot disease in many economically important vegetables and tubers such as carrot, cabbage, potato, onion, cucumber, eggplant, garlic, bell pepper, radish, sweet potato, squash and tomato. The use of chemical bactericides is not suitable to control soft rot bacteria due to their toxic side effects and the emergence of resistance in bacterial populations. The use of bio-control bacteria to inhibit bacterial pathogens such as Pcc is a practical and useful method to overcome the issue. In this study, in order to control this disease with the help of bacterial biological control agents, some bacteria were isolated from rhizosphere of vegetables and potato. The aim of this study was to find bacteria with different bio-control mechanisms which are able to inhibit different isolates of Pcc. On the other hand, these bacteria (with different bio-control mechanisms) were combined to investigate the possibility of increasing their bio-control ability in a mixed manner.

12 rhizospheric bacteria were isolated from vegetables and potato. Antibacterial properties were evaluated using chloroform vapor method. Also, 12 bacteria were isolated from rhizosphere of vegetables and potato by minimal medium to evaluate the quorum quenching (QQ) ability. Chromobacterium violaceum CV026 biosensor was used to assess QQ activity in bacteria. There were two types of bio-control tests: the first case was the application of bacteria individually and the second case was the mix of bacteria with different bio-control mechanisms, antibacterial property and QQ activity, and its application against two strains of Pcc including PccK and PccM. At first, before doing the second bio-control test, interaction test between bio-control bacteria was performed by culturing two bacteria perpendicular to each other on solid medium. Bio-control test of Pcc on plant tissues (potato tubers, carrots and bell peppers) was performed by calculating the weight of rotting tissue in comparison with the control (containing Pcc alone). Classical and molecular bacteriological tests were performed to detect these bacteria.

Among rhizospheric bacteria with antibacterial properties, A10 isolate had a large inhibitory halo, 16.67 mm, against one strain of Pcc (PccK) and it had the ability to bio-control of PccK on vegetables and potato tubers. Among bacteria, only SS5 isolate showed QQ ability in both biosensor and bio-control tests. A10 and SS5 were detected as Pantoea and Pseudomonas sp. respectively. Pseudomonas SS5 had moderate bio-control activity against two Pcc strains with QQ mechanism as inhibition percentage on potato tubers rot for PccM and PccK were 53.92 % and 68 %, respectively. Pantoea A10 with antibacterial activity which had low or high bio-control properties against Pcc strains as inhibition percentage on potato tubers rot for PccM and PccK were 5.81 % and 91.33 %, respectively. The bio-control effect of A10 and SS5 bacteria against Pcc strains on carrots and bell peppers was better than potatoes. The interaction of two bacteria on solid medium, SS5 and A10, was not visible as a transparent area at their intersection, so they did not show an inhibitory effect against each other. Therefore, it is possible to use combination of two bacteria, A10 and SS5, for bio-control tests. The results showed that the bio-control ability of these two bacteria in the mixed state was much higher than the single application of each bacterium. In mix of two bio-control bacteria, disease was further reduced at least for PccM, in other words, the bio-control property was increased. In the case of the combination of two bacteria against PccM, compared to SS5 and A10 application alone, it produced 2.21 and 4.53 times less rot tissue, respectively.

There are two main bio-control mechanisms in bio-control bacteria for Pcc rot reduction including bacteria with antibacterial properties and bacteria with QQ abilities. In this study, for the first time, it was found that two bacteria with different bio-control mechanisms, including Pantoea and Psudomonas sp., are able to control Pcc separately and in combination. For the first time, the improvement of the bio-control properties of these two bacteria in combination with each other was identified. It was also found that the effect of these different bio-control bacteria on the control of each Pcc isolate and even each host of vegetables or potato tuber could be different, so that one Pcc isolate was sometimes completely inhibited and the other Pcc isolate was not affected by the bio-control bacteria. Because QQ mechanism disrupts the basic common physiological pathways in all Pcc isolates. So, this mechanism in bio-control bacteria apparently has a more general effect than the antibacterial effect.

Bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis, Cmm, is one of the limiting diseases of tomato cultivation. Because of the chemical control problems, biological control methods are used to control this disease in the recent investigation. Basil (Ocimum basilicum L.) is an annual herbaceous plant from the Lamiaceae family, and various endophytic bacteria have been reported from this plant. Also, many studies have shown that endophytic bacteria have the ability to stimulate growth and control many plant diseases. Therefore, in recent research, it was tried for the first time to test endophytic bacteria isolated from basil plant against tomato bacterial canker.

In this research, 16 endophytic bacteria isolated from basil. They were tested for their antibacterial properties against tomato bacterial canker in laboratory conditions using chloroform vapor method. Also, the effect of these bacteria on increasing total phenol and growth factors, including fresh weight, dry weight and seedling height were investigated. In the next step, the best bacteria in terms of growth factors were used to control bacterial canker, on tomato seedlings, under greenhouse conditions. Next, the physiological changes related to total phenol, catalase and peroxidase were investigated. Finally, molecular identification was done by PCR using universal 16SrRNA gene-specific primers ,27f and 1492r, DNA sequencing and BlastN in NCBI Genebank.

None of the endophytic bacteria showed antibacterial properties against bacterial canker under laboratory conditions. ReA1 isolate showed the highest growth stimulation and total phenol increasing compared to other bacteria therefore, it was used against tomato bacterial canker and reduced the disease by nearly fifty percent. Also, the growth factors were increased compared to the infected and healthy controls after inoculation with ReA1. In addition, the amount of peroxidase, catalase and total phenol increased in plants treated with ReA1 isolate compared to the control. So that the amount of total phenol, peroxidase and catalase, respectively from the highest to the lowest, was related to the application of ReA1 and Cmm, ReA1 alone, Cmm alone and the control without bacteria. ReA1 isolate was detected as Enterobacter sp. by DNA sequencing results.

Recent research showed that Enterobacter sp. strain ReA1 isolated from a plant belonging to the Lamiaceae family (basil) was tested on tomato plants from the Solanaceae family, which was able to reduce tomato bacterial canker disease without direct antibacterial effect. The main possible effects of this bacterium are related to the phenomenon of induced resistance in the plant and the increase of defensive and antimicrobial compounds in the plant. This control effect on the different plant species belonging to Solanaceae, the possibility that this bacterium could be used as a biocontrol agent on many kinds of plants and against a wide range of diseases. It also stimulated the growth of the plant, which makes possible the simultaneous use of this bacterium as a fertilizer and biocontrol agent.

Erwinia amylovora, the cause of apple and pear fire blight disease, is one of the main factors limiting the production of these fruits in the world. Since only a limited number of copper compounds have a protective effect in controlling this disease, increasing attention has been paid to investigating the effect of plant essential oils on this pathogen.

The effect of different concentrations of carvacrol (the active ingredient of thyme, oregano, etc.) and trans-anethole (the active ingredient of fennel essential oil) on the growth of E. amylovora colony, was investigated in laboratory conditions, and their minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were determined. The inhibitory effect of MIC of these substances alone and the mixture of one-half of their MIC and MBC concentrations was investigated by disc method on nutrient agar medium on the growth of E. amylovora colony. The effect of the three best mixing ratios of these materials on the fruit tissue of the pear Spadona variety was evaluated on the severity of fire blight disease.

Carvacrol and trans-anethole both had antibacterial effects against E. amylovora, but the effect of carvacrol was greater than the other. Mixing carvacrol with trans-anethole increased its effectiveness. Among the three mixing ratios, 47 µg/ml of carvacrol + 141 µg/ml of trans-anethole caused the greatest reduction in disease severity in pear fruit tissue.

The use of a mixture of carvacrol and trans-anethole can be considered for the management of fire blight disease.

Microgreens are immature vegetables with green leaves that can be harvested and consumed shortly after the appearance of the first true leaves. Basically, the part that appears above the root is used as a fresh vegetable for salads. They are more nutritious than their adult plants. In this study, basil (Ocimum basilicum) seeds were used to produce microgreens. Pythium spp. such as Pythium ultimum is one of the diseases that limits the cultivation of many microgreens.

Ten endophytic bacteria were isolated from the roots of some adult plants of the Lamiaceae family (including basil, Phlomis aucheri and rosemary). Basil seeds were disinfected with 1% sodium hypochlorite for two minutes and then washed five times with distilled water and dried at room temperature. For each treatment, 20 seeds with three replications were placed in two layers of sterile filter paper inside petri dishes and transferred to an incubator with a temperature of 21 ° C and a humidity of 65 to 70%. In the initial pretest to select the best bacteria in terms of growth stimulus characteristics, three traits of germinated seeds percentage, seedling height and wet weight at a common bacterial concentration (5×107 cfu/ml) were evaluated. Antifungal activity against P. ultimum was performed in mix of PDA and NA medium. Bacterium was detected using 16S rRNA region primers: 27F and 1492r. Several growth factors and biochemical changes including total phenol, peroxidase and catalase were investigated.

None of the bacteria showed a direct antifungal effect against P. ultimum in the laboratory. Among the bacteria, isolate GF1 showed more effects of growth stimulation than other bacteria. All growth traits showed a significant increase in the level of 5% probability in both bacterial isolate GF1 concentrations compared to the control, so that the least observed changes in the increase of each growth factor was above 50%. The highest increase in both concentrations was related to seed length vigor and germination vigor. In some cases, a significant difference was observed between the two concentrations of GF1 isolate. None of the bacteria showed a direct antifungal effect against P. ultimum in the laboratory. Sequencing of 16S region of ribosomal genes showed that the bacterium is Rhizobium sp. . This bacterium (in two concentrations of 5×107 and 5×106) had a significant effect of improving the weight loss of microgreens, the disease severity and disease incidence caused by P. ultimum in comparison with the control. Biochemical analysis showed that total phenol, catalase and peroxidase were significantly increased in microgreens inoculated with this bacterium. The results related to changes in total phenol, catalase and peroxidase after 14 days and at the same time with microgreen harvest showed that the bacteria in both concentrations were able to increase the amount of total phenol and defense enzymes in the microgreens. The concentrations used by the bacteria showed a significant difference in all cases, but the low concentration was also able to increase the amount of total phenol and enzymes compared to the control. Changes in total phenol, catalase and peroxidase in almost all cases from highest to lowest included the following treatments: high concentration of bacteria and fungus, low concentration of bacteria and fungus, high concentration of bacteria, low concentration of bacteria, fungus alone and the control which did not contain any microbial treatment. However, in the case of catalase, the changes resulting from inoculation of the pathogenic fungus alone and the changes resulting from the use of bacteria at lower concentrations were almost equal and did not show a significant difference.

In this study, unlike many similar studies that aim at biological control. The initial screening of bacteria with useful potential in agriculture was not based on antifungal properties. Rather, the primary goal was to increase germination and microgreen growth factors. Since the goal of biological control is ultimately to increase efficiency of crop yield by disease control, it makes sense to prioritize growth-promoting characteristics that directly affect the efficiency. This study showed that the endophytic bacterium, Rhizobium sp. GF1, without direct antifungal effect using inducing resistance mechanism and increasing the plant's defense compounds is able to significantly control the disease. In this study, bacterium was obtained that have a dual useful ability in the fields of plant protection and vegetable growing.

Microgreens are young plants that have gained popularity among consumers due to their high nutritional value. In the present study, the shelf life and nutritional properties of beet microgreens subjected to silicon and nano-silica in the post-harvest period have been investigated. Silicon and nano-silica treatments were carried out at two levels (Control and 100 mg/L as foliar spraying), after which the microgreens were harvested and kept at 4°C for 12 days. The results showed that the activity of polyphenol oxidase enzyme increased with the increase of storage time and as a result the browning of the tissue also increased. Also, with increasing storage time, the percentage of weight loss, hydrogen peroxide, malondialdehyde and carotenoid increased and chlorophyll a, b and total, total phenol, flavonoid, antioxidant capacity and vitamin C decreased. The use of silicon and nano-silica treatment decreased the activity of polyphenol oxidase enzyme and browning of the microgreen tissue compared to the control. Application of silicon increased total chlorophyll (33.75%), total phenol (2.49%) and total antioxidant capacity (39.7%) of microgreens in the post-harvest period. Also, the use of nano-silica increased the amount of total phenol (5.73%), antioxidant capacity (55%) and vitamin C (38.77%) compared to the control. In general, the results of this research showed that the pre-harvest application of silicon and nano-silica improves the nutritional characteristics of beet microgreens in the post-harvest period.

Pseudomonas tolaasii is one of the most important pathogenic bacteria of the edible fungus (Agaricus bisporus) in Iran and the world. In this study, comparing the severity of pathogenicity of the isolates showed that the highest pathogenicity was related to isolate Pt 4. Antibacterial effect of 10 plant essential oils against isolate Pt 4 was measured by disk diffusion method and based on the diameter of the inhibitory halo, basil and thyme essential oils had the highest antibacterial effect, respectively. The results of chemical analysis of gas chromatography showed that methyl chavicol with 67.05% was the highest monoterpene compound of basil essential oil, which together with two well-known compounds of thyme including carvacrol and thymol showed a controlling effect against P. tolaasii. The results showed that the best effect in controlling the disease on the fungal cap at minimum inhibitory concentration (MIC) was for thymol, carvacrol and methyl chavicol, respectively, but at some concentrations higher than MIC, thymol and carvacrol caused symptoms of cap blackening. Methyl chavicol had no adverse effect against Agaricus bisporus in all concentrations used, despite having less effect on reducing pathogenicity. Therefore, the use of methyl chavicol combination can be a good option to prevent bacterial brown blotch disease in mushroom cultivation halls.

Fire blight, caused by Erwinia amylovora, is one of the important bacterial diseases of pome fruit trees. It causes the blight of different organs of the tree (blossoms, shoots, leaves, fruits, and limbs) and exudates' production. The main symptoms of fire blight infection are the burnt-like appearance of infected tissues, bacterial ooze released from the infected tissues, shepherd’s crook, wilting, and water-soaked appearance. Recently, it has been included in the top 10 plant pathogenic bacteria. This study aimed to collect, identify and characterize E. amylovora isolates from provinces of Semnan in Iran and determine their pathogenicity factor and the current situation of fire blight disease in this province.

In the present study, 68 strains isolated from pear, quince, and apple hosts were identified in Semnan Provinces. Phenotypic, nutritional, and biochemical tests were performed on strains. The molecular identification of isolates was conducted using specific primers A and B of the plasmid pEA29. A pathogenicity test of the bacterial isolates on the immature pear fruit of the Spadona pear cultivar was performed, and the most pathogenic bacterium isolate was selected for further evaluation. Pathogenicity test was carried out on immature fruits and blooms of eight pear cultivars. Pathogenicity characteristics such as amylovoran, biofilm, and siderophore were studied in most virulence isolates.

The isolates were gram and oxidase negative, catalase-positive, and anaerobic. The isolates were able to produce a hypersensitive response in tobacco and levan production, but none of them could produce fluorescent pigment on King B medium and growth at 39 °C. The results suggested that of 68 strains collected, 52 isolates belong to Erwinia amylovora bacteria. The virulent isolate (D 43) was collected from Dibaj city. All 52 isolates under study proliferated 1000 base pairs. These tests, along with sequencing of PCR products, proved they were Erwinia amylovora species. Studying pathogenicity factors showed an almost direct relationship between all pathogenicity factors (other than biofilm production) and the severity of disease symptoms on tissues. The most resistant blooms tissues to D 43 isolate were shown to be related to Dargazi and Spadona pear cultivars; Duchess and Chini Hesar pear cultivars had the most resistant immature fruits tissues to D 43. It is therefore concluded that the susceptibility tissues to isolates of Erwinia amylovora were different in various cultivars. This point could help control fire blight disease and programs of breeding cultivars in the future.

Quorum sensing (QS) is a process of cell to cell communication that allows bacteria to be aware about cell density. In this phenomenon bacteria communicate with each other through signaling molecules such as N-acylhomoserine lactones (AHLs). Also, virulence gene expression in many pathogenic bacteria like Pectobacterium is under control of QS. Some bacteria can degrade AHLs molecules by a process called anti quorum sensing or quorum quenching (QQ); therefore, QQ can be used in biocontrol of plant pathogenic bacteria. The main purpose of this research was to detect QQ genes in strong QQ-based biocontrol bacteria.

In this investigation, QQ bacteria were isolated from rhizosphere and phyllosphere of some agricultural and non-agricultural plants using minimum media containing AHL. QQ bacteria were identified using biosensors CV026 and VIR07. Mechanism of QQ action was determined by heating, proteinase treatment and filtering methods. Afterwards, in order to find acylase and lactonase genes in these bacteria, homology searches were performed using BLASTn and BLASTp in NCBI. Then, several primers were designed by several softwares including CLC main workbench 5.5, Primer Premier 6, Primer3, Oligo7 and primer BLAST.

The most abundant and strongest QQ isolates were shown enzymatic activity. Main twenty-seven isolates with high QQ activity were detected. Four genera and species including Pseudomonas chlororaphis, Pseudomonas putida, Acinetobacter sp. and Bacillus sp. were found as the bacteria with highest QQ and biocontrol ability against Pectobacterium. As a results of this study, in the Bacillus Aiia lactonase gene;in P. chlororaphis three acylase genes of pvdQ، quiP and hacB; in P.putida two acylase genes of  pvdQ and quiP  and in Acinetobacter one acylase genes were traced.

According to the results of this investigation, Pseudomonas genus was determined as the frequent QQ bacteria. Also, P. chlororaphis species with the strongest QQ activity, were divided in to two groups; the first isolates with two acylase genes (pvdQ and quiP) and the second isolates containing extra homologous acylase hacB. Isolates of the latter group with the most detected genes involved in QQ, were identified as the best QQ-based biocontrol bacteria against Pectobacterium.

One hundred seventhy seven quorum quenching bacteria were isolated from Solanaceae family using two methods of minimal medium containing synthetic acyl homoserine lactone signal 3-oxo-C6-HSL and sole common culture medium. Eleven isolates of bacteria, includingPseudomonas, Enterobacter, Acinetobacter and Bacillus were identified as bacteria with high quorum quenching ability, especially the genus Pseudomonas showed the highest quorum quenching activity. Investigating quorum quenching mechanisms among these bacteria revealed that enzymes and non-enzymatic small compounds with quorum quenching activity were involved in this process. However, most of the bacteria showing enzymatic activity were confirmed by HPLC. Results also showed that one isolate of Acinetobacter calcoaceticus obtained by the second method, had non-enzymatic quorum quenching activity. The quorum quenching bacteria mainly reduced the biofilm and pathogenicity of Pectobacterium carotovorum subsp. carotovorum on potato and cactus. Therefore, there was a direct relationship between increasing quorum quenching activity and biocontrol ability against thispathogen. However, there were some exceptions. In addition, some isolates of Pseudomonas as the best biocontrol agents were identified, which were effective against the pathogen based on quorum quenching.

Leaf spot symptoms on maize were observed in autumn 2011 in Shiraz (Fars province, Iran). In order to identify the causal agent, the leaves of infected plants were squashed in sterilized water, and cultured on EMB medium. Green colonies were observed after 2-3 days. Following purification, the biochemical tests including Gram, oxidase, O/F, hypersensitivity reaction on tobacco, culturing on YDC medium, production of indole, acetoin, citrate, production of acid from salicin, arbutin, melobiose, raffinose, sucrose and pathogenicity test on maize seedlings were performed. The 16S rRNA of a representative isolate was partially amplified and sequenced. Based on the disease symptoms, phenotypic and pathogenicity tests and sequencing data, the isolates were identified as Pantoea stewartii subsp. indologenes. This is the first report of the disease and the causal bacterium in Iran.