فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 47 (پاییز 1402)

Toxic heavy metal contamination of industrial water is a significant universal problem. They accumulate in living tissues throughout the food chain which has humans at its top. These toxic metals can cause accumulative poisoning, cancer, and brain damage. Uranium is one of the most serious heavy metals because of its high toxicity and radioactivity. Excessive amounts of uranium have found their way into the environment through the activities associated with the nuclear industry (1). Conventional methods for removing uranium from wastewater include precipitation, evaporation, ion exchange, membrane processing, and adsorption. Nevertheless, these methods have several disadvantages, such as high installation and operating costs, the requirement of preliminary treatment steps, the difficulty of treating the subsequently generated solid waste, and low efficiency at low metal concentration (2,3). Owing to an increase in environmental awareness, there has been an emphasis on the development of new environmentally friendly ways to decontaminate waters using low-cost methods and materials. In this study, microbial biomass has emerged as a complementary, economic, and eco-friendly device for controlling the mobility and bioavailability of metal ions (2,4). The present work evaluates the performance of the Citrobacter freundii biomass to remove uranium ions from aqueous solutions. The effect of pH, temperature, initial concentration, and sorbent dose on biosorption capacity is also studied.

Materials: Citrobacter freundii bacteria used in this research with PTCC No. 1772 was purchased from the Scientific and Industrial Research Organization of Iran. Uranyl nitrate salt (UO2(NO3)2.6H2O) was obtained from the Research Institute of Nuclear Sciences and Technologies. Nutrient Broth culture medium, sulfuric acid, and sodium hydroxide and other materials used in this research were supplied from the Merck Company.Preparation of uranium solutions and biomass: A stock solution containing 1000 mg L-1 of U(VI) was prepared of UO2(NO3)4.6H2O. The working solutions were prepared daily from stock solutions. In this study, the biomass of Citrobacter freundii bacteria was heat treated in an autoclave at a temperature of 121°C for 15 minutes at a pressure of 1.5 atmospheres. Experimental design and batch biosorption studies: The design of the experiment was done using the response surface method by Design Expert software. Four variables, including initial uranium concentration (mg/l), temperature (°C), pH, and biosorbent dose (g/l), in five levels α-, -1, 0, +1, α+, 1 were used to design the experiment (Table 1). Therefore, 27 experiments were presented using a central composite design. The values of the variables and the obtained answers are shown in Table 2. Uranium biosorption experiments were performed by adding specified amounts of bacterial biomass in 20 ml Erlenmeyer flasks containing uranium solution with the concentration and pH corresponding to each experiment, with the specified temperature in the Shaker. After 90 minutes, each sample was centrifuged at 4500 rpm for 15 minutes at 4°C. Then, the remaining uranium in the solution was measured by ICP (Perkin Elmer/Optima 7300DV). The percentage of uranium removal (R) was calculated by equation 1:  Where C0 and Cf are the initial and the final concentrations of the metal ion solutions (mg/l), respectively.Table 1- Variables and Levels of the Central Composite Design Method

By using the RSM-CCD method, the optimization of the biosorption process was carried out. Table 2 shows the experimental results based on each point of the experimental design. Then, using analysis of variance (ANOVA), the obtained results were evaluated.The equation obtained for the biosorption efficiency of uranium by Citrobacter freundii is expressed as follows:Removal= +68.97045-1.43160 * C (ppm)+12.81296 * pH+1.08935 * T (0C)+2.89856 * M (g/l)+0.55737 * C (ppm) * pH+0.011459* C (ppm)* T (0C)+0.014961* C (ppm)* M (g/l)-0.32111 * pH * T (0C)-0.62783 * pH * M (g/l)-0.037633* T (0C) * M (g/l)-6.93488E-003* C (ppm)2-4.06361 * pH2Table 2- Values of Variables and Experimental Responses in the Response Surface Method.

The F-value and p-value of the proposed model are equal to 5.03 and 0.0027, respectively, reflecting the accuracy of the proposed model. This model with R2 equal to 0.81 shows that the proposed model can well predict the experimental values. The results showed that the factor of initial uranium concentration and pH statistically affect (p-value‹ 0.05) the uranium biosorption process. In contrast, temperature and sorbent dose factor (p-value› 0.05) have no statistically effect on uranium removal by Citrobacter freundii. With increasing uranium concentration from 10 mg/l to 77.5 mg/l, the removal increases from %66.5 to %99/92. Then, with increasing uranium concentration from 77.5 mg/l to 100 mg/l, the removal decreases to %97.34. On the other hand, one of the most important effective parameters in biosorption is the pH of the solution. With increasing the pH from 2 to 5, the removal decreased from %96.82 to %79.01 due to the formation of uranyl complexes (5). In this research, the results indicated that the pre-treated biomass under the conditions suggested by Design Expert software (19.84 g/l of biomass, temperature 28.92 OC, pH 2.89 and initial uranium concentration 53.71 mg/l) is able to remove approximately 99.99 percent of uranium from the contaminated area, which shows valuable potential Citrobacter freundii in bioremediation applications of uranium from contaminated wastewaters.

The escalating consumption of chemical fertilizers has imposed severe environmental and economic burdens on the society. Studies have shown the detrimental effects of these chemical agents on human health, necessitating a shift towards alternative fertilization practices. Research has focused on harnessing the potential of naturally occurring soil microorganisms as a sustainable and environmentally friendly solution to enhance plant growth. Biofertilizers, soil amendments enriched with beneficial microorganisms such as fungi, protozoa, actinomycetes, or algae, collectively known as plant growth-promoting bacteria (PGPB), represent a promising alternative to chemical fertilizers. Studies have shown that Bacillus-based biofertilizers can enhance nutrient uptake and regulate phytohormone metabolism within plants. Moreover, Bacillus strains exhibit remarkable resilience under environmental stress and secrete enzymes that disrupt the cell walls of pathogenic bacteria, viruses, fungi, and nematodes, thereby protecting plants from disease and promoting healthy growth and yield. In light of the aforementioned benefits, this study aims to isolate and molecularly identify native Bacillus strains with plant growth-promoting properties. The potential of these strains for incorporation into biofertilizers derived from Iranian native strains will be investigated.

In this study, rhizospheric and rhizoplane Bacillus strains from Iranian agricultural soils with Nitrogen-fixing ability were isolated and characterized for their plant growth-promoting properties. Strains with higher growth capacity were screened by 5% overnight culture in 100 ml NFB medium completed with 0.5% yeast extract. The strains were streaked onto Pikovskaya agar containing insoluble tricalcium phosphate. Quantitative analysis of phosphate solubilization was performed by culturing the strains in the mentioned liquid medium.  For the screening of (IAA) producer strains, the overnight culture of bacteria inoculated to a 250 ml Erlenmeyer-containing nutrient broth fortified with L-tryptophan. Then 2 ml of the supernatant was mixed with 2 ml Salkowski reagent. The optical absorbance was measured at 535 nm and the produced IAA was measured by standard curve graph. Production of ammonia was assayed by inoculation e of strains into peptone water. Nessler’s reagent was added to the medium. The development of yellow color showed the production of ammonia by the strain. The ability of HCN production was assayed by overnight culture of strain inoculated in nutrient agar slant fortified with glycine. The color change of the paper filter soaked in 0.5% picric acid and 2% sodium carbonate from yellow to brown indicated the production of HCN. Evaluation of antifungal activity against aspergillus niger, verticillium dahlia, and fusarium graminearum was performed on potato agar (MPA) plates, and calculated according to the following formula I= [(C-T)/C]*100. Quantitative measurement of ACC-deaminase activity of bacillus isolates was carried out using a modified NFB-ACC medium. siderophore production from cultural variables analyzed by CAS agar plate in reference to the change of color of CAS medium when microorganisms were grown in CAS agar plates. Phylogenetic relationships among the strains were established using 16S rDNA gene sequencing.  GraphPad Prism 6 software was used for statistical analysis. Data analysis was performed using one-way ANOVA and t-test and statistically significant was considered as p<0.05.

In the intricate realm of soil microbiology, the rhizosphere and rhizoplane stand out as vibrant microbial hotspots, teeming with life and playing pivotal roles in plant health and nutrition. The rhizosphere, the narrow zone of soil surrounding plant roots, is a dynamic environment enriched in root exudates, serving as a nutrient-rich haven for a diverse array of microorganisms. Within this intricate ecosystem, nitrogen-fixing bacteria emerge as crucial players, converting atmospheric nitrogen into a form usable by plants, thereby supporting their growth and productivity. Nitrogen-fixing growth-promoting bacteria isolated from the rhizosphere play a crucial role in regulating soil nitrogen availability for plants. Among the nitrogen-fixing bacteria that inhabit the rhizosphere, Bacillus strains have garnered attention for their exceptional ability to fix nitrogen and promote plant growth. These beneficial microbes possess a suite of plant growth-promoting traits, making them valuable assets for sustainable agricultural practices.

This study focused on isolating nitrogen-fixing bacteria. Over 80% of the isolated nitrogen-fixing bacilli were obtained from the rhizoplane, likely due to the high concentration of root exudates and microbial accumulation in this region. Despite previous studies indicating higher bacterial diversity in the rhizosphere, our findings suggest a distinct microbial niche within the rhizoplane supporting nitrogen-fixing bacteria. To select Bacillus strains with the highest plant growth-promoting potential, we screened them based on their growth ability, resulting in the selection of five promising strains. Phosphorus, a critical nutrient for plant growth, is often limiting. Bacillus strain S3 demonstrated the highest phosphate dissolution capacity (278.3 µg/mL). Bacilli secrete organic acids and produce siderophores that chelate iron bound to mineralized phosphates, making them accessible to plants. Moreover, studies have shown that bacilli can convert insoluble inorganic and organic phosphorus compounds into soluble forms via ACC deaminase enzyme production. Most strains exhibited ACC deaminase activity, enabling them to utilize 1-aminocyclopropane-1 as a nitrogen source. Siderophores, known for their biocontrol potential against plant pathogens, were produced by all strains except S3. Indole-3-acetic acid, a key plant hormone that promotes plant growth and germination, was produced in significant amounts by Bacillus strain S3. IAA production varies among species depending on nutrient availability. Bacillus indirectly enhances root growth by producing ammonia, a nitrogen source. Bacillus strains S3 and S5 exhibited the highest ammonia production, corroborating previous studies. Additionally, all strains demonstrated over 70% growth inhibition against fungal strains. Collectively, the obtained results underscore the promising potential of the native Bacillus strains isolated in this study for biofertilizer development. Their diverse plant growth-promoting traits, including nitrogen fixation, phosphate solubilization, ACC deaminase activity, siderophore production, IAA synthesis, ammonia production, and antifungal activity, make them valuable candidates for sustainable agricultural practices.

The simplest living things containing chlorophyll are algae, which use photosynthesis to create and release oxygen. The environment with the highest concentration of algae is water. These organisms contain vital chemicals and are rich in different types of proteins and carbohydrates. Their cultivation and purification have been developed as a result. It is feasible to research growing methods, habitats and the nutritional value of algae as well as to purify beneficial species for use in aquaculture by correctly identifying algae. One phylum of green algae is Dunaliella. The cells are biflagellate and motile. Blue-green algae are thought to be the earliest photoautotrophic animals. They are unicellular or filamentous creatures that contain phycocyanin and chlorophyll. Fish breeding ponds frequently use these kinds of creatures. Diatoms are eukaryotic microalgae that play a significant role in the ocean's food chain and can fix carbon dioxide. Diatoms are used by aquatic species as food as well. Additionally, they serve as unique sources of several fuels and oils. Because of their high sensitivity to salinity, acidity and pollution, they are referred to as biological indicators. One of Iran's oligotrophic lakes, Lake Urmia is situated in West Azerbaijan province. The population of Artemia has declined as a result of the rise in salinity of the water and the fall in Dunaliella algae. Thus, one of the main objectives of this study was to examine the variety of unicellular algae present in Lake Urmia

Lake water was sampled in the final month of each of the four seasons that were conducted. The pH and salinity were measured, simultaneously. Microalgae were tallied using a hemocytometer slide. Water samples from each station were collected independently and they were cultured in 250 ml flasks by Walne culture media for unicellular algae at two salinities of 30 and 100 grams per liter. There was a constant flow of aeration and 100 candelas of fluorescent light. Samples were detected using a light microscope after an algal bloom that lasted for roughly ten days. Two direct approaches were employed to purify the algae: streak culture (using agar culture medium) and inverted microscope (using Johnson's culture medium). Dunaliella species were identified molecularly using the ITS sequence and the 18srDNA gene. The CTAB method was also utilized for DNA extraction. A pair of specific primers was used to amplify the 18srDNA gene, containing the forward sequence 5'-cgg gat ccg tag tca tat gct tgt ctc-3' and the reverse sequence 5'-cgg aat tcc ttc tgc agg ttc acc-3'. Additionally, a pair of forward specific primers was used to amplify the region ITS, consisting of the sequence 5-aat cta tca ata acc aca ccg-3 and the sequence 5-ttt cat tcg cca tta cta agg-3. Amplification of the 18srDNA gene and ITS region requires PCR reagents, which are as follows: 4.2 µl of deionized distilled water, 1 µl buffer, 1.4 µl MgCl2, 0.8 µl dNTP and 0.2 µl primer for each reaction. Next, 0.2 µl of reverse primer, 0.2 µl of Tag DNA polymerase and 2 µl of sample-extracted DNA were taken into consideration and a total volume of 25 µl was used for the PCR process. The temperature cycle that was employed similarly consists of initial annealing at 95°C for one minute, final annealing at 95°C for one minute, primer binding at 52°C for one minute, new strand production at 72°C for two minutes and twenty seconds and final extension at 72°C for four minutes. This cycle was repeated in 33 cycles. The final PCR product was detected by electrophoresis technique. Primer binding took place at 57°C for 50 seconds, first annealing took place at 95°C for 5 minutes, final annealing took place at 94°C for 1 minute and 72°C It was utilized for one minute to create a new strand and for 10 minutes at 72 degrees Celsius to complete the 35 cycles of final strand expansion. Ultimately, a comparison was made between the sequencing results and the sequences found in the GenBank gene database.

Comparing freshwater and ocean waters to Lake Urmia, the latter two have more diversity and abundance of unicellular algae due to their oligotrophic nature. Identification of microalgae by morphological and physiological traits is insufficiently precise. Seven species of the Dunaliella genus are identified by a study based on the morphological traits of Dunaliella isolated from eleven distinct areas of Urmia Lake. Morphological analyses revealed that D. salina was the predominant species, with D. acidophilia and D. parva species isolated exclusively in the Shahid Kalantari transit area. D. salina and D. tertiolecta were the species found in the Hydar-Abad region. Due to the region's declining rainfall, retreating sea level and rising salinity, a study of Dunaliella species in the area reveals a low species diversity. The algae species isolated from Golmankhane port are D. bardawil, D. salina and D. tertiolecta. Research revealed that the Agh-Gol region is home to the isolated species D. bardawill, D. salina and D. tertiolecta. Two species of Dunaliella were isolated in the Zanbil mountain area. D. primulecta, D. salina and more species found in the Bari region. In the vicinity of Shahid Kalantari, six species of Dunaliella were isolated: D. salina, D. tertiolecta, D. bardawil, D. parva, D. ruineniana and D. acidophilia.

The findings indicated that the Shahid Kalantari neighborhood of Dunaliella has the highest level of diversity. Algal diversity was higher in the spring than it was in the summer. The drop in diversity during the summer may have been brought on by rising temperatures, which also increased salinity and exacerbated the drought. The links between several strains of this species of algae have been better understood through the application of molecular techniques. It is conclusively known that at least four species of D. salina, D. tertiolecta, D. viridis and D. bardawil, as well as four new strains, have been identified in Lake Urmia by concurrent investigations of morphological and molecular traits.

Pathogenic microorganisms are one of the important factors in contaminating and endangering the safety of foods for consumers, and continuous control of foods in terms of the presence of pathogenic microorganisms can guarantee the health of foods. Yersinia entrocolitica is a foodborne pathogenic bacterium. Raw milk and fresh traditional cheese may be contaminated with this bacterium. Generally, Iranians tend to consume traditional and homemade foods more than industrial products. This issue is especially relevant to milk products such as cheese, butter, and local cream. The purpose of this study was to determine the contamination of raw milk and traditional cheeses produced in Chaharmahal and Bakhtiari province with Yersinia entrocolotica.

A total, of 100 raw milk and 50 traditional cheese samples were obtained randomly from dairy plants and milk collection stations, as well as retail shops. The initial enrichment of the samples was performed in the TSB medium. Then, 100 microliters of the medium was centrifuged and 25 microliters of its sediment was cultured on the special medium of Cefsoludin Irgazan Novobiocin agar (CIN) with supplement (containing Cefsoludin 7.5 mg, Irgasan 0.2 mg and Novobiocin 1.25 mg). Suspicious colonies (red colonies) were identified on the CIN agar and biochemical tests including IMViC, Ureas, and H2S production.DNA extraction was performed by boiling method and polymerase chain reaction (PCR) was performed for the identification of Ali and Yst genes using specific primers (Table 1). Table 1. Characteristics of Primers Used to Identify Yersinia Enterocolitica Genes Antibiotic resistance test was performed by the disk diffusion method on Muller-Hinton agar according to the Clinical and Laboratory Standards Institute (CLSI). Resistance of Yersinia enterocolitica isolates to 7 commonly used antibiotics in treatment including cefixime (5µg), gentamicin (10µg), tryptoprim (5µg), ciprofloxacin (5µg), amoxicillin (10µg), tetracycline (30µg), and sulfamethoxazole (300µg).

Out of 100 samples of raw milk and 50 samples of traditional cheese, 20 samples (13.3%) suspected Yersinia enterocolitica bacteria were isolated. of these, 14 samples (14%) were related to raw milk samples and 6 samples (12%) were related to traditional cheese samples. The results of the PCR test showed that 5 isolates (3.3%) were Y. enterocolitica bacteria, 4 isolates (4%) related to raw milk, and 1 isolate (2%) related to traditional cheese. In addition, in the evaluation of virulence genes, it was found that out of 4 raw milk isolates, 1 isolate carried the Yst gene, 2 isolates carried the Ail gene, and 1 isolate had both genes. Also, 1 isolate of Y. enterocolitica isolated from traditional cheeses carried the Ail gene (Table 2). Table 2. Percentage of Contamination of Raw Milk and Traditional Cheese with Yersinia entrocolitica, (%) The results of antibiogram tests on Yersinia enterocolitica isolates showed the highest resistan.ce to Gentamicin, Trimethoprim, Ciprofloxacin, and Sulfamethoxazole antibiotics, and moderate resistance to cefixime and amoxicillin (Table 3).  Table 3. Antibiotic Resistance Pattern of Yersinia Entrocolitica Isolated from Raw Milk and Traditional Cheese.

In general, the results of the present study and other studies in Iran and the world show that raw milk and traditional cheeses, contaminated with Yersinia enterocolitica, which carry virulence genes and are resistant to some common antibiotics, can endanger the health of consumers.

The genus Xanthomonas belongs to the Xanthomonadaceae family and the Gammaproteobacteria class, which includes short rod-shaped gram-negative bacteria that cause disease in more than 400 different plant hosts and are the most important pathogen in a wide range of plants, which results in a decrease in productivity in the agricultural industry.Pathogenic bacteria in plants including Xanthomonas campestris pv. campestris are one of the most important factors that reduce the productivity of agricultural products and are responsible for major economic losses in the agricultural industry. This bacterial strain is pathogenic in a wide range of plants such as rice, wheat, citrus fruits, tomatoes, peppers, cabbage, melons, bananas, and seeds such as beans. The methods of dealing with the disease that has been used against them so far involve environmental damage, the accumulation of toxins in the soil, and bacterial resistance. Therefore, it seems that more effective methods are needed. Bacteriophages, Viruses infect bacteria, which are exclusive to their host attack, and as new, safe, and effective inhibitory agents against plant pathogenic bacteria, they have been much considered in recent studies.In the studies conducted on isolated phages effective against Xanthomonas bacteria, promising results have been obtained in the field of plant disease management in laboratory and field conditions. In this regard, the identification and complete knowledge of the biological characteristics of phages effective against this bacterium is very crucial for the development of effective biological control products.

In the present study, isolation and identification of lytic bacteriophage effective on strains of Xanthomonas campestris bacteria were investigated to inhibit two forms of planktonic and biofilm bacteria so that it can be used in the future to fight the contamination of agricultural products with this pathogenic bacterium. Under sterile conditions and with a sterile loop, a single plaque was completely removed from the selective plate containing phage plaques and autoclaved in 5 ml of YMB medium and 10 μl of fresh suspension of the bacterial strain X. campestris DSM 1706 was added and incubated for 24 hours in the incubator was incubated at a temperature of 28 degrees Celsius and aeration at 150 rpm. Then the solution was centrifuged for 10 minutes at a speed of 6000 rpm and the supernatant was filtered with a 0.45 micron needle filter.Lytic phage is effective on the strain Xanthomonas campestris pv. campestris DSM1706. It was separated from the water of the Karun River by the two-layer agar method. The phage host range to the staining method was checked. Then, phage morphology through transmission electron microscopy (TEM) imaging, phage stability against pH and different temperatures, one-step proliferation curve, and the effect of phage on removing and inhibiting bacterial biofilm were investigated.

In this study, clear phage plaques, caused by the proliferation of lysing phages of the desired strain, were observed. Phage isolated on the morphological characteristic was based on the tectonic family. In addition to the strain Xanthomonas campestris pv. campestris DSM 1706, it had a lytic effect on three other pathogenic isolates obtained from infected cabbage fields including SAM 4209, SAM 4211, and SAM 4212. This phage had high stability in the temperature range of 20 °C to 50 °C and was inactivated at -70 °C. It also had good lytic activity in the range of pH 5 to 10 and became inactive at pH 3. According to the one-step proliferation curve, the phage proliferation cycle lasts about 70 minutes which includes a 25–30-minute incubation phase and a 40-minute phage release phase. Phage showed an 86% inhibitory effect and 93% elimination effect on the biofilm of this bacterium.In the present study, phage stability against different temperatures and acidity was studied as two main factors. The results showed that the phage has good lytic activity in acidity between 5 and 9, its performance decreases in alkaline acidity more than 9 and it becomes inactive in acidity 3 and does not show any lytic activity against the target bacteria. The best performance and the highest lytic activity were observed at an acidity of about 7.

Since the first discovery of phages in this study, it has been proven that phages as a biological control strategy are promising alternatives with many advantages for agricultural chemicals and antibiotics. Based on the results of this research, the obtained lytic phage has stability in the spectrum in laboratory conditions (a wide range of pH and temperatures) and was able to lyse the host bacteria in planktonic cell form and inhibit and remove the bacterial biofilm with a high percentage. As a result, it can be a biological agent with high potential in controlling plant diseases and replacing combat methods as a common and suitable candidate for phage therapy.In several studies in this field, it has been shown that the use of different compounds mixed with solutions containing phage increases the persistence of phage on leaf surfaces. For example, in one study, the use of skim milk and this combination led to improved control of tomato bacterial spot disease compared to standard treatment with chemical bactericides.In line with this study, it is suggested to investigate the effect of phage on other pathogenic strains of Xanthomonas bacteria and also on their biofilm in different ways. Among other things, specific lytic bacteriophages of Xanthomonas bacteria can be used individually or in combination with other isolated bacteriophages along with antibiotics or other control agents such as copper compounds in preventive and therapeutic measures as phage therapy against bacterial infections.

Biological control is an efficient method in the management of plant diseases, which has a lower risk for human health and the environment. In this research, the biocontrol fungus Trichoderma harangue was used to control Fusarium wilt disease to lemon verbena with the scientific name Lippiacitridora Kunth (a medicinal plant from the Verbenaceae family). Fusarium wilt disease of lemon verbena is considered one of the most important plant diseases in the world and it reduces the quantity, quality, and nutritional value of the product. Symptoms of the disease first appear on the lower leaves and usually after the appearance of flowers and fruit formation. As a result of the activity of the fungus F. oxysporum, brown and blocked wood vessels and symptoms of yellowing and wilting similar to water deficiency are observed in the plant. Fusariumredolens species is mostly observed with root rot of different plant species in warm and temperate regions. Like F. oxysporum, this species has a special form that host specificity. Trichoderma spp. species are mainly soil-borne and exert biological control against plant pathogens indirectly through competition for food and space, changing environmental conditions, or promoting plant growth and plant defense mechanisms and antibiotics or they do it directly through mechanisms such as mycoparasitism.

Samples of pathogenic fungi were collected from the fields, isolated, purified and identified, and the sample of antagonistic fungus was prepared in pure from the fungi collection of the Mycology Laboratory of Faculty Agriculture, Lorestan University under the access code OQ702632 and cultivated on PDA culture medium.Molecular identification of fungal isolates: Genomic DNA extraction of the fungi was done using the Zhang and Stephenson method with some modifications. For this purpose, the mycelium mass was used of five to seven-day-old fungi. The PCR test was performed in a final volume of 25 microliters with the help of a pair of specific reverse primers related to the TEF-1α gene region and using the PCR Master Mix kit of the British PCR Bio System company and according to the company's instructions. In the negative control sample, sterile distilled water was added instead of DNA. Pathogenic interactions and biocontrol by Dual-culture method The biocontrol effect of Trichoderma on the Fusarium pathogen was carried out using the dual-culture method in 9 cm Petri plates containing PDA in the form of a completely randomized design and in three replications. In order to investigate the cause of Lemon verbena wilting and its biological control, samples were taken from Khorramabad county and 45 samples were transferred to the laboratory for isolation and identification. The antagonist isolate of T. harzianum against pathogenic fungi was used in laboratory conditions as dual-culture and the effect of volatile substances and in greenhouse conditions in the form of a completely randomized design and in three replications.Inhibitory effect of Trichoderma isolate on Fusarium wilt disease of lemon verbena under greenhouse conditions: The Mohammadi method was used in order to observe the effect of the antagonist fungus T. harzianum on the plant growth indicators of lemon verbena (height, fresh, and dry weight) in infected and non-infected conditions with the pathogenic fungus and in comparison with the infected and non-infected control, and to prepare the inoculum for Trichoderma isolates. After planting the lemon verbena and applying the treatments, the greenhouse was visited daily and the health and disease status of the plants were examined including yellowing, drying, and wilting of the plants. At the end of the experiment, 30 to 40 days after inoculation, plant parameters were recorded such as wet and dry weight and plant height.

Based on the morphological and molecular characteristics, Fusarium isolates were separated and classified into two species: F. oxysporum and F. redolens. In the laboratory, the isolation of T. harzianum inhibited the mycelium growth of F. oxysporum and F. redolens pathogens, but it had the most inhibitory effect on the pathogenic species of F. oxysporum, and in cross-culture methods and the effect of volatile compounds, respectively. It controlled this pathogen by 71.5 and 61.5%. While the control of F. redolens was 56.5% and 50.5% in the two methods. The results of greenhouse experiments showed that T. harzianum biocontrol, in addition to increasing the growth factors of the plant, reduced the disease indicators and controlled the disease. The effect of biological agent T. harzianum on the severity of F. oxysporum after 30 days was equal to 73.86% and in F. redolens was 74.16%. In terms of growth factors, T. harzianum treatment had the highest and F. redolens treatment had the lowest growth indicators.
Discussion of .

Both species of Fusariumoxysporum and F. redolens were used in the test to prove pathogenicity in the greenhouse by impregnating the roots of the seedlings in the spore suspension of the fungi causing the disease, and both caused damage and were pathogenic. In the macroscopic examination of Trichoderma isolates and Fusarium species using dual culture, it was observed that T. harzianum isolates limited the growth of both pathogenic fungi F. oxysporum and F. redolens. The highest percentage of growth inhibition was 71.5% in the simultaneous confrontation of T. harzianum isolates with pathogenic agent F.oxysporum and the lowest growth inhibition percentage of 56.5 was observed in the simultaneous confrontation of T. harzianum isolate with the pathogenic agent F. redolens. It was found that the biocontrol fungus T. harzianum controlled the pathogenic fungus F. oxysporum better than F. redolens and further hindered its growth. In the present study, it was found that adding T. harzianum isolates to the soil around seedlings in greenhouse conditions increases the growth indicators (height, fresh weight, and dry weight of shoots and roots) and also reduces the severity of the disease and symptoms of yellowing and wilting in lemon verbena seedlings. It is infected with F. oxysporum and F. redolens.