Morphological and Molecular Investigation of Fusarium Wilt of Lemon Verbena and its Biological Control by Trichoderma Harzianum in Khorramabad County

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.