التكامل بين المضادات الحيوية Gliotoxin و Trichodermin والعزلات المنتجة لها من الفطر Trichoderma spp في تصنيع مبيد حيوي ضد مسببات مرض تعفَن البذور وموت بادرات القطن

Integration of the Antibiotics Gliotoxin and Trichodermin and their produced isolates of Trichoderma spp in the manufacture of a biological preparation against the pathogens of seed rot and the damping-off disease of cotton seedlings

This study was conducted in the laboratories and fields of the College of Agriculture, Karbala University, during the agricultural season 2021-2022. With the aim of manufacturing a biocidal from different isolates of the fungus Trichoderma spp. supplemented with microsclerotia and the mycotoxins produced from them, to combat pathogens, including the pathogens of seeds decay and damping-off disease of cotton. Ten isolates of the fungus Trichoderma spp, diagnosed in previous studies, were tested, four of which were selected for biocidal manufacturing after testing their antagonistic ability and production of mycotoxins, and testing the possibility of overlap between them.
The biocide, which was called (FOUR.T) was synthesized by a compatibility synthesis of four isolates of the biological fungi T.viride, T.pseudokoningii, T.reesei, and T.koningiopsis after proving their ability to produce stone bodies (Micro sclerotia) on the medium. The liquid, and its production of mycotoxins (Trichodermin and Gliotoxin), and the fortification of the biocidal with them, to increase the effectiveness of the antagonistic pesticide against pathogens, and its biological efficiency under the conditions of storage and drought.
While a number of fungi associated with infected cotton seeds and seedlings were isolated in four different areas that were cultivated in Karbala governorate, the two fungi, Fusarium spp, and Rhizoctonia spp achieved the highest incidence of 75% for each of them, followed by the appearance of Pythium spp. and Alternaria sp. and Verticillum sp. The results of the pathogenicity test showed the virulence of Fusarium brachygibbosum, Fusarium solani and Alternaria alternata in attacking cotton seeds and seedlings and reducing the percentage of germination and seedling growth. It achieved a significant significant effect in reducing the germination rate, which amounted to 0.0% and an inhibition rate of 100%.
Fungal isolates isolated from moldy cottonseeds and infected seedlings were phenotypically diagnosed based on microscopic characteristics, and the most virulent isolates were molecularly identified by analyzing the DNA base sequences of the ITS region after they were sent to the South Korean Macrogen Company for the purpose of determining the nucleotide sequence. The deposit number of these isolates was in the gene bank (ON738702), (ON738704), (ON738701) for isolates F. brachygibbosum, F. solani, and A.alternata respectively, and comparing each isolate with globally identified isolates in the gene bank.
The treatment of cotton seeds with isolates of the fungus Trichoderma spp. It has proven highly efficient in reducing infection rates and increasing cotton seedling germination rates. It also showed high efficiency against the pathogens F.brachygibbosum, F.solani, and A.alternata, where the percentage of inhibition ranged (40.62-94.44%), and the highest antagonistic ability reached 94.44% for the isolate T.pseudokoningii against the pathogenic fungus F.solani. The results of extracting toxins from the filters of Trichoderma spp. isolates used in the study revealed the presence of the mycotoxins Trichodermin and Gliotoxin in large quantities, and these toxins had a great role in inhibiting the growth of pathogenic fungi in the laboratory, where the percentage of inhibition ranged (69.44-86.11), with the highest percentage of inhibition reaching 86.11% T.viride isolate against the pathogenic fungus A.alternata.
While the results of field experiments showed the role of isolates of the fungus Trichoderma spp. The biocide (FOUR.T) in controlling pathogens showed a significant increase in the percentage of germination of cotton seeds and a decrease in the percentage of seed rot and seedling death compared to the comparison treatment of pathogenic fungi. The highest percentage of germination was 100%, the percentage of inhibition was 0.00%, and the percentage of infection was 3.33%. When using the biocide (FOUR.T) prepared from isolates (T. viride, T.pseudokoningii, T.koningiopsis and T. reesei) against pathogens.
The isolates of the fungus Trichoderma spp. And the biocide (FOUR.T) significantly in plant growth parameters, where the results showed an increase in the fresh and dry weight of the vegetative and root masses of cotton seedlings, as the fresh weight ranged between (3-6 g), compared to the treatment of isolates F. brachygibbosum, F. solani, A. alternata which was 0.5, 1 and 1gm, respectively.
The results of induction of systemic resistance showed that isolates of Trichoderma spp, including the biocidal combination (FOUR.T) had a significant effect in inducing systemic resistance in plants in increasing the activity of peroxidase enzyme, polyphenol oxidase and phenols and an increase in the value of total chlorophyll, which urged plants to develop systemic resistance. Against the pathogens F.brachygibbosum F.solani and A.alternate, the value of chlorophyll ranged (0.72-0.86) units/gm fresh weight, and the activity of peroxide enzyme ranged 0.390-0.528 (units/gm fresh weight), and the value of phenols ranged 0.32-0.46. (mg/gm fresh weight), the highest activity of peroxide enzyme was 0.528 in the treatment of the biological preparation, as well as for chlorophyll and phenols, and it was 0.86, 0.46, respectively, while the activity of polyphenol oxidase enzyme ranged (1.29-1.65), and the highest effectiveness was in the treatment of isolate the fungus T. .viride, which reached 1.65 (units/gm fresh weight) against the pathogen F.brachygibbosum, and the treatment with isolate of the fungus T. reesei against the pathogen F.solani.