Real-time PCR early detection of Trichoderma treatments efficiency against cotton charcoal rot disease

Abstract

The charcoal rot (CRD) disease agent, the soil fungus Macrophomina phaseolina, survives by developing reproductive units (spores and pycnidia) and infects various host plants. In cotton plants, the disease can lead to dehydration and death of the host plant at late-season stages. Pima cv. cotton plants, the leading cotton crop in Israel, are particularly susceptible to this disease. Developing biological pesticides against soil diseases is at the forefront of scientific research globally and their importance is increasing due to the world's trend towards a reduction in the use of chemical fungicides. In this work, eight Trichoderma isolates were tested under laboratory conditions against M. phaseolina. Two T. longibrachiatum isolates (T7507 and T7407) and a T. asperellum isolate (P1) achieved promising results. The bioprotective properties of these isolates' secreted metabolites were evaluated in solid and liquid cultures. The T7407 strain was the most influential in the solid medium with 55% inhibition capacity; the P1 strain excelled in the liquid medium with 62% inhibition capacity. The three M. phaseolina isolates were then tested in seedlings (up to 42 days) against controls: non-infected plants, infected unshielded plants, and plants treated with non-influencing Trichoderma isolate (O.Y. 14707). The bio-shielding agents were added directly to the seeds with the sowing in this growth room pathogenicity assay. At the experiment's end, the T. longibrachiatum (T7407) treatment markedly improved the plants' wet weight (45%), height (32%) and phenological development (56%) compared to the non-influencing Trichoderma species control. Since the disease is commonly latent in sprouts, statistical differences in the plants' growth parameters are challenging to reach, as occurred here. Still, the real-time PCR tracking of the pathogen DNA inside the plants' roots revealed dramatic changes. The pathogen DNA dropped to near-zero levels in the T. longibrachiatum- and T. asperellum-treated plants. Interestingly, the O.Y. 14707 isolate, which showed no bioprotective properties in the lab tests or plants' growth indices, had a similar significant repression impact on the pathogen roots infection. The results of this work demonstrate the importance of the early molecular assessment of preventive treatment effectiveness against M. phaseolina before a full growing season evaluation.