Biological control of head rot and damping-off of sunflower using yeasts, Bacillus spp. and Trichoderma spp.

Abstract

The soil-borne fungi Sclerotinia sclerotiorum, Sclerotium rolfsii and Rhizoctonia solani are ubiquitous plant pathogens with a wide host range. They are among the most widespread pathogens, and cause destructive diseases of many crops, including sunflower (Helianthus annus L.), an economically important oilseed crop. Chemical fungicides are available for control of seedling damping-off caused by these pathogens, but there are environmental concerns and the possibility of developing tolerance in pathogen populations, which have led to a drastic reduction in their usage and increased the pressure to find alternative means of disease control. Additionally, there are no registered fungicides that effectively control Sclerotinia head rot of sunflower caused by S. sclerotiorum in South Africa. Successes in biological control (biocontrol) and plant growth promotion research have led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of three strains of Bacillus spp., one yeast and one commercial strain of Trichoderma spp., and their respective combinations, on Sclerotinia head rot on sunflower. An additional commercial strain of Trichoderma spp. was also evaluated for the control of damping-off. In vitro biological control and growth promotion studies were carried out under greenhouse conditions with the use of foliar spray treatment as the method of application for head rot, and seed and soil drench treatments for damping-off. In vitro screening was undertaken to select the best Bacillus and yeast isolates from 136 Bacillus spp. and 100 yeasts isolated from local wild sunflower heads. Dual-culture bioassays were undertaken and isolates were assessed for antagonism by examining the radial growth of S. sclerotiorum mycelium. A scale was used to group the isolates, based on their inhibition ability in order to select the best isolates to screen in vivo. Seventeen Bacillus isolates achieved a Class 3 rating (≥70% inhibition of pathogen mycelial growth), while only 4 yeast isolates achieved a Class 2 rating (41-69% inhibition). The isolates, along with T. atroviride strain 77 (T77), were further screened in vivo under greenhouse conditions for antagonistic activity against Sclerotinia head rot of sunflower cv. PAN7080 plants, when plants were at the R6 reproductive stage.

Disease incidence was recorded 14 days after inoculation with BCAs and S. sclerotiorum, and grain was harvested, dried and weighed 85-115 days after planting. A total of 20 yeast and Bacillus isolates were screened against S. sclerotiorum and 4 Bacillus isolates and 1 yeast isolate reduced disease incidence by ≥50%, compared to the disease control. Bacillus B16 resulted in complete disease suppression, followed by B24, B26 and T77, which reduced disease incidence to 12.5%. Seven of the 20 yeast and Bacillus isolates, along with T77, significantly improved grain yield. B16 resulted in the highest grain yield, followed by T77. The effect of inoculum concentration was evaluated for the best performing yeast and Bacillus spp. isolates. A concentration of 1 x 108 cells mL-1 for yeast Y79, and 1 x 109 cfu mL-1 for B16, B24 and B26 caused the greatest disease suppression and improvement in grain yield. In comparison to the Bacillus isolates, Y79 was the poorest performing biocontrol agent (BCA), reducing the incidence of head rot the least. In addition, it was not as effective at improving grain yield and failed to perform consistently between the first, second and third greenhouse screening. Sunflower heads treated with single and combined inoculations of T77, Y79 and B16, B24 and B26 exhibited improved grain yield. Combined inoculations of B16 + B26 and B26 + B24 provided over 10.0% increase in grain yield (12.8% and 15.5%, respectively) over the disease-free control. Y79, when inoculated in combination with B16 and B24, scored reduced disease incidences of 62.5% and 37.5% as well as improved grain yields of 15.8 g and 36.0 g, respectively. In vitro dual-culture assays carried out with T. asperellum strain kd (Tkd) showed effective antibiosis activity and marked mycoparasitism of S. sclerotiorum, R. solani and S. rolfsii, despite the BCA performing poorly according to the Bell rating scale in dual culture plates. Greenhouse trials were carried out in Speedling 24® trays, and Tkd was applied as a seed treatment alone and/or a monthly-bimonthly soil drench. Various other greenhouse trials were set up to evaluate the potential of Tkd to suppress damping-off of sunflower caused by the three pathogens, and several growth parameters were measured. Seed treatment in combination with a monthly or bimonthly soil drench significantly increased seedling, shoot, root and head dry weight, along with root area, when tested against all three pathogens- effectively reducing disease incidence. Reduced disease

incidence and enhanced seedling and plant growth were also achieved when Tkd was applied as a seed treatment alone, drench at planting alone, and drench at planting + bimonthly drench, but at lower levels. A number of methods were adapted from studies carried out in other parts of the world with the objective of finding a fast and reliable method of inducing sclerotia of S. sclerotiorum to germinate carpogenically and produce ascospores. However, none of the published techniques worked under the conditions tested. Only one method, adapted from a study conducted by a fellow South African researcher, resulted in stipe formation, but not in ascospore production. The failure of these published techniques to work under the local conditions may be attributed to the fungus having stringent requirements for environmental conditioning before it will sporulate carpogenically. It appears that these requirements vary with the geographic source of the sclerotia, and that effective conditioning parameters in one place may not work in other geographic locations. The results presented in this dissertation confirm the concept of biological control by Trichoderma spp. and Bacillus spp. as a viable disease control strategy to manage S. sclerotiorum of sunflower. Furthermore, this dissertation forms a basis for further Trichoderma-Bacillus-Yeast interaction studies to determine whether strains of these three organisms could be combined to enhance biocontrol and plant growth promotion.