Preharvest management of botrytis cinerea on leucospermum ‘ayoba sun’ flowers using bacterial isolates.

Abstract

Ornamental plants and fresh-cut flowers play a crucial role in the South African economy, with over 80% of fynbos exported to the USA, Europe, England, Japan, China, and other countries. However, ornamental plants are prone to leaf spots, poor budding and small-sized flower development at preharvest. This is a result of Botrytis blight disease caused by B. cinerea which presents an enormous challenge for the fresh-cut flower industry. One of the ornamental plants, Leucospermum ‘Ayoba Sun’ flower, a new fynbos cultivar recently bred for disease resistance, growth vigor and its bright attractive color is prone to Botrytis blight disease at preharvest affecting the consumer acceptability and market value at postharvest. Preharvest treatments such as fungicides are used to manage B. cinerea. However, fungicides such as benzimidazole have been effective to manage B. cinerea with some limitations that include fungicides resistance, residues, environmental effects, and health concerns in humans. Furthermore, biological control agents such as Bacillus brevis, Pseudomonas sp. and Bacillus subtilis have been reported to be effective against B. cinerea. Hence, there is a need to evaluate other control strategies since few chemical and biological control methods have been effective against B. cinerea on ornamentals. Therefore, the aim of this research was to evaluate the effect of bacterial biological control agents to manage B. cinerea on Leucospermum ‘Ayoba Sun’ at preharvest. A total of 96 bacterial isolates were isolated from leaves of Tetradenia riparia, Vernonia amygdalina, Plantago lanceolata L., Ipomoea batatas (L.) Poir, Cymbopogon marginatus, anthers of maize (Zea mays L.), flowers of Plantago lanceolata L. and heads of wheat Triticum aestivum L., and soil samples collected at different locations in KwaZulu-Natal where macadamia nut trees (Macadamia integrifolia, C. marginatus) were planted. During in vitro screening, the six best isolates were selected Triticum as the most promising antagonists against B. cinerea based on their inhibition zones (65% to 82%) and used in the in vitro secondary screening. Furthermore, these bacterial isolates suppressed the mycelial growth of B. cinerea by shrinking, deformation, cutting, and bending of the pathogen hyphae when observed under the scanning electron microscope (SEM). Moreover, all the isolates were identified as Bacillus amyloliquefaciens strains when their sequences were subjected to Blast analyses. The potential of the commercial product (CP) Double Nickel 55, a preventative bacterial inoculant with B. amyloliquefaciens (D747) and B. amyloliquefaciens strains A5 and SWL were evaluated on Leucospermum ‘Ayoba Sun’ leaves and buds under greenhouse conditions. The concentration of B. amyloliquefaciens SWL and B. amyloliquefaciens A5 suspension was adjusted to 1×108 cells/ml and the concentration of B. cinerea was adjusted to 1×105 conidia/ml using a hemocytometer. Double Nickel 55 was applied at 5×1010 CFU per gram. The leaves treated with B. amyloliquefaciens A5 had the lowest disease severity (13.2%), followed by B. amyloliquefaciens SWL (19.8%) and Double Nickel 55 CP (22.67%) compared to the control inoculated with the pathogen-only (76%). Double Nickel 55 (CP) treated leaves had a percentage disease incidence of 25.18%, B. amyloliquefaciens A5 (10.44%), B. amyloliquefaciens SWL (21.57%) and control (69.39%). The effect Double Nickel 55 (CP), B. amyloliquefaciens A5 and B. amyloliquefaciens SWL on Leucospermum ‘Ayoba Sun’ plant quality was scored 6, 3 and 5, respectively, compared to the control with a score of 0. Moreover, Double Nickel 55 (CP) treated buds had the lowest disease severity (3,3%) followed by B. amyloliquefaciens A5 (21,3%), B. amyloliquefaciens SWL (50%) and control (70%). Hence, B. amyloliquefaciens A5 treatment had the lowest disease severity and percentage disease incidence on the leaves and the least effect on plant phytotoxicity while Double Nickel 55 resulted in the lowest disease severity on the buds. Leucospermum ‘Ayoba Sun’ leaves treated with Double Nickel 55, B. amyloliquefaciens A5, B. amyloliquefaciens SWL and water (control) were collected at 7, 14, and 35 days post-inoculation to determine the effect of B. cinerea on peroxidase activity (POD), total phenolic content (TPC) and total flavonoids (TFC). On day 35, the effect of Double Nickel 55 (CP) on TFC of Leucospermum ‘Ayoba Sun’ was 28.56 GAE/g dry matter, B. amyloliquefaciens A5 (13.56 GAE/g dry matter), B. amyloliquefaciens SWL (24.87 GAE/g dry matter) and control (26.63 GAE/g dry matter). The effect of Double Nickel 55 (CP) on TPC in Leucospermum ‘Ayoba Sun’ was 407 GAE/g dry matter, B. amyloliquefaciens A5 (410 GAE/g dry matter), B. amyloliquefaciens SWL (395.3 GAE/g dry matter) and control (441 GAE/g dry matter). The effect of Double Nickel 55 (CP) on POD in Leucospermum ‘Ayoba Sun’ was 0.2 U/min/g dry matter, B. amyloliquefaciens A5 (0.23 U/min/g dry matter), B. amyloliquefaciens SWL (0.26 U/min/g dry matter) and control (0.38 U/min/g dry matter). Therefore, at 35 days post-inoculation the control and Double Nickel 55 treatments exhibited the highest TFC of 26,03 GAE/g dry matter and 28,56 GAE/g dry matter, respectively. The control (441 GAE/g dry matter) and B. amyloliquefaciens A5 (410 GAE/g dry matter) had the highest TPC. Moreover, the control (0,38 U/min/g dry matter) and B. amyloliquefaciens SWL (0,26 U/min/g dry matter) had the highest POD enzyme activity on day 35 compared to other treatments. Therefore, B. amyloliquefaciens A5 resulted in the lowest disease severity and disease incidence, high TPC, low TFC and POD at 35 days post-inoculation. While B. amyloliquefaciens, SWL resulted in lower disease severity and disease incidence but high POD, low TPC and TPC. Hence, the disease incidence and disease severity are indirectly proportional to all plant enzyme and antioxidant activity. B. amyloliquefaciens A5 and B. amyloliquefaciens SWL enhanced TPC and POD in Leucospermum ‘Ayoba Sun’ leaves, respectively. Moreover, an increase in TPC and POD content in Leucospermum ‘Ayoba Sun’ plants reduces the spread of B. cinerea. The findings suggest that B. amyloliquefaciens A5 has the potential to be used as a biological control agent against B. cinerea infecting the leaves and the buds of Leucospermum ‘Ayoba Sun’.