Inhibitory effect ofBacillus subtilisWL-2 and its IturinA lipopeptides againstPhytophthora infestans

Abstract

15Potato late blight triggered by Phytophthora infestans ((Mont.) de Bary) represents a 16 great food security threat worldwide and is difficult to control. Currently, Bacillus spp. 17 have been considered biocontrol agents to control many fungal diseases. Here, 18 Bacillus subtilis WL-2 was selected as the antifungal strain with the most potential 19 against P. infestans mycelium growth. Additionally, the functional metabolites 20 extracted from WL-2 were identified as IturinA-family cyclic lipopeptides (CLPs) via 21 2 high-performance liquid chromatography (HPLC) and electrospray ionization mass 22 spectrometry (ESI-MS). Analyses using scanning and transmission electron 23 microscopy (SEM and TEM) revealed that IturinA caused a change in the mycelial 24 surface and damage to the internal cell structure, including cell membrane disruption 25 and irregular organelle formation. Moreover, propidium iodide staining and nucleic 26 acid and protein release were detected to clarify the cell membrane damage caused by 27 IturinA. Additionally, IturinA triggered reactive oxygen species (ROS) generation and 28 malondialdehyde (MDA) production. Mitochondrial membrane potential (MMP), 29 mitochondrial respiratory chain complexes activity (MRCCA), respiratory control rate 30 (RCR), and oxidative phosphorylation efficiency (P/O) assays indicated that P. 31 infestans mitochondria affected by IturinA were so seriously damaged that the MMP 32 and MRCCA declined remarkably and that mitochondrial ATP production ability was 33 weakened. Therefore, IturinA induces cell membrane damage, oxidative stress, and 34 dysfunction of mitochondria, resulting in P. infestans hyphal cell death. As such, the 35 results highlight that B. subtilis WL-2 and IturinA have great potential as candidates for 36 inhibiting P. infestans mycelium growth and controlling potato late blight. 37 IMPORTANCE 38Potato (Solanum tuberosum L.) is the fourth most common global food crop, and 39 its planting area and yield increase yearly. Notably, in 2015, China initiated a potato 40 staple food conversion strategy, and by 2020, approximately 50% of potatoes will 41 be consumed as a staple food. The plant pathogen fungus Phytophthora infestans 42 ((Mont.) de Bary) is the culprit of potato late blight; however, biological agents 43 3 rather than chemicals are highly necessary to control this threatening disease. In 44 this study, we discovered an antifungal substance, IturinA, a lipopeptide produced 45 by Bacillus subtilis WL-2. Moreover, our research revealed the actual mechanism 46 of IturinA against P. infestans mycelium growth and clarified the potential of B. 65Behind rice, wheat, and corn, potato (Solanum tuberosum L.) is the fourth most 66 stable food crop in the world. However, potato production is often endangered by 67 many pathogens. Worryingly, late blight triggered by Phytophthora infestans ((Mont.) 68 de Bary) could directly reduce or even eliminate potato production, and an outbreak 69 of this disease could even result in a grievous ...

Bacillus megaterium WL-3 Lipopeptides Collaborate Against Phytophthora infestans to Control Potato Late Blight and Promote Potato Plant Growth

“…de Bary] W101 was obtained from China General Microbiological Culture Collection Center (CGMCC 3.19919) and cultured on Rye (R) solid medium at 20°C ( Wang et al, 2017 ). B. megaterium WL-3 (MK241789) was isolated from Capsicum frutescens leaves and cultured on Luria Bertani (LB) solid medium at 35°C ( Wang, 2018 ). P. infestans mycelium disk (diameter = 7 mm) was applied to the R solid medium (diameter = 9 cm) and cultured for 3 days, at which time the prepared B. megaterium WL-3 Living cell (LC), Cell suspension (CS, 1 × 10 7 CFU/mL, 100 μL), and Cell-free supernatant (CFS, 100 μL) were evaluated for their inhibitory effect using the plate dual culture method ( Kunova et al, 2016 ).…”

“…Phytophthora infestans mycelium was collected and oscillated to expose sporangium (1 × 10 7 CFU/mL), and zoospores (1 × 10 7 CFU/mL) were collected after release at 10°C for 3 h ( Wang, 2018 ). The sensitive variety of potato, “Bintje” plant (60 days), was used to produce tubers (2.0 cm × 2.0 cm × 0.5 cm) and leaflets for in vitro experiments ( Jiang et al, 2017 ).…”

“…de Bary], is the most destructive and intractable disease ( Schepers et al, 2018 ). At present, except for a selection of disease-resistant varieties, potato crops are predominantly treated with chemicals in order to control the development of late blight ( Caulier et al, 2018 ; Wang, 2018 ). The excessive use of chemical pesticides has resulted in increasingly serious environmental pollution and has accelerated the physiological differentiation and variation of P. infestans ( Jin et al, 2017 ; Fukue et al, 2018 ).…”

“…However, no studies as yet have determined or compared the individual contributions of particular CLPs such as surfactins, fengycins, and iturins against the oomycete P. infestans ( Gong et al, 2015 ). In addition, synergism between CLPs to control potato late blight is still unknown ( Wang, 2018 ; Zhang and Sun, 2018 ), and it is also not known how changes in environmental factors, such as temperature, humidity, and microorganism interference might affect the antagonistic effects ( Ongena et al, 2005 ; Parthipan et al, 2017 ). Therefore, it is worth investigating CLPs for their biocontrol effect against late blight in field trials.…”

“…Therefore, it is worth investigating CLPs for their biocontrol effect against late blight in field trials. Some reports have suggested that CLPs induce resistance in plants as their antagonistic mechanism against plant pathogens ( Wang, 2018 ). In addition, the intrinsic promotion of potato plant growth by CLPs is also a worthwhile aspect of protection to investigate ( Perez et al, 2017 ).…”