Cyclic lipopeptide diversity and biocontrol versatility of Pseudomonas species associated with the cocoyam rhizosphere

Abstract

Fluorescent Pseudomonas bacteria are commonly found in the rhizosphere of plants and produce a broad spectrum of antibiotics and biosurfactants including cyclic lipopeptides (CLPs). CLPs are beneficial to the producing strain in biofilm formation, swarming motility, and antibiosis. The role of CLPs in the biological control of plant pathogens has been attributed to their ability to interact with the cell membrane causing structural distortion, cell lysis and abnormal growth, thereby leading to death of target cells. Cocoyam is an important food crop for more than 400 million people worldwide. Cocoyam genotypes include red, white and yellow varieties. Cocoyam yield is often affected by cocoyam root rot disease (CRRD) caused by Pythium myriotylum. Studies have shown the capacity of suppressive soil of Boteva in Cameroon to curtail CRRD while conducive soils like that of Ado-Ekiti and Umudike in Nigeria only enhance disease severity. Myriads of biotic and abiotic factors including plant genotype, soil properties and plant age, affect the microbial diversity and activities in the rhizosphere of plants. In this thesis, we investigated the drivers of fluorescent Pseudomonas and CLP diversity in the cocoyam rhizosphere in Cameroon and Nigeria. Fluorescent Pseudomonas isolates obtained from the rhizosphere of red and white cocoyam from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. Fifty percent of the isolates produced CLPs including xantholysin, WLIP, lokisin, entolysin, putisolvin, and eight novel CLPs, termed N1 to N8. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. These results indicate that plant genotype affects CLP diversity rather than taxonomic diversity. Consequently, we showed the effect of soil quality and cocoyam age on the taxonomic and CLP diversity of Pseudomonas strains isolated at two weeks, one month and two months timepoints, respectively, from the rhizosphere of tissue culture-derived white cocoyams planted in a soil suppressive to CRRD, at Boteva, Cameroon, and two soils which are conducive to CRRD, obtained from Ado-Ekiti and Umudike, Nigeria. Initial drop collapse test revealed that isolates recovered from the Boteva soil produce biosurfactants at two weeks, one month and two months timepoints, respectively; while isolates obtained from Ado-Ekiti and Umudike, produce biosurfactants at two weeks only. Strains from Ado-Ekiti and Umudike soils were affiliated with the P. fluorescens and P. putida groups, and produced only viscosinamide and putisolvin III. On the other hand, strains from Boteva soil were associated with the P. koreensis, P. fluorescens and Pseudomonas protegens groups, and produced viscosinamide, cocoyamide, bananamides, orfamide, sessilin, and novel CLPs. These novel CLPs were structurally characterized as new members of the amphisin group and termed botevamides. This study revealed that the higher the soil quality the more diverse the Pseudomonas and CLPs produced, and that age could also affect the discovery of specific CLPs such as viscosinamide, orfamide, novel botevamides and sessilin. Moreover, soil quality appeared to be linked to the presence of isolates belonging to the P. koreensis group and the ability to produce cocoyamide, independent of genotype and plant age. Furthermore, rice is a staple for about half of the world’s population. However, rice production is affected by several abiotic and biotic factors. Prominent among the biotic factors is rice blast mediated by Magnaporthe oryzae. There is therefore the need to search for potent CLPproducing Pseudomonas strains with potential to control CRRD and rice blast. In this thesis, we investigated the capacity of CLP-producing Pseudomonas strains and their crude CLP extracts to control rice blast caused by M. oryzae. In planta biocontrol assays showed that lokisin-, WLIP-, entolysin- and CLP N3-producing strains, and crude extracts of lokisin, WLIP and entolysin induced resistance to M. oryzae VT5M1. In contrast, a xantholysin-producing strain and crude extracts of CLP N3, xantholysin and orfamide did not display ISR against the rice blast disease. WLIP mutants’ analysis using rice pathosystem confirmed the role of WLIP in triggering ISR to rice blast. Crude extracts of CLP N3, lokisin, WLIP, entolysin and orfamide reduced blast severity on rice when mixed with M. oryzae before foliar inoculation. In vitro microscopic assays showed the capacity of all CLPs tested in this study to inhibit appressoria formation by M. oryzae (chapter 5). Using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR), we elucidated the structures of WLIP, xantholysin, lokisin, entolysin, putisolvin III, and novel CLP N1 (now termed cocoyamide, chapter 3), novel members of the amphisin group, designated botevamide A-E (chapter 4), and the novel bananamide-type CLP N3, now termed bananamide D-G (chapter 6). Moreover, Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamide D and E. Purified compounds of these CLPs displayed in vitro inhibition against the mycelia of P. myriotylum , N1 (cocoyamide), N2, N8 and lokisin caused hyphal leakage, while xantholysin, entolysin, WLIP and putisolvin III caused hyphal leakage and branching. Purified botevamide A-D (chapter 4) and bananamide D-G (chapter 6) caused extensive hyphal branching and distortions. Bananamide D-G also inhibited M. oryzae and caused hyphal branching. Bananamide D-G-producing strain COW3 displayed antibiosis and mycophagy against Pythium myriotylum, while it only showed mycophagy on M. oryzae (chapter 6). We elucidated the biosynthetic gene cluster of COW3, COR10 and NSE1, which encodes novel bananamide D-G, Lokisin and WLIP, respectively. Putting all together, this thesis showed that cocoyam genotype, soil quality and cocoyam age could affect Pseudomonas and CLPs diversity, but soil quality seems to be the major influencer of diversity in this thesis. KMD, Genome mining, LC-MS and NMR are complementary methods for the discovery and characterization of novel CLPs. Pseudomonas spp. and CLPs could control CRRD by direct antagonism; and rice blast by direct antagonism and/ or induced systemic resistance. Structural diversity of novel botevamide A-D and bananamides D-G influenced their antifungal and anti-oomycetes activities. This thesis presents a rich collection of CLP-producing Pseudomonas strains and new CLPs, which could contribute to global food securit