Pseudomonas cyclic lipopeptides suppress the rice blast fungus Magnaporthe oryzae by induced resistance and direct antagonism

Beneficial Pseudomonas spp. produce an array of antimicrobial secondary metabolites such as cyclic lipopeptides (CLPs). We investigated the capacity of CLP-producing Pseudomonas strains and their crude CLP extracts to control rice blast caused by Magnaporthe oryzae, both in a direct manner and via induced systemic resistance (ISR). In planta biocontrol assays showed that lokisin-, white line inducing principle (WLIP)-, entolysin- and N3-producing strains successfully induced resistance to M. oryzae VT5M1. Furthermore, crude extracts of lokisin, WLIP and entolysin gave similar ISR results when tested in planta. In contrast, a xantholysin-producing strain and crude extracts of N3, xantholysin and orfamide did not induce resistance against the rice blast disease. The role of WLIP in triggering ISR was further confirmed by using WLIP-deficient mutants. The severity of rice blast disease was significantly reduced when M. oryzae spores were pre-treated with crude extracts of N3, lokisin, WLIP, entolysin or orfamide prior to inoculation. In vitro microscopic assays further revealed the capacity of crude N3, lokisin, WLIP, entolysin, xantholysin and orfamide to significantly inhibit appressoria formation by M. oryzae. In addition, the lokisin and WLIP biosynthetic gene clusters in the producing strains are described. In short, our study demonstrates the biological activity of structurally diverse CLPs in the control of the rice blast disease caused by M. oryzae. Furthermore, we provide insight into the non-ribosomal peptide synthetase genes encoding the WLIP and lokisin biosynthetic machineries

Effects of different substrates on the morphological characteristics and proximate content of Pleurotus pulminarius

Morphological characteristics and proximate content of Pleurotus pulminarius cultivated on different substrates were investigated. The substrates used are cotton waste, sawdust, palm fruit waste, paper, mixture of sawdust and palm fruit waste and mixture of cotton waste and paper (1:1) were sterilized and inoculated with Pleurotus pulminarius spawn (sorghum-produced) aseptically, incubated under controlled temperature and humidity before exposure to sunlight. Length and, diameter of stalk and pileus were measured during stages of harvest. Protein, fat, carbohydrate, crude fibre, ash and moisture content were determined by standard methods. Pleurotus pulminarius appeared on saw dust and cotton waste; paper, combination of saw dust and palm fruit waste, mixture of paper and cotton waste; and palm fruit waste after 2 days, 3 days and 5 days respectively. For the first stage of harvest, Saw dust had the highest length of stalk (cm) of 5.40 ± 0.63 and the lowest value of 2.70 ± 0.14 was obtained from palm fruit waste only. Mixture of paper waste and cotton waste has the highest diameter of stalk (3.25 ± 0.61 cm) (second harvest), the lowest diameter of stalk (0.4 ± 0.1 cm) was recorded for palm fruit (third harvest). Cotton waste had the highest diameter of pileus (7.88 ± 1.29 cm) (first harvest) while the lowest value (1.78 ± 0.63 cm) was obtained from mushroom harvested on the mixture of paper and cotton waste (second harvest). Pleurotus pulminarius harvested on mixture of paper and cotton waste has the highest percentage protein of 40.54% while the least value 28.79% protein was obtained from mushroom harvested on saw dust. Mushroom harvested on saw dust has the highest percentage fat content of 0.99% and palm fruit waste gave the lowest percentage fat of 0.74%. Palm fruit waste displayed the highest percentage ash and crude fibre contents of 4.38% and 4.81% respectively while cotton and paper wastes had the least ash and crude fibre content of 1.81% and 0.47% respectively. Saw dust gave the highest percentage carbohydrate of 62.54%s. Moisture content of 0.51% was obtained for mushrooms harvested on Palm fruit waste, Sawdust and mixture of sawdust and palm fruit waste as the least value while the mixture of paper and cotton waste has the highest moisture content of 3.49%. The study concluded that mushroom can serve as cheap source of food supplements such as protein, crude fibre, ash (micro and macro nutrients), fat, carbohydrate and moisture with beneficial bioconversion of agricultural and other wastes materials in the environment. Key words: Pleurotus pulminarius, cotton, sawdust, palm fruit, paper, wast

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium)

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams 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. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam

Biosynthesis and antimicrobial activity of pseudodesmin and viscosinamide cyclic lipopeptides produced by pseudomonads associated with the cocoyam rhizosphere

Pseudomonascyclic lipopeptides (CLPs) are encoded non-ribosomally by biosynthetic gene clusters (BGCs) and possess diverse biological activities. In this study, we conducted chemical structure and BGC analyses with antimicrobial activity assays for two CLPs produced byPseudomonasstrains isolated from the cocoyam rhizosphere in Cameroon and Nigeria. LC-MS and NMR analyses showed that thePseudomonassp. COR52 and A2W4.9 produce pseudodesmin and viscosinamide, respectively. These CLPs belong to the Viscosin group characterized by a nonapeptidic moiety with a 7-membered macrocycle. Similar to other Viscosin-group CLPs, the initiatory non-ribosomal peptide synthetase (NRPS) gene of the viscosinamide BGC is situated remotely from the other two NRPS genes. In contrast, the pseudodesmin genes are all clustered in a single genomic locus. Nano- to micromolar levels of pseudodesmin and viscosinamide led to the hyphal distortion and/or disintegration ofRhizoctonia solaniAG2-2 andPythium myriotylumCMR1, whereas similar levels of White Line-Inducing Principle (WLIP), another member of the Viscosin group, resulted in complete lysis of both soil-borne phytopathogens. In addition to the identification of the biosynthetic genes of these two CLPs and the demonstration of their interaction with soil-borne pathogens, this study provides further insights regarding evolutionary divergence within the Viscosin group

Cyclic lipopeptide‐producing Pseudomonas koreensis group strains dominate the cocoyam rhizosphere of a Pythium root rot suppressive soil contrasting with P. putida prominence in conducive soils

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P . koreensis and P . putida (sub)groups, with representations in the P . fluorescens , P . chlororaphis , P . jessenii and P . asplenii (sub)groups. In contrast, P . putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P . koreensis group, being the most abundant. However, putisolvin III‐V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives—putisolvin III‐V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD

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

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