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.

Involvement of lytic enzymes and secondary metabolites produced by Trichoderma spp. in the biological control of Pythium myriotylum.

This investigation was aimed to evaluate the antimicrobial activities and involvement of extracellular lytic enzymes produced by four strains of Trichoderma in the inhibition of Pythium myriotylum. Antagonistic effects were tested by dual culture. Activities of lytic enzymes were evaluated from the filtrate of each strain after cultivation in selected media. Organic extracts were obtained from liquid media subsequent to the cultivation of Trichoderma in potato dextrose broth (PDB). Non-volatile organic compounds such as polyphenols and flavonoids were evaluated spectrophotometrically while volatile organic compounds (VOCs) were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). The antimicrobial activity of the organic extracts was determined using the poisoning method. Results have shown that all the strains were antagonists against P. myriotylum. T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11) that were found to produce cellulase, protease, and xylanase. Over 20 compounds were identified in each extract, including esters, lactones, and organic acids. The organic extracts also contained high amounts of polyphenolic compounds and flavonoids and significantly inhibited the mycelial growth of P. myriotylum. The minimal inhibition concentrations were 80 µg/µL, 40 µg/µL, 20 µg/µL, and 10 µg/µL, for extracts obtained from T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11), respectively. There was significant correlation between the production of total polyphenol and flavonoid content and the antagonistic effects of the tested strains.

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.

Phenazines and biosurfactants interact in the biological control of soil-borne diseases caused by Pythium spp.

In this study, the putative role of phenazines and rhamnolipid-biosurfactants, antagonistic metabolites produced by Pseudomonas aeruginosa PNA1, was tested in the biological control of Pythium splendens on bean (Phaseolus vulgaris L) and Pythium myriotylum on cocoyam (Xanthosoma sagittifolium L Schott). A rhamnolipid-deficient and a phenazine-deficient mutant of PNA1 were used either separately or jointly in plant experiments. When the mutants were applied separately, no disease-suppressive effect was observed, although both mutants still produced one of the antagonistic compounds (phenazines or rhamnolipids). When the mutants were concurrently introduced in the soil, the biocontrol activity was restored to wild-type levels. Bean seeds developed significantly less pre-emergence damping-off caused by P. splendens when treated with a mixture of purified phenazine-1-carboxamide and rhamnolipids than with any of the chemicals alone. When phenazines and rhamnolipids were combined at concentrations that had no observable effects when the metabolites were applied separately, mycelial growth of P. myriotylum was significantly reduced. In addition, microscopic analysis revealed substantial vacuolization and disintegration of Pythium hyphae after incubation in liquid medium amended with both metabolites. Results of this study indicate that phenazines and biosurfactants are acting synergistically in the control of Pythium spp.

Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity.

AIM: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. MATERIAL AND RESULTS: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. CONCLUSION: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens.

Intraspecific variability of Pythium myriotylum isolated from cocoyam and other host crops.

Intraspecific variability within 51 isolates of Pythium myriotylum from cocoyam (Xanthosoma sagittifolium) and other host crops was analysed using optimum growth temperature, esterase banding patterns, AFLPs, rDNA-ITS sequencing, and virulence to cocoyam. P. myriotylum isolates virulent to cocoyam could easily be differentiated from other isolates of P. myriotylum by their optimum growth temperature. Isolates from cocoyam grew best at 28 degrees C with no growth at 37 degrees C, while P. myriotylum isolates from other host crops had their optimum growth temperature at 37 degrees C. Esterases produced consistent zymograms with 18 discrete esterase markers, but no monomorphic markers were produced for isolates virulent to cocoyam. Isozyme profiles based on esterase analysis showed that isolates that infect cocoyam plantlets formed a related group, irrespective of their geographic origin. P. myriotylum isolates from other host plants also grouped together, but could clearly be distinguished from the cocoyam cluster. AFLPs produced 189 scorable bands for the cocoyam isolates, of which 77% are monomorphic. Phenetic analysis of AFLP data grouped all isolates originating from cocoyam together except for the isolates C103-04, CMR17, CMR22, and CMR25. These isolates regrouped with isolates of Pythium myriotylum from other host crops or the outgroup and were found not to be pathogenic for cocoyam. ITS sequences of isolates of P. myriotylum from cocoyam were 99.1-99.7% identical to sequences deposited in GenBank. However, alignments of ITS sequences revealed a base transition at position 824 from adenine in typical isolates of P. myriotylum to guanine in isolates that could infect cocoyam plantlets. In a limited pathogenicity test, all isolates from cocoyam having guanine at position 824 were able to infect tissue culture derived cocoyam but not those exhibiting adenine. This study demonstrates for the first time, molecular evidence that isolates of P. myriotylum that infect cocoyam are distinct from P. myriotylum isolates from other crops and have developed a certain degree of host adaptation.

Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum.

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.