Fungi associated with black mould on baobab trees in southern Africa.

There have been numerous reports in the scientific and popular literature suggesting that African baobab (Adansonia digitata) trees are dying, with symptoms including a black mould on their bark. The aim of this study was to determine the identity of the fungi causing this black mould and to consider whether they might be affecting the health of trees. The fungi were identified by sequencing directly from mycelium on the infected tissue as well as from cultures on agar. Sequence data for the ITS region of the rDNA resulted in the identification of four fungi including Aureobasidium pullulans, Toxicocladosporium irritans and a new species of Rachicladosporium described here as Rachicladosporium africanum. A single isolate of an unknown Cladosporium sp. was also found. These fungi, referred to here as black mould, are not true sooty mould fungi and they were shown to penetrate below the bark of infected tissue, causing a distinct host reaction. Although infections can lead to dieback of small twigs on severely infected branches, the mould was not found to kill trees.

Three traditional fermented baobab foods from Benin, Mutchayan, Dikouanyouri, and Tayohounta: preparation, properties, and consumption.

Forest food resources contribute significantly to food supply in areas where they grow. Three fermented baobab foods were studied: Dikouanyouri (from seeds, pH = 6.5); Tayohounta (from seed kernels, pH = 7), and Mutchayan (from baobab pulp and sorghum, pH = 4.2). Bacillus spp. (8.5 and 9.5 Log cfu /g) and lactic acid bacteria (8.9 and 8.4 Log cfu /g,) dominate in Dikouanyouri and Tayohounta, respectively. In Mutchayan, lactic acid bacteria (8.1 Log cfu/g) and yeasts (7.2 Log cfu/g) predominated. The arbitrary index of protein cleavage increases from 2.3% (unfermented products) to 13.7% in Dikouanyouri and 21.3% in Tayohounta, indicating significant protein degradation. Mutchayan is the most frequently consumed product.

Seven new species of the Botryosphaeriaceae from baobab and other native trees in Western Australia.

In this study seven new species of the Botryosphaeriaceae are described from baobab (Adansonia gibbosa) and surrounding endemic tree species growing in the Kimberley region of northwestern Australia. Members of the Botryosphaeriaceae were predominantly endophytes isolated from apparently healthy sapwood and bark of endemic trees; others were isolated from dying branches. Phylogenetic analyses of ITS and EF1-alpha sequence data revealed seven new species: Dothiorella longicollis, Fusicoccum ramosum, Lasiodiplodia margaritacea, Neoscytalidium novaehollandiae, Pseudofusicoccum adansoniae, P. ardesiacum and P. kimberleyense.

Rapid screening of starter cultures for maari based on antifungal properties.

Forty Bacillus isolates obtained from maari (used as condiment in Burkina Faso) including 17 B. subtilis, 4 B. circulans, 7 B. pumilus and 6 B. licheniformis were investigated for use as starter cultures in maari production. The isolates were screened by PCR for the sfp gene responsible for the production of the lipopeptide biosurfactant, surfactin. The sfp gene was detected in all of the seventeen B. subtilis isolates, in 2 out of 7 B. pumilus, in 4 out of 6 B. licheniformis whereas no B. circulans was positive for the sfp gene by PCR screening. Furthermore, all the 40 Bacillus spp. were screened for biosurfactant production and inhibitory activity against Aspergillus flavus, A. niger, A. versicolor and Rhizopus oryzae. Results demonstrated a relationship between the presence of the sfp gene and the antifungal activity and biosurfactant production of Bacillus isolates. In addition, molecular typing of the 17 B. subtilis isolates by Multilocus Sequence Typing (MLST) resulted in 15 Sequence Types, one of them included three strains. Randomly Amplified Polymorphic DNA-PCR (RAPD-PCR), used for B. licheniformis, B. megaterium, B. circulans and B. pumilus revealed that the inhibitory activity and biosurfactant production were strain-dependent. Finally, the detection of chitinase (chi) and ß-glucanase (glu) biosynthesis genes was found to be associated with the antifungal activity for 16 B. subtilis isolates. The present work provides a greater understanding of the antifungal activity and biosurfactant production ability within the Bacillus spp. isolated from maari and contributes to the selection of Bacillus isolates to be used as starter cultures for controlled production of maari.

Phylogenetic species recognition and hybridisation in Lasiodiplodia: A case study on species from baobabs.

Lasiodiplodia species (Botryosphaeriaceae, Ascomycota) infect a wide range of typically woody plants on which they are associated with many different disease symptoms. In this study, we determined the identity of Lasiodiplodia isolates obtained from baobab (Adansonia species) trees in Africa and reviewed the molecular markers used to describe Lasiodiplodia species. Publicly available and newly produced sequence data for some of the type strains of Lasiodiplodia species showed incongruence amongst phylogenies of five nuclear loci. We conclude that several of the previously described Lasiodiplodia species are hybrids of other species. Isolates from baobab trees in Africa included nine species of Lasiodiplodia and two hybrid species. Inoculation trials with the most common Lasiodiplodia species collected from these trees produced significant lesions on young baobab trees. There was also variation in aggressiveness amongst isolates from the same species. The apparently widespread tendency of Lasiodiplodia species to hybridise demands that phylogenies from multiple loci (more than two and preferably four or more) are compared for congruence prior to new species being described. This will avoid hybrids being incorrectly described as new taxa, as has clearly occurred in the past.

Characteristics and phylogeny of Bacillus cereus strains isolated from Maari, a traditional West African food condiment.

Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 10(5)cfu/ml in DW, and ranged between 6.5×10(3) and 1.2×10(4)cfu/g in potash, 10(9)-10(10)cfu/g in seed mash during fermentation and 10(7) - 10(9) after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8h fermentation. The "emetic" type B. cereus were present in DW, the seed mash at 48-72h of fermentation and in the final product, while the remaining isolates (PanC type IV) were detected in ash, at 48-72h fermentation and in the final product. This work sheds light on the succession and pathogenic potential of B. cereus species in traditional West African food condiment and clarifies their phylogenetic relatedness to B. cereus biovar anthracis. Future implementation of GMP and HACCP and development of starter cultures for controlled Maari fermentations will help to ensure a safe product.

Inhibition of Bacillus cereus growth by bacteriocin producing Bacillus subtilis isolated from fermented baobab seeds (maari) is substrate dependent.

Maari is a spontaneously alkaline fermented food condiment made from baobab tree seeds. Due to the spontaneous nature of maari fermentations growth of the opportunistic human pathogen Bacillus cereus is occasionally observed. Bacillus subtilis strains are important for alkaline seed fermentations because of their enzymatic activities contributing to desirable texture, flavor and pH development. Some B. subtilis strains have antimicrobial properties against B. cereus. In the present work, three bacteriocin producing B. subtilis strains (B3, B122 and B222) isolated from maari were tested. The production of antimicrobial activity by the three strains was found to be greatly influenced by the substrate. All three B. subtilis strains produced antimicrobial activity against B. cereus NVH391-98 in BHI broth as determined by the agar well diffusion assay, whereas no antimicrobial activity was detected in whole cooked baobab seeds and in 10% (w/v) grinded baobab seeds. Incorporation of BHI with up to 5% (w/w) grinded baobab seeds enhanced the antimicrobial activity of B. subtilis compared with pure BHI in a strain dependent manner. Incorporation of BHI with 50% (w/w) baobab grinded seeds decreased the antimicrobial activity. Addition of the inorganic salts FeCl3, MgSO4 and MnSO4 has previously been reported to increase bacteriocin production of B. subtilis, but the addition of these salts to 10% (w/v) grinded baobab seed broth did not cause antimicrobial activity. Survival of B. cereus NVH391-98 in co-culture with B. subtilis was tested in BHI broth, 10% (w/v) grinded baobab seed based broth and during baobab seed fermentation to produce maari. B. cereus NVH391-98 grew well in all three substrates in mono-culture. All the 3 B. subtilis strains were able to decrease B. cereus NVH391-98 to levels below the detection limit (<10 CFU/ml) in BHI, but not in baobab seed based substrates, even though the outgrowth of B. cereus NVH391-98 was delayed by up to 40 h. In conclusion, production of antimicrobial activity by the investigated B. subtilis strains is highly substrate-specific and strain-specific. The three B. subtilis strains delayed but did not prevent B. cereus outgrowth during baobab seed fermentations. Maari is produced through mixed microbial population fermentations. B. subtilis co-starter culture candidates originating from maari which are able to prevent pathogen outgrowth remain to be identified.

Microorganisms associated with Maari, a Baobab seed fermented product.

A microbiological study was carried out on Baobab fermented seeds (Maari) obtained from 4 different production sites in Burkina Faso (Mansila, Toulfé, Ouagadougou and Gorgadji). A total of 390 representative isolates comprising 251 aerobic mesophilic bacteria (AMB) and 139 lactic acid bacteria (LAB) were isolated and identified to species level using a combination of pheno- and genotypic methods including conventional morphological analysis, carbohydrate fermentation profiling, rep-PCR ((GTG)(5)-fingerprinting) and 16S rRNA gene sequencing. The fermentation of Baobab seeds was initiated by the AMB identified as Bacillus subtilis (82% of AMB isolates) and Staphylococcus sciuri (18% of AMB isolates). No lactic acid bacteria were isolated at the beginning of the process. After 24h fermentation time, Enterococcus faecium appeared in the fermenting seeds and remained until the end of the fermentation, as the predominant LAB. In Maari collected from retail outlets the AMB count ranged from 6.7log10CFU/g to 10log10CFU/g while the LAB load ranged from 4.4log10CFU/g to 9.9log10CFU/g. The AMB were identified as belonging to genus Bacillus (12 species), Staphylococcus (3 species) and one species of Aerococcus, Macrococcus, Leifsonia, Kurthia, Proteus, Acinetobacter and Globicatella, respectively. A putatively novel, previously undescribed Corynebacterium sp. was also found. E. faecium was the dominant LAB in all investigated retail samples except one sample dominated by Pediococcus acidilactici.