Occurrence, distribution and contamination levels of heat-resistant moulds throughout the processing of pasteurized high-acid fruit products.

Heat-resistant moulds (HRMs) are well known for their ability to survive pasteurization and spoil high-acid food products, which is of great concern for processors of fruit-based products worldwide. Whilst the majority of the studies on HRMs over the last decades have addressed their inactivation, few data are currently available regarding their contamination levels in fruit and fruit-based products. Thus, this study aimed to quantify and identify heat-resistant fungal ascospores from samples collected throughout the processing of pasteurized high-acid fruit products. In addition, an assessment on the effect of processing on the contamination levels of HRMs in these products was carried out. A total of 332 samples from 111 batches were analyzed from three processing plants (=three processing lines): strawberry puree (n = 88, Belgium), concentrated orange juice (n = 90, Brazil) and apple puree (n = 154, the Netherlands). HRMs were detected in 96.4% (107/111) of the batches and 59.3% (197/332) of the analyzed samples. HRMs were present in 90.9% of the samples from the strawberry puree processing line (1-215 ascospores/100 g), 46.7% of the samples from the orange juice processing line (1-200 ascospores/100 g) and 48.7% of samples from the apple puree processing line (1-84 ascospores/100 g). Despite the high occurrence, the majority (76.8%, 255/332) of the samples were either not contaminated or presented low levels of HRMs (<10 ascospores/100 g). For both strawberry puree and concentrated orange juice, processing had no statistically significant effect on the levels of HRMs (p > 0.05). On the contrary, a significant reduction (p < 0.05) in HRMs levels was observed during the processing of apple puree. Twelve species were identified belonging to four genera - Byssochlamys, Aspergillus with Neosartorya-type ascospores, Talaromyces and Rasamsonia. N. fumigata (23.6%), N. fischeri (19.1%) and B. nivea (5.5%) were the predominant species in pasteurized products. The quantitative data (contamination levels of HRMs) were fitted to exponential distributions and will ultimately be included as input to spoilage risk assessment models which would allow better control of the spoilage of heat treated fruit products caused by heat-resistant moulds.

Sugarcane genes differentially expressed in response to Puccinia melanocephala infection: identification and transcript profiling.

Brown rust caused by the fungus Puccinia melanocephala is a major disease of sugarcane (Saccharum spp.). A sugarcane mutant, obtained by chemical mutagenesis of the susceptible variety B4362, showed a post-haustorial hypersensitive response (HR)-mediated resistance to the pathogen and was used to identify genes differentially expressed in response to P. melanocephala via suppression subtractive hybridization (SSH). Tester cDNA was derived from the brown rust-resistant mutant after inoculation with P. melanocephala, while driver cDNAs were obtained from the non-inoculated resistant mutant and the inoculated susceptible donor variety B4362. Database comparisons of the sequences of the SSH recombinant clones revealed that, of a subset of 89 non-redundant sequences, 88% had similarity to known functional genes, while 12% were of unknown function. Thirteen genes were selected for transcript profiling in the resistant mutant and the susceptible donor variety. Genes involved in glycolysis and C4 carbon fixation were up-regulated in both interactions probably due to disturbance of sugarcane carbon metabolism by the pathogen. Genes related with the nascent polypeptide associated complex, post-translational proteome modulation and autophagy were transcribed at higher levels in the compatible interaction. Up-regulation of a putative L-isoaspartyl O-methyltransferase S-adenosylmethionine gene in the compatible interaction may point to fungal manipulation of the cytoplasmatic methionine cycle. Genes coding for a putative no apical meristem protein, S-adenosylmethionine decarboxylase, non-specific lipid transfer protein, and GDP-L-galactose phosphorylase involved in ascorbic acid biosynthesis were up-regulated in the incompatible interaction at the onset of haustorium formation, and may contribute to the HR-mediated defense response in the rust-resistant mutant.

Analysis of expressed sequence tags derived from a compatible Mycosphaerella fijiensis-banana interaction.

Mycosphaerella fijiensis, a hemibiotrophic fungus, is the causal agent of black leaf streak disease, the most serious foliar disease of bananas and plantains. To analyze the compatible interaction of M. fijiensis with Musa spp., a suppression subtractive hybridization (SSH) cDNA library was constructed to identify transcripts induced at late stages of infection in the host and the pathogen. In addition, a full-length cDNA library was created from the same mRNA starting material as the SSH library. The SSH procedure was effective in identifying specific genes predicted to be involved in plant-fungal interactions and new information was obtained mainly about genes and pathways activated in the plant. Several plant genes predicted to be involved in the synthesis of phenylpropanoids and detoxification compounds were identified, as well as pathogenesis-related proteins that could be involved in the plant response against M. fijiensis infection. At late stages of infection, jasmonic acid and ethylene signaling transduction pathways appear to be active, which corresponds with the necrotrophic life style of M. fijiensis. Quantitative PCR experiments revealed that antifungal genes encoding PR proteins and GDSL-like lipase are only transiently induced 30 days post inoculation (dpi), indicating that the fungus is probably actively repressing plant defense. The only fungal gene found was induced 37 dpi and encodes UDP-glucose pyrophosphorylase, an enzyme involved in the biosynthesis of trehalose. Trehalose biosynthesis was probably induced in response to prior activation of plant antifungal genes and may act as an osmoprotectant against membrane damage.

Fluorescent pseudomonads as biocontrol agents for sustainable agricultural systems.

The highly diverse genus Pseudomonas contains very effective biocontrol agents that can increase plant growth and improve plant health. Biocontrol characteristics, however, are strain-dependent and cannot be clearly linked to phylogenetic variation. Isolate screening remains essential to find suitable strains, which can be done by testing large local collections for disease suppression and plant-growth promotion exemplified in a case study on forage legumes in Uruguay or by targeted screening for Pseudomonas spp. which produce desirable secondary metabolites, as demonstrated in a case study on cocoyam in Cameroon. In both case studies, access to reference strains from public and private collections was essential for identification, phylogenetic studies and metabolite characterization.

An efficient method for the extraction of high-quality fungal total RNA to study the Mycosphaerella fijiensis-Musa spp. Interaction.

Efficient RNA isolation is a prerequisite for gene expression studies and it has an increasingly important role in the study of plant-fungal pathogen interactions. However, RNA isolation is difficult in filamentous fungi. These organisms are notorious for their rigid cell walls and the presence of high levels of carbohydrates, excreted from the fungal cells during submerged growth, which interferes with the extraction procedures. Although many commercial kits are already available for RNA isolation, they do not provide, in most cases, enough amount of pure RNA to be used in upstream applications. In the present work, we propose an easy and efficient protocol for isolating total RNA from the filamentous fungus Mycosphaerella fijiensis, the most important foliar pathogen of Musa spp. varieties worldwide. In addition, we applied the proposed protocol to the isolation of total RNA from banana leaves infected with the pathogen. Our methodology was developed based on the SDS method with modifications including a carbohydrate precipitation step. The protocol resulted in high-quality total RNA, from fungal mycelium grown in PDB medium and infected banana leaves, suitable for further molecular studies. The proposed methodology is also applicable to the ascomycete fungus Passalora fulva (syn. Cladosporum fulvum).