The exploration of potato-associated bacteria in the Central Andean Highlands and their application in integrated crop management systems

Potato is the most important food crop after wheat and rice. A changing climate, coupled with a heightened consumer awareness of how food is produced and legislative changes governing the usage of agrochemicals, means that alternative more integrated and sustainable approaches are needed for crop management practices. Bioprospecting in the Central Andean Highlands resulted in the isolation and in vitro screening of 600 bacterial isolates. The best performing isolates, under in vitro conditions, were field trialled in their home countries. Six of the isolates, Pseudomonas sp. R41805 (Bolivia), Pseudomonas palleroniana R43631 (Peru), Bacillus sp. R47065, R47131, Paenibacillus sp. B3a R49541, and Bacillus simplex M3-4 R49538 (Ecuador), showed significant increase in the yield of potato. Using – omic technologies (i.e. volatilomic, transcriptomic, proteomic and metabolomic), the influence of microbial isolates on plant defence responses was determined. Volatile organic compounds of bacterial isolates were identified using GC/MS. RT-qPCR analysis revealed the significant expression of Ethylene Response Factor 3 (ERF3) and the results of this study suggest that the dual inoculation of potato with Pseudomonas sp. R41805 and Rhizophagus irregularis MUCL 41833 may play a part in the activation of plant defence system via ERF3. The proteomic analysis by 2-DE study has shown that priming by Pseudomonas sp. R41805 can induce the expression of proteins related to photosynthesis and protein folding in in vitro potato plantlets. The metabolomics study has shown that the total glycoalkaloid (TGA) content of greenhouse-grown potato tubers following inoculation with Pseudomonas sp. R41805 did not exceed the acceptable safety limit (200 mg kg-1 FW). As a result of this study, a number of bacteria have been identified with commercial potential that may offer sustainable alternatives in both Andean and European agricultural settings.

Identification and analysis of mechanisms involved in a broad-spectrum disease resistant mutant of the winter wheat cv. Guardian

M66 an X-ray induced mutant of winter wheat (Triticum aestivum) cv. Guardian exhibits broad-spectrum resistance to powdery mildew (Blumeria graminis f. sp. tritici), yellow rust (Puccinia striiformis f. sp. tritici), and leaf rust (Puccinia recondita f. sp. tritici), along with partial resistance to stagnonospora nodorum blotch (caused by the necrotroph Stagonosporum nodorum) and septoria tritici blotch (caused by the hemibiotroph Mycosphaerella graminicola) compared to the parent plant ‘Guardian’. Analysis revealed that M66 exhibited no symptoms of infection following artificial inoculation with Bgt in the glasshouse after adult growth stage (GS 45). Resistance in M66 was associated with widespread leaf flecking which developed during tillering. Flecking also occurred in M66 leaves without Bgt challenge; as a result grain yields were reduced by approximately 17% compared to ‘Guardian’ in the absence of disease. At the seedling stage, M66 exhibited partial resistance. M66, along with Tht mutants (Tht 12, Tht13), also exhibit increased tolerance to environmental stresses (abiotic), such as drought and heat stress at seedling and adult growth stages, However, adult M66 exhibited increased susceptibility to the aphid Schizaphis graminum compared to ‘Guardian’. Resistance to Bgt in M66 was characterized with increased and earlier H2O2 accumulation at the site of infection which resulted in increased papilla formation in epidermal cells, compared to ‘Guardian’. Papilla formation was associated with reduced pathogen ingress and haustorium formation, indicating that the primary cause of resistance in M66 was prevention of pathogen penetration. Heat treatment at 46º C prior to challenge with Bgt also induced partial disease resistance to Blumeria graminis f. sp. tritici in ‘Guardian’ and M66 seedlings. This was characterized by a delay in primary infection, due to increased production of ROS species, such as hydrogen peroxide, ROS-scavenging enzymes and Hsp70, resulting in cross-linking of cell wall components prior to inoculation. This actively prevented the fungus from penetrating the epidermal cell wall. Proteomics analysis using 2-D gel electrophoresis identified primary and secondary disease resistance effects in M66 including detection of ROS scavenging enzymes (4, 24 hai), such as ascorbate peroxidase and a superoxidase dismutase isoform (CuZnSOD) in M66 which were absent from ‘Guardian’. Chitinase (PR protein) was also upregulated (24 hai) in M66 compared to ‘Guardian’.Monosomic and ditelosomic analysis of M66 revealed that the mutation in M66 is located on the long arm of chromosome 2B (2BL). Chromosome 2BL is known to have key genes involved in resistance to pathogens such as those causing stripe rust and powdery mildew. The TaMloB1 gene, an orthologue of the barley Mlo gene, is also located on chromosome 2BL. Sanger sequencing of part of the coding sequence revealed no deletions in the TaMloB1 gene between ‘Guardian’ and M66.

Evaluation of microbial adjuncts and their effect on the ripening of cheddar cheese

A bacteriocin-producing strain of Lactobacillus paracasei DPC 4715 was used as an adjunct culture in Cheddar cheese in order to control the growth of “wild” nonstarter lactic acid bacteria. No suppression of growth of the indicator strain was observed in the experimental cheese. The bacteriocin produced by Lactobacillus paracasei DPC 4715 was sensitive to chymosin and cathepsin D and it may have been cleaved by the rennet used for the cheese manufactured or by indigenous milk proteases. A series of studies were performed using various microbial adjuncts to influence cheese ripening. Microbacterium casei DPC 5281, Corynebacterium casei DPC 5293 and Corynebacterium variabile DPC 5305 were added to the cheesemilk at level of 109 cfu/ml resulting in a final concentration of 108 cfu/g in Cheddar cheese. The strains significantly increased the level of pH 4.6-soluble nitrogen, total free amino acids after 60 and 180 d of ripening and some individual free amino acids after 180 d. Yarrowia lipolytica DPC 6266, Yarrowia lipolytica DPC 6268 and Candida intermedia DPC 6271 were used to accelerate the ripening of Cheddar cheese. Strains were grown in YG broth to a final concentration of 107 cfu/ml, microfluidized, freeze-dried and added to the curd during salting at level of 2% w/w. The yeasts positively affected the primary, secondary proteolysis and lipolysis of cheeses and had aminopeptidase, dipeptidase, esterase and 5’ phosphodiestere activities that contributed to accelerate the ripening and improve the flavor of cheese. Hafia alvei was added to Cheddar cheesemilk at levels of 107 cfu/ml and 108 cfu/ml and its contribution during ripening was evaluated. The strain significantly increased the level of pH 4.6-soluble nitrogen, total free amino-acids, and some individual free amino-acids of Cheddar cheese, whereas no differences in the urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms of the cheeses were detected. Hafia alvei also significantly increased the level of some biogenic amines. A low-fat Cheddar cheese was made with Bifidobacterium animalis subsp. lactis, strain BB-12® at level of 108 cfu/ml, as a probiotic adjunct culture and Hi-Maize® 260 (resistant high amylose maize starch) at level of 2% and 4% w/v, as a prebiotic fiber which also played the role of fat replacer. Bifidobacterium BB-12 decreased by 1 log cycle after 60 d of ripening and remained steady at level of ~107 cfu/g during ripening. The Young’s modulus also increased proportionally with increasing levels of Hi-maize. Hencky strain at fracture decreased over ripening and increased with increasing in fat replacer. A cheese based medium (CBM) was developed with the purpose of mimicking the cheese environment at an early ripening stage. The strains grown in CBM showed aminopeptidase activity against Gly-, Arg-, Pro- and Phe-p-nitroanalide, whereas, when grown in MRS they were active against all the substrates tested. Both Lb. danicus strains grown in MRS and in CBM had aminotransferase activity towards aromatic amino acids (Phe and Trp) and also branched-chain amino acids (Leu and Val). Esterase activity was expressed against p-nitrophenyl-acetate (C2), pnitrophenyl- butyrate (C4) and p-nitrophenyl-palmitate (C16) and was significantly higher in CBM than in MRS.

Tooth wear in Irish teenagers: a laboratory and epidemiological study

Aim: To investigate the characteristics, development and determinants of toothwear among Irish schoolchildren. Methods: A cross-sectional (examination at 16-years-old) and longitudinal (examinations at 5-,12-,14-years) study were conducted. Two indices were used to measure toothwear, children/parents completed a demographic profile and questionnaire on oral hygiene and dietary practices, health, and lifestyle in both studies. Saliva was collected from consenting 16-year-olds. The explanatory variables for the cross-sectional and longitudinal study were derived from children/parents responses. Differences in salivary profiles were determined for subsets; the protein concentration was determined with Bradford protein assay and protein carbonyl concentration (a protein oxidation marker) was determined spectrophotometrically. Gel-electrophoresis and mass spectrometry determined proteins and ion chromatography inorganic ions. Statistical significance was accepted at p<0.05. Results: At 16-years-old the prevalence of toothwear with dentine visible was 44%. No difference in salivary flow rates existed. In unstimulated saliva a higher mean, protein carbonyl (p<0.0001) and total calcium concentration (p<0.002) existed for the group with moderate toothwear. In stimulated saliva the moderate toothwear group had a lower mean protein concentration(p<0.0001). The 2-DE protein spots prepared for a sub-group differed between those with toothwear and without. Mass spectrometry, identified one of the different proteins as IgA. For 16-year-olds, the self-reported factors indicated that brushing after breakfast was associated with lower toothwear scores(p<0.03). Nail-biting, being asthmatic or reporting a dry mouth were associated with higher toothwear scores(all p<0.05). Eating an apple daily or less was associated with less toothwear(p<0.002). In the longitudinal study toothwear into dentine at age five or 12-years was associated with more toothwear at age 14(all p<0.05). Discussion: The results illustrate the multifactorial aetiology of toothwear. The biochemical and physical correlates of saliva with toothwear requires further research. Conclusion: The impact of previous toothwear, salivary, dietary and personal factors on toothwear in the early permanent dentition is demonstrated.