Effect of pre-harvest sanitizer treatments on Listeria survival, sensory quality and bacterial community dynamics on leafy green vegetables grown under commercial conditions.

Leafy green vegetables (LGVs) have large surface areas and can be colonized by various microorganisms including pathogens. In this study, we investigated the effect of pre-harvest sanitizer treatments on the survival of inoculated proxy pathogen Listeria innocua ATCC 33090 and the natural microbial community of mizuna, rocket (arugula), red chard and spinach grown under commercial conditions. Electrolyzed water (e-water), peracetic acid (PAA), and 1-bromo-3-chloro-5-dimethylhydantoin (BCDMH) were tested against water controls. We also observed the subsequent sensorial changes of harvested, bagged LGV leaves over a period of 12 days within chill storage alongside the growth, diversity and structure of bacterial populations determined using 16S rRNA gene amplicon sequencing and total viable counts (TVC). Treatment with PAA resulted in the highest reductions of L. innocua (2.4-5.5 log units) compared to the other treatments (0.25-2.5 log units). On day 0 (24 h after sanitizer application), the TVC on sanitizer treated LGVs were significantly reduced compared to water controls, except for rocket. During storage at 4.5 (±0.5)°C sanitisers only hindered microbial growth on LGVs initially and did not influence final bacterial population levels, growth rates or changes in LGV sample colour, decay, odour and texture compared to water controls. Shelf-life was not extended nor was it reduced. The community structure on LGV types differed though a core set of bacterial amplicon sequence variants (ASV) were present across all samples. No significant differences were observed in bacterial diversity between sanitizer treatments, however sanitizer treated LGV samples had initially reduced diversity compared to water treated samples. The bacterial compositions observed at the end point of storage considerably differed from what was observed at initial point owing to the increase in abundance of specific bacterial taxa, mainly Pseudomonas spp., the abundance and growth responses differing between LGV types studied. This study provides a better understanding on the microbiology and sensory impact of pre-harvest applied sanitiser treatments on different LGVs destined for commercial food use.

Aqueous peat extract exposes rhizobia to sub-lethal stress which may prime cells for improved desiccation tolerance.

Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.

Physiological changes in rhizobia after growth in peat extract may be related to improved desiccation tolerance.

Improved survival of peat-cultured rhizobia compared to survival of liquid-cultured cells has been attributed to cellular adaptations during solid-state fermentation in moist peat. We have observed improved desiccation tolerance of Rhizobium leguminosarum bv. trifolii TA1 and Bradyrhizobium japonicum CB1809 after aerobic growth in water extracts of peat. Survival of TA1 grown in crude peat extract was 18-fold greater than that of cells grown in a defined liquid medium but was diminished when cells were grown in different-sized colloidal fractions of peat extract. Survival of CB1809 was generally better when grown in crude peat extract than in the control but was not statistically significant (P > 0.05) and was strongly dependent on peat extract concentration. Accumulation of intracellular trehalose by both TA1 and CB1809 was higher after growth in peat extract than in the defined medium control. Cells grown in water extracts of peat exhibit morphological changes similar to those observed after growth in moist peat. Electron microscopy revealed thickened plasma membranes, with an electron-dense material occupying the periplasmic space in both TA1 and CB1809. Growth in peat extract also resulted in changes to polypeptide expression in both strains, and peptide analysis by liquid chromatography-mass spectrometry indicated increased expression of stress response proteins. Our results suggest that increased capacity for desiccation tolerance in rhizobia is multifactorial, involving the accumulation of trehalose together with increased expression of proteins involved in protection of the cell envelope, repair of DNA damage, oxidative stress responses, and maintenance of stability and integrity of proteins.

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea.

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.