The principal objective of this study was to isolate and identify the microorganisms present in commercial kefir grains, a novel kefir-fermented coconut water (CWK) and a novel coconut water kefir-fermented sourdough using phenotypic identification and Sanger sequencing and examine the microbial diversity of CWK and CWK-fermented sourdough throughout the fermentation process using the MiSeq Illumina sequencing method. The phenotypic characterisation based on morphology identified ten isolates of LAB, five AAB and seven yeasts from kefir (K), CWK and CWK-fermented sourdough (CWKS). The results confirm the presence of the LAB species Limosilactobacillus fermentum, Lactobacillus. plantarum, L. fusant, L. reuteri and L. kunkeei; the AAB species Acetobacter aceti, A. lovaniensis and A. pasteurianus; and the yeast species Candida kefyr, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, C. guilliermondii and C. colliculosa. To the best of our knowledge, the identification of Rhodotorula from kefir is being reported for the first time. This study provides important insights into the relative abundances of the microorganisms in CWKS. A decrease in pH and an increase in the titratable acidity for CWK- and CWK-fermented sourdough corresponded to the increase in D- and L-lactic acid production after 96 h of fermentation. Significant reductions in the pHs of CWK and CWKS were observed between 48 and 96 h of fermentation, indicating that the kefir microorganisms were able to sustain highly acidic environments. There was also increased production of L-lactic acid with fermentation, which was almost twice that of D-lactic acid in CWK.

Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in biotic and abiotic stress responses, as well as development. Arabidopsis thaliana CPK3 (AtCPK3) is involved with plant signaling pathways such as stomatal movement regulation, salt stress response, apoptosis, seed germination and pathogen defense. In this study, AtCPK3 and its orthologues in relatively distant plant species such as rice (Oryza sativa, monocot) and kiwifruit (Actinidia chinensis, asterid eudicot) were analyzed in response to drought, bacteria, fungi, and virus infections. Two orthologues were studied in O. sativa, namely OsCPK1 and OsCPK15, while one orthologue—AcCPK16—was identified in A. chinensis. Reverse-transcriptase quantitative PCR (RT-qPCR) analysis revealed that OsCPK1 and AcCPK16 exhibit similar responses to stressors to AtCPK3. OsCPK15 responded differently, particularly in bacterial and fungal infections. An increase in expression was consistently observed among AtCPK3 and its orthologues in response to virus infection. Overexpression mutants in both Arabidopsis and kiwifruit showed slight tolerance to drought, while knockout mutants were slightly more susceptible or had little difference with wild-type plants. Overexpression mutants in Arabidopsis showed slight tolerance to virus infection. These findings highlight the importance of AtCPK3 and its orthologues in drought and pathogen responses and suggest such function must be conserved in its orthologues in a wide range of plants.

To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method (Bioanalyzer); and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.