Bacterial diversity, community structure and function in association of potato scabby tubers during storage in northern Thailand.

Potato scab is a common potato tuber disease that affects quality and cost in the marketplace, shortening storage, and increasing the chance for secondary infection. The tubers with disease severity of 1 to 4 are accepted and stored in potato storage for cheap selling in Thailand. However, there are few studies of the bacterial community of the scabby tuber during storage. Thus, we aim to elucidate the diversity, structure, and function of the bacterial community of 30-day storage potato scabby tubers stored in different temperatures using 16S amplicon metagenomic sequencing. Bacterial communities of storage potato scabby tubers (Spunta cultivar) collected from different storage temperatures, 4 °C (MEP1) and 6 °C (MEP2), were characterized using 16S rRNA amplicon metagenomic sequencing. The alpha-diversity abundance in the bacteriome of the scabby tubers stored at 6 °C was higher than in those stored at 4 °C. Actinobacteria (34.7%) was a dominant phylum in MEP1, while Proteobacteria (39.9%) was predominant in MEP2. The top 10 genera of both communities were Rhizobium group, Streptomyces, Pectobacterium, Ruminococcus, Cellulomonas, Promicromonospora, Prevotella, Enterobacter, Pedobacter, and Paenarthrobacter. Moreover, functional profile prediction of both communities reveals essential genes in the pathosystem: nos, bglA, and cebEFG-msiK for potato scab disease and phc and peh operons for rot disease. Our findings are the first study to explore details of the bacteriome of the accepted potato scabby tubers for selling during storage in Thailand and strongly indicate that although potatoes were stored at low temperatures, diseases still occur by secondary pathogens.

Identification, pathogenicity and effects of plant extracts on Neopestalotiopsis and Pseudopestalotiopsis causing fruit diseases.

Pestalotiopsis and related genera, including Neopestalotiopsis and Pseudopestalotiopsis have damaged many plants for many decades; however, there is little available information about the fungi on tropical fruit in Thailand. This study isolated and characterized pestalotioid fungi on tropical fruit, investigated host specificity, and screened whether plant extracts could control the fungi. In total, 53 diseased fruit samples were sampled from eight types of fruit trees (jackfruit, rose apple, mangosteen, plum, snake fruit, rambutan, strawberry, and avocado). Based on morphological characteristics, 44 isolates were classified as belonging to pestalotioid taxa. Of these isolates, seven with distinct characteristics were selected for identification using molecular analysis, and six isolates were identified as Neopestalotiopsis and one as Pseudopestalotiopsis. In the cross-inoculation experiment, the isolates exhibited nonhost specificity and could infect at least two host plants. The isolates were used to screen for a potential biocontrol resource using six crude plant extracts (clove, ginger, lemongrass, mangosteen, roselle, and turmeric). All crude extracts except mangosteen could inhibit the growth of Neopestalotiopsis and Pseudopestalotiopsis. Application of crude plant extracts could be a potential treatment to control these diseases on tropical fruit.

Phytophthora infestans Sporangia Produced in Artificial Media and Plant Lesions Have Subtly Divergent Transcription Profiles but Equivalent Infection Potential and Aggressiveness.

Sporangia of the potato late blight agent Phytophthora infestans are often used in studies of pathogen biology and plant responses to infection. Investigations of spore biology can be challenging in oomycetes because their sporangia are physiologically active and change in response to environmental factors and aging. Whether sporangia from artificial media and plant lesions are functionally equivalent has been a topic of debate. To address these issues, we compared the transcriptomes and infection ability of sporangia from rye-sucrose media, potato and tomato leaflets, and potato tubers. Small differences were observed between the mRNA profiles of sporangia from all sources, including variation in genes encoding metabolic enzymes, cell-wall-degrading enzymes, and ABC transporters. Small differences in sporangia age also resulted in variation in the transcriptome. Taking care to use sporangia of similar maturity, we observed that those sourced from media or plant lesions had similar rates of zoospore release and cyst germination. There were also no differences in infection rates or aggressiveness on leaflets, based on single-spore inoculation assays. Such results are discordant with those of a recent publication in this journal. Nevertheless, we conclude that sporangia from plant and media cultures are functionally similar and emphasize the importance of using "best practices" in experiments with sporangia to obtain reliable results.

Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora spp. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this cis spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.

A MADS-box transcription factor regulates a central step in sporulation of the oomycete Phytophthora infestans.

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.

How Does Phytophthora infestans Evade Control Efforts? Modern Insight Into the Late Blight Disease.

The infamous oomycete Phytophthora infestans has been a persistent threat to potato and tomato production worldwide, causing the diseases known as late blight. This pathogen has proved to be remarkably adept at overcoming control strategies including host-based resistance and fungicides. This review describes the features of P. infestans that make it such a daunting challenge to agriculture. These include a stealthy lifestyle that helps P. infestans evade plant defenses, effectors that suppress host defenses and promote susceptibility, profuse sporulation with a short latent period that enables rapid dissemination, and a genome structure that promotes the adaptive evolution of P. infestans by fostering genetic diversity. Nevertheless, there is reason to be optimistic that accumulated knowledge about the biology of P. infestans and its hosts will lead to improved management of late blight.

Selective determination of homocysteine levels in human plasma using a silver nanoparticle-based colorimetric assay.

The first use of silver nanoparticles (AgNPs) for the rapid, simple, and selective determination of homocysteine (Hcy) levels in human plasma was studied. Hcy and five other amino acids, including cysteine (Cys), could be distinguished by their different aggregation kinetics, which caused a change in the visible color and a shift in the UV-vis absorption spectra. The difference in the cross-linking (aggregation) rate between Hcy and Cys was used as the basis for developing a selective probe for Hcy and allowed the detection of Hcy in the linear range of 2-12 µM (R(2)=0.9936). The limits of detection and quantification were found to be 0.5 µM and 1.7 µM, respectively. To investigate its selectivity and potential applicability, this AgNP-based method was successfully applied for the determination of Hcy levels in actual biological (human plasma) samples, where the determined levels of Hcy were within the error range of the measured level using the traditional chemiluminescence microparticle immunoassay (CMIA). Thus, the use of AgNPs is a feasible and potentially reliable method for the determination of Hcy levels in biological samples.