Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4'',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (≤ 500 μL) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeño peppers.Download video file.^{(57M, mov)}     

Influence of crop background on natural enemies of aphids on Brussels sprouts

The most abundant natural enemies of aphids on Brussels sprout crops were Syrphidae, different species being attracted differentially to weedy or weed-free plots according to whether they were more host-plant-orientated (e.g. Melanostoma spp., Platycheirus spp.) and thus affected directly by the background, or more aphid-orientated (e.g. Syrphus balteatus) and so less affected by background than by aphid numbers. Oviposition by Melanostoma spp. was usually much greater in weedy sprout crops than on sprouts in bare soil, and their eggs were also very abundant on weeds. Eggs of other syrphid species were scarcer on weeds. In contrast to Melanostoma, Platycheirus spp. usually oviposted preferentially on sprouts growing in bare soil. Oviposition by S. balteatus was in response to aphid abundance and thus tended to become greater on sprouts in bare soil. Notably more adults of non-aphido-phagous Syrphidae were caught over weedy than over non-weedy Brussels sprout plants. Anthocoris nemorum nymphs and adults were very common on sprout plants and weeds in the weedy crop but were scarce on sprouts in bare soil; A. nemorum oviposited on white and yellow charlock (Raphanus raphanistrum and Sinapis arvensis) occasionally. Parasitism of Brevicoryne brassicae by Diaeretiella rapae appeared to be related to aphid numbers and was only indirectly influenced by the crop background. Field experiments with green and brown cloth backgrounds showed that some syrphids were attracted to green; A. nemorum was relatively scarce over both artificial backgrounds. It is concluded that decreased natural enemy action is partly responsible for the initially greater abundance of B. brassicae in a weed-free crop of Brussels sprouts than in a weedy crop.     

不同生境下景宁玉兰灌丛萌株形态及其生殖特征

景宁玉兰是萌生能力很强的灌木树种,为了解不同生境下景宁玉兰萌枝形态及其生殖特征,选择了灌丛(SH)、黄山松林(PTF)、落叶阔叶林(DBF)、杉木林(CLF)和林缘(FE)等5种坡向一致的生境类型,通过典型样地调查,比较分析了5种不同生境下景宁玉兰萌枝数量、大小、高度、枝系、开花及结实等性状。结果表明:(1)景宁玉兰在黄山松林下的分布密度最高,但5种生境下景宁玉兰每丛的萌枝数量没有差异,每丛最大萌枝基径对每丛萌枝数有一定影响,但最大萌枝高度与每丛萌枝数没有关系。在所有调查的景宁玉兰居群中,大于2根以上萌枝的灌丛达82.5%,说明萌生更新在种群繁衍中发挥着重要作用。(2)在落叶阔叶林下的萌枝基径显著小于其他生境(P0.05),黄山松林下一级枝粗度和长度显著小于其他生境(P0.05);灌丛中的总分枝率和逐步分枝率最高,灌丛和林缘生境的逐步分枝率(SBR2:3)高于其他生境。(3)生殖萌枝基径(r=0.320,P0.05)和高度(r=0.349,P0.05)与花量均呈显著正相关关系。杉木林下景宁玉兰生殖萌枝的花量显著高于黄山松林、落叶阔叶林和林缘生境(P0.05)。虽然黄山松林下景宁玉兰萌枝开花率及开花萌枝比例最低,但其果实大小、单果种子粒数及每丛结实率却较高(P0.05),(4)景宁玉兰对环境变化极为敏感,生境类型和海拔对萌枝形态和生殖性状均有一定影响。研究表明,景宁玉兰萌生特征主要受其内在生物学特性所控制,而萌枝形态及生殖特征则与其所处环境条件更为密切。     

Evidences for Microbial Precipitation of Calcite in Speleothems from Krem Syndai in Jaintia Hills,Meghalaya, India

Speleothems from Krem Syndai, Meghalaya in Northeast India were studied for their microbial diversity using 16S rDNA-based phylogenetic approach and conventional microbiological techniques along with geochemistry, mineralogy and in vitro experiments to understand participation of microorganisms in CaCO₃ precipitation. Speleothems imaged by scanning electron microscopy showed round coccoid-like, sporangia-like and spinose calcified structures, numerous broken cocci shells with spotted interiors inside a calcite crystal, honeycomb long reticulate, smooth, flat, twisted, ribbon-like, tubular, beaded, microbe-mineralized filaments and extracellular polymeric substances (EPS). Fourier spectroscopy indicated the presence of various organic compounds. δ¹³C and δ¹⁸O isotopic ratios of speleothems ranged from ?4.65 to ?7.34‰ and ?3.06 to ?6.80‰, respectively. Total number of microbial cells using SYBR Gold was high. Fluorescence in situ hybridization (FISH) indicated approximately 3 × 10⁵ to 5 × 10⁵ cells g sed^–1 in the speleothems out of which the number of microbes belonging to Eubacteria ranged from 1.8 × 10⁵ to 3.6 × 10⁵ cells, g sed^–1. FISH showed ～45% active microbial cells of the total cell number in samples. DNA-based high-throughput amplicon sequencing revealed 19 bacterial phyla in the speleothem. Approximately 42% of the sequences were similar to Proteobacteria (Alphaproteobacteria: 22.4%, Betaproteobacteria: 8.9%, Gammaproteobacteria: 8.6%). Sequences similar to Nitrospiraceae (22.8%) had the highest proportion of sequences belonging to a single family. Bacterial strains isolated from the speleothems raised alkalinity and precipitated calcite in the laboratory cultures which was confirmed by X-ray diffraction (XRD) analyses. These isolates belonged to Bacillus spp., Actinomycetes spp., Streptomyces spp., Pseudomonas spp., Micrococcus spp., Staphylococcus spp., Xanthobacter spp. and Arthrobacter spp. Overall, the results showed unequivocal evidence of bacterial fingerprints during CaCO₃ precipitation in the cave.     

SPATIAL DISTRIBUTION AND DEVELOPMENT OF ROOT SPROUTS IN FAGUS GRANDIFOLIA (FAGACEAE)

Root sprouts around 31 Fagus grandifolia parent trees, some declining due to beech bark disease, were studied to describe the pattern of sprout distribution, the ecological importance of this pattern, and the relationship between sprout patterns and parent vigor. Microtome sections of roots were studied to determine the histological origin of sprouts. Sprout distribution was circular and usually centered on the parent tree. Most sprouts were within 8 m of the parent and remained attached to the parent root system even after the sprouts reached 10 yrs of age. Root sprouting in F. grandifolia may be effective in replacing dead parent trees, but the potential for clones to spread is limited. Spatial distribution of sprouts was mostly unaffected by tree vigor. Root sprouts originated from callus tissues associated with wounds on superficial woody roots.     

Quantitative assessment of toxic and nontoxic <Emphasis Type="Italic">Microcystis</Emphasis> colonies in natural environments using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization and flow cytometry

Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.     

Hermetia illucens L. larvae–associated intestinal microbes reduce the transmission risk of zoonotic pathogens in pig manure

Black soldier fly (BSF) larvae are considered a promising biological reactor to convert organic waste and reduce the impact of zoonotic pathogens on the environment. We analysed the effects of BSF larvae on Staphylococcus aureus and Salmonella spp. populations in pig manure (PM), which showed that BSF larvae can significantly reduce the counts of the associated S. aureus and Salmonella spp. Then, using a sterile BSF larval system, we validated the function of BSF larval intestinal microbiota in vivo to suppress pathogens, and lastly, we isolated eight bacterial strains from the BSF larval gut that inhibit S. aureus. Results indicated that functional microbes are essential for BSF larvae to antagonise S. aureus. Moreover, the analysis results of the relationship between the intestinal microbiota and S. aureus and Salmonella spp. showed that Myroides, Tissierella, Oblitimonas, Paenalcalignes, Terrisporobacter, Clostridium, Fastidiosipila, Pseudomonas, Ignatzschineria, Savagea, Moheibacter and Sphingobacterium were negatively correlated with S. aureus and Salmonella. Overall, these results suggested that the potential ability of BSF larvae to inhibit S. aureus and Salmonella spp. present in PM is accomplished primarily by gut-associated microorganisms.     

Influence of crop background on aphids and other phytophagous insects on Brussels sprouts

More Brevicoryne brassicae and other alate aphids were caught in yellow water-traps in a weed-free crop of Brussels sprouts than in a crop with a weedy background. More B. brassicae colonized Brussels sprout plants in bare soil than in weeds; very few alatae were attracted to cruciferous weeds in the crop. Results in 1 yr suggest that initially larger populations on the weed-free sprouts became smaller than populations on the weedy sprouts because the larger aphid population attracted more natural enemies. Aleyrodes brassicae and certain Lepidoptera were also more abundant on sprout plants in bare soil than on sprouts surrounded by weeds; more adult A. brassicae were caught in water traps over the bare soil. More A. brassicae were present on sprout plants surrounded by a green than by a brown cloth background but the differences were not significant (P < 0–05). Numbers of B. brassicae on sprout plants with green and brown backgrounds varied greatly and did not differ significantly. In field cages, B. brassicae alatae were more attracted to potted sprout plants surrounded by bare soil than to ones surrounded by rings of living or cut grass or by artificial green rings. This effect was greater with small than with large sprout plants surrounded by grass rings. The maintenance of a limited weed cover is considered potentially useful in integrated control of some brassica pests.     

Differences in Growth of Salmonella enterica and Escherichia coli O157:H7 on Alfalfa Sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log₁₀ on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log₁₀. The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

The inhibition of potato sprout growth by light.

When potato seed tubers (Solanum tuberosum cv. Pentland Javelin) were stored in darkness or diffuse daylight at 9°C and transferred at intervals to conditions suitable for sprouting, their capacity for sprout growth was unaffected by the presence or absence of light during previous storage. When similar tubers were stored at 10°C, 18°C or 25°C, sprout growth commenced earliest at 25°C, but the date was unaffected by fluorescent light. It was concluded that light did not affect the length of the dormant period, but only the rate of sprout elongation after that period had ceased. When tubers with growing sprouts at 10°C or 18°C were transferred from darkness into fluorescent light, sprout growth virtually ceased. Transfer from light into darkness resulted in immediate sprout growth, at a rate comparable with tubers stored continuously in the dark. Tubers of three Peruvian cultivars, stored in farm-scale diffuse-daylight stores, grew progressively shorter sprouts with increasing daily exposure to light from 30 min to 12 h. Storage of cv. Wilja under 21 Wm^-2 (total) of white fluorescent light for 10 h per day maintained the sprouts at the same length as ten times this light intensity for 1 h per day. In a subsequent experiment with cv. Bintje the 10 h, low-intensity light regime gave slightly shorter sprouts. It appeared that the total light energy falling on the tubers was the dominant factor controlling sprout growth.     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

Florida reef sponges harbor coral disease-associated microbes

Sponges can filter large volumes of seawater and accumulate highly diverse and abundant microbial communities within their tissue. Culture-independent techniques such as fluorescent in situ hybridization (FISH), 16S small subunit (SSU) rRNA gene analyses, and transmission electron microscopy (TEM) were applied to characterize the presence and distribution of microbes within sponges abundant on south Florida reefs. This study found that coral disease-associated bacteria (CDAB) are harbored within Agelas tubulata and Amphimedon compressa. FISH probes detected several potential bacterial pathogens such as Aurantimonas coralicida, Cytophaga sp., Desulfovibrio spp, Serratia marcescans, and Vibrio mediterranei within A. compressa and A. tubulata host sponges. Spatial differences in the distribution of targeted bacteria were seen within sponge hosts. Transmission electron microscopy of A. compressa indicated there was a higher concentration of bacteria in the choanosome compared to the ectosome. These observed spatial distributions support the presence of internal sponge niches, which could play a role in the location of the CDAB within the sponges.     

Rapid and simple detection of the invA gene in Salmonella spp. by isothermal target and probe amplification (iTPA)

Aims: Salmonella spp. are an important cause of food‐borne infections throughout world, and the availability of rapid and simple detection techniques is critical for the food industry. Salmonella enterica serovars Enteritidis and Typhimurium cause the majority of human gastroenteritis infections, and there are a reported 40 000 cases of salmonellosis in the United States each year. Methods and Results: A novel rapid and simple isothermal target and probe amplification (iTPA) assay that rapidly amplifies target DNA (Salmonella invA gene) using a FRET‐based signal probe in an isothermal environment was developed for detection Salmonella spp. in pre‐enriched food samples. The assay was able to specifically detect all of 10 Salmonella spp. strains without detecting 40 non‐Salmonella strains. The detection limit was 4 × 10¹ CFU per assay. The iTPA assay detected at an initial inoculum level of <10 CFU in the pre‐enriched food samples (egg yolk, chicken breast and peanut butter). Conclusions: This detection system requires only a water bath and a fluorometer and has great potential for use as a hand‐held device or point‐of‐care‐testing diagnostics. The iTPA assay is sensitive and specific and has potential for rapid screening of Salmonella spp. by food industry.     

Genetic mapping of QTLs conditioning soybean sprout yield and quality

Soybean sprouts have been used as a food in the Orient since ancient times. In this study, 92 restriction fragment length polymorphism (RFLP) loci and two morphological markers (W1 and T) were used to identify quantitative trait loci (QTLs) associated with soybean sprout-related traits in 100 F₂-derived lines from the cross of ’Pureunkong’×’Jinpumkong 2’. The genetic map consisted of 76 loci which covered about 756 cM and converged into 20 linkage groups. Eighteen markers remained unlinked. Phenotypic data were collected in 1996 and 1997 for hypocotyl length, percentage of abnormal seedlings, and sprout yield 6 days after germination at 20°C. Hypocotyl length was determined as the average length from the point of initiation of the first secondary root to the point of attachment of the cotyledons. The number of decayed seeds and seedlings, plus the number of stunted seedlings (less than 2-cm growth), was recorded a s abnormal seedlings. Seed weight was determined based on the 50-seed sample. Sprout yield was recorded as the total fresh weight of soybean sprouts produced from the 50-seed sample divided by the dry weight of the 50-seed sample. Four QTLs were associated with sprout yield in the combined analysis across 2 years. For the QTL linked to L154 on the Linkage Group (LG) G the positive allele was derived from Pureunkong (R ² = 0.19), whereas at the other three QTLs (A089 on LG B1, A668n on LG K and B046 on LG L) the positive alleles were from Jinpumkong 2. QTLs conditioning seed weight were linked to markers A802n (LG B1), A069 (LG E), Cr321 (LG F) and A235 (LG G). At these four markers, the Jinpumkong allele increased seed weight. Markers K011n on LG B1, W1 on LG F and A757 on LG L were linked to QTLs conditioning hypocotyl length; and Bng119, K455n and K418n to QTLs conditioning the abnormal seedlings. The QTLs conditioning sprout yield were in the same genomic locations as the QTLs for seed weight identified in this population or from previously published research, indicating that QTLs for sprout yield are genetically linked to seed-weight QTLs or else that seed-weight QTLs pleiotropically condition sprout yield. These data demonstrate that effective marker-assisted selection may be feasible for enhancing sprout yield in a soybean. The transgressive segregation of sprout yield, as well as the existence of two QTLs conditioning greater than 10% of the phenotypic variation in sprout yields provides an opportunity to select for progeny lines with a greater sprout yield than currently preferred cultivars such as Pureunkong. Received: 23 August 2000 / Accepted: 23 January 2001     

Coiled-sprout in the potato: the effect of various storage and planting conditions

The incidence of coiled-sprout was determined in Scotch and local-grown Arran Pilot and Duke of York seed tubers which had been stored at 10°, 4° and 15° C. and in a farm store with no temperature control. All four tuber types were planted in the field and, in addition, the two types of Duke of York were planted in Perlite at 7°, 10° and 15° C. In the field, and when maintained at 7° and 10° C, the percentage of sprouts coiling and the intensity of coiling was greater in tubers stored at 10° and 15° C. than at 4° C. There was no coiling when the Duke of York tubers were planted at 15° C. In a further experiment tubers were stored at 20° C. in the light and dark and samples were planted monthly for 3 months at temperatures of 7°, 10° and 15° C. During the following 3-month period only light-stored were planted because of the excessive amount of tip-death in the tubers stored in the dark. There was very little coiling in the dark-stored tubers. In the first two plantings of the light-stored tubers there was virtually no coiling of those planted at 15° C. There was some, however, at 7° and 10° C. In subsequent plantings there was more coiling and no effect of planting temperature. Attempts to isolate Verticiculum nubilum from sprouts were successful in only a small percentage of attempts and it was not possible to demonstrate any difference between its distribution on coiled and normal sprouts. It was not possible to induce coiling by infection of sprouts with spores of V. nubilum. Over a wide range of sprout sizes the amount of coiling was a function of sprout size at planting. However, the parts which coiled were those in the apical bud of the sprout at the time of planting and hence contributed only a small amount to the total sprout size. It is likely, therefore, that the correlation between coiling and sprout size reflects the changing metabolism of the elongating regions of sprouts with their increase in length, these regions developing in such a way as to produce a greater tendency to coiling. The internal reactions concerned in these changes, however, are not known.     

The inheritance of node number and rate of node production in Brussels sprouts

Summary The total vegetative node number, rate of node production and number of sprouts over 13 mm diameter were recorded for 10 F₁ Brussels sprout cultivars and 45 progenies derived by intercrossing and selfing them. Significant differences, resulting from additive gene action, were found between the 10 cultivars and between their progenies for both characters. For total node number there was also evidence of dominant gene action. Total node number and rate of node production were closely correlated as were total node number and the number of harvested sprouts. The factors causing differences in rate of node production are indicated and the relationship of this character to other Brussels sprout yield components is outlined.     

Improved DNA‐FISH for cytometric detection of Candida spp

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l^?1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.     

Spatial structure in the “Plastisphere”: Molecular resources for imaging microscopic communities on plastic marine debris

Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the “Plastisphere”—the thin layer of life on the surface of PMD—has been technology‐limited. Research into microbe–microbe and microbe–substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI‐FISH (combinatorial labelling and spectral imaging – fluorescence in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria, Alpha‐, and Gammaproteobacteria) and four newly designed probes (targeting Bacteroidetes, Vibrionaceae, Rhodobacteraceae and Alteromonadaceae) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell‐count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro‐metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.     

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars

Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO₃ ^–-N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH₄ ⁺ and NO₃ ^–. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH₄ ⁺ and NO₃ ^– with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar.