Reconfiguring the health-promoting hospital: the role of chaplaincy in England.

This paper seeks to explore how hospitals can be reconfigured to adopt more 'health-promoting' approaches and values. Specifically, the paper focuses on the role of hospital chaplaincy and argues that spiritual care should be considered alongside other health domains. Using semi-structured interviews, the aim of the paper is to explore the experiences of patients who accepted (n = 10) and declined (n = 10) hospital chaplaincy services. Data were analysed drawing on principles of interpretative phenomenological analysis (IPA). The findings suggested that participants who accessed chaplaincy services reported using the chaplains for pastoral, religious and spiritual care which contributed positively to their sense of well-being. This included religious rituals and supportive conversations. The majority of these participants had existing links with a faith institution. Participants who declined chaplaincy services reported having personal religious or spiritual beliefs. Other reasons cited, included: that the offer was made close to discharge; they had different support mechanisms; they were unaware of what the chaplaincy service offered. Participants identified a number of skills and attributes they associated with chaplains. They perceived them as being religious but available to all, somebody to talk to who was perceived as impartial with a shared knowledge and understanding. The paper concludes by highlighting the important role of chaplaincy as part of a holistic health-promoting hospital. This has implications not only for the design, delivery and promotion of chaplaincy services but also for health promotion more broadly to consider spiritual needs.

Real-Time PCR Assays for Races of the Spinach Fusarium Wilt Pathogen, Fusarium oxysporum f. sp. spinaciae.

Fusarium wilt of spinach, caused by Fusarium oxysporum f. sp. spinaciae, is a significant limitation for producers of vegetative spinach and spinach seed crops during warm temperatures and/or on acid soils. Identification of isolates of F. oxysporum f. sp. spinaciae, and distinction of isolates of the two known races, entails time-intensive pathogenicity tests. In this study, two real-time PCR assays were developed: one for a candidate effector gene common to both races of F. oxysporum f. sp. spinaciae, and another for a candidate effector gene unique to isolates of race 2. The assays were specific to isolates of F. oxysporum f. sp. spinaciae (n = 44) and isolates of race 2 (n = 23), respectively. Neither assay amplified DNA from 10 avirulent isolates of F. oxysporum associated with spinach, 57 isolates of other formae speciales and Fusarium spp., or 7 isolates of other spinach pathogens. When the assays were used to detect DNA extracted from spinach plants infected with an isolate of race 1, race 2, or a 1:1 mixture of both races, the amount of target DNA detected increased with increasing severity of wilt. Plants infected with one or both isolates could be distinguished based on the ratio in copy number for each target locus. The real-time PCR assays enable rapid diagnosis of Fusarium wilt of spinach and will facilitate research on the epidemiology and management of this disease, as well as surveys on the prevalence of this understudied pathogen in regions of spinach and/or spinach seed production.

Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

A Quantitative PCR Assay for Detection and Quantification of Fusarium sambucinum.

Fusarium sambucinum is an ascomycete that has been isolated from a broad range of plant hosts, including hop (Humulus lupulus L.), where it acts as a causal agent of Fusarium canker, a disease that can impact cone quality and yield in severe cases. Current diagnostic methods rely on isolation of the fungus from plant tissue, a time- and resource-intensive process with limited sensitivity, complicated by the potential presence of other Fusarium spp. that have been reported on hop. Our objective was to develop a rapid and sensitive diagnostic tool to detect and quantify F. sambucinum in plant tissues. Using a modified random amplified polymorphic DNA PCR assay, we identified a F. sambucinum-specific marker that serves as the target in a TaqMan (hydrolysis) probe quantitative PCR (qPCR) assay that can be used to detect F. sambucinum DNA in a background of plant DNA. When used to screen 52 isolates of F. sambucinum and isolates representing 13 other Fusarium spp., the assay was robust in detecting F. sambucinum while discriminating between F. sambucinum and closely related Fusarium spp., including F. venenatum. Furthermore, this assay reliably detects as little as 1 pg of F. sambucinum DNA in a background of total DNA from plant tissue. Within-sample comparisons of this qPCR assay with traditional cultural isolation methods demonstrated the greater sensitivity of the qPCR-based method for detection of F. sambucinum. When used to screen 220 asymptomatic stem samples, the qPCR assay detected F. sambucinum in 100 samples (45.5%); by comparison, F. sambucinum was detected in only 3 samples (1.4%) by culturing methods. Moreover, quantification of F. sambucinum DNA was possible for 60 of these samples, indicating the utility of the qPCR assay for early detection. This assay should be useful in diagnostic and epidemiological applications to detect and quantify F. sambucinum from multiple hosts and environmental samples.

Phytophthora cactorum causing bleeding canker of Acer x freemanii in southern Idaho.

Since 2018, bleeding cankers have been observed on maple trees in multiple home gardens in southwest Idaho. The cankers ooze a dark sap and and are approximately 10 cm to 35 cm in diameter. Cankers typically occur on the main trunk but are also present on scaffold branches in severe infecrions. Symptoms of foliar chlorois, branch dieback, and premature autumn senescence were also associated with the disease. Phytophthora DNA was detected in symptomatic material from five trees using real-time PCR (Miles et al., 2017). In July 2019 recovery of a causal agent from a symptomatic Acer x freemanii tree was attempted. Excisions were made from the interface of healthy and diseased tissue around the cankers using a chisel. The tissue was then placed in sealed plastic ziplock bags at 4°C for 7 days. Hyphae were then removed with forceps and placed onto potato dextrose agar (PDA) amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Colonies resembling Phytophthora cactorum were consistently observed after 5 days at 21°C. Tentative P. cactorum identification was based on the presence of abundant papillate and caducous sporangia on a short pedicel; sporangia were approximately 30 µm long and 26 µm wide (Bush et al., 2006; Hudler, 2013). Individual hyphal tips were transferred to fresh PDA plates and sequencing of both the rDNA ITS region and Cytochrome c oxidase subunit I (COI) was completed for a representative isolate (D19-130). DNA extraction, PCR and sequencing were as previously described (Woodhall et al. 2013; Robideau et al., 2011). The resulting DNA sequences for rDNA ITS (MW315449) and COI (MW881040) were both 100% identical (723/723 bp and 728/728 bp) with sequences from cultures previously identified as P. cactorum (MH171627 and MH136858). To determine pathogenicity, 14 month-old maple (A. x freemanii) trees in individual containers with potting mix were wounded 15 mm above the soil line with a single 10 mm incision using a sterile razor blade and inoculated by placing a 10 mm2 fully colonized PDA plug of isolate D19-130 on the wound. The inoculum and wound were then covered with a damp cotton ball that was secured loosely with parafilm. Control plants consisted of uninoculated plants and wounded plants inoculated with a PDA agar plug. Each treatment was replicated five times and placed in a controlled environment chamber set at 24ºC and 90% relative humidity. All treatments were sprayed with water daily to ensure the cotton balls remained damp. After 8 weeks, black lesions, up to approximately 25 mm above the soil line, were observed on the stem base of all P. cactorum-inoculated plants. No black lesions were observed on non-inoculated plants or plants inoculated with a PDA agar plug. P. cactorum was isolated from lesions, as described above, except polystyrene foam boxes containing moist paper towels were used instead of bags. This report confirms P. cactorum as a causal agent of bleeding canker of maple in Idaho for the first time. It has been shown that several Phytophthora species can infect maple (Jung and Burgess, 2009; Huddler, 2013). P. cactorum has a wide host range but certain strains have been associated with lethal bleeding stem cankers in maple and other deciduous trees worldwide (Huddler, 2013). Knowledge of the causal agent of bleeding canker on maple will help determine appropriate disease management practices.

Yield Losses and Control by Sedaxane and Fludioxonil of Soilborne Rhizoctonia, Microdochium, and Fusarium Species in Winter Wheat.

Soilborne Rhizoctonia, Microdochium, and Fusarium species are major causal agents of seedling and stem-base diseases of wheat. Currently, seed treatments are considered the most effective solution for their control. Rhizoctonia solani anastomosis groups (AGs) 2-1 and 5, R. cerealis, Microdochium, and Fusarium spp., were used in series of field experiments to determine their capability to cause soilborne and stem-base disease and to quantify their comparative losses in the establishment and yield of wheat. The effectiveness and response to seed treatment formulated with 10 g sedaxane and 5 g fludioxonil 100 kg-1 against these soilborne pathogens were also determined. Our results showed that damping-off caused by soilborne R. cerealis was associated with significant reductions in the emergence and establishment, resulting in stunted growth and low plant numbers. The pathogen also caused sharp eyespot associated with reductions in the ear partitioning index. R. solani AG 2-1 and AG 5 were weakly pathogenic and failed to cause significant damping-off, root rot, and stem-base disease in wheat. Fusarium graminearum and F. culmorum applied as soilborne inoculum failed to cause severe disease. Microdochium spp. caused brown foot rot disease and soilborne M. nivale reduced wheat emergence. Applications of sedaxane and fludioxonil increased plant emergence and reduced damping-off, early stem-base disease, and brown foot rot, thus providing protection against multiple soilborne pathogens. R. cerealis reduced the thousand grain weight by 3.6%, whereas seed treatment including fludioxonil and sedaxane against soilborne R. cerealis or M. nivale resulted in a 4% yield increase.

Rhizoctonia solani AG2-1 causing root rot of wasabi (Eutrema japonica) in the UK.

In 2014, glasshouse-grown wasabi (Eutrema japonica) grown in a compost based media displayed symptoms of poor growth and wilting. Visual assessment of the roots showed that 25% of the symptomatic plants sampled had raised black lesions on the roots affecting between 5 and 20% of the total root area. To isolate the causal agent, affected material (approximately 5 mm3) was surface disinfested in sodium hypochlorite (2%) for 30 s, rinsed twice in sterile water and plated on to water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Plates were incubated at 20ºC until fungal colonies were visible. After three days, colonies of Rhizoctonia solani were identified based on the presence of septate hyphae with right-angle branching, a pure culture was obtained through hyphal tip transfer onto a new plate of PDA. DNA was extracted from a 7-day old plate of the isolate (WAS1) as described previously (Woodhall et al., 2013). The AG of WAS1 was determined as AG2-1 using a subgroup specific real-time PCR assay (Budge et al., 2009b) and confirmed by DNA sequencing as described previously (Lekuona Gomez et al., 2015). The sequence was 100% identical (587/587bp) to a previously identified AG2-1 isolate 1971 (GenBank accession FJ435126) (Budge et al., (2009a). Pathogenicity of the isolate was confirmed by inoculating three healthy one-year-old wasabi plants grown in loam based compost (John Innes No.3) each with four 5 mm fully colonised PDA plugs of isolate WAS1 placed at approx. 40 mm depth in the soil. Four sterile PDA plugs were place in each of three control plants. All six plants were placed in a greenhouse at 21°C, 18h:6h light: dark and watered as required. After 21 days, multiple black root lesions typically 3-5mm in length were observed on the roots of all inoculated plants. No lesions were observed on the control plants. From three lesions per plant, isolations were attempted as described above. Rhizoctonia solani was recovered from all isolations and the resulting cultures all tested positive for AG2-1 using the real-time PCR assay. Isolations were attempted from the roots of healthy control plants but Rhizoctonia was not recovered. Here we demonstrate that R. solani AG2-1 is associated with root necrosis of Eutrema japonica. Rhizoctonia solani AG2-1 has been reported previously in various Brassica crops in the UK (Budge et al., 2009a) and on Matthiola incana (Lekuona Gómez et al., 2015). It has also been reported causing disease in potatoes and as widely present in UK field soils (Woodhall et al., 2013). Although R. solani AG1 and AG4 of R. solani have been reported to infect Eutrema japonica in Japan (Takeuchi et al., 2003; 2008), this is the first finding that identifies AG2-1 as the causal agent. The potential presence of AG2-1 in soil and/or as plant debris should be considered prior to planting susceptible hosts.

First report of rubbery rot of potato caused by Geotrichum candidum in the United States.

Rubbery rot of potato caused by Geotrichum candidum Link is characterized by symptoms of damp, flaccid tubers that feel rubbery when squeezed (Humpreys-Jones 1969), similar in consistency to potato diseases such as pink rot (caused by Phytophthora erythroseptica) and Pythium leak (caused by species of Pythium). In November 2019, several symptomatic tubers of potato variety 'Ciklamen' that had been held in storage since harvest and originated from an over-head irrigated, sandy-loam production field in Bingham county, Idaho were submitted to the University of Idaho for diagnosis. Shipping-point inspection records indicated 4-9% of tubers were affected. External symptoms included irregularly shaped, randomly located sunken black-colored lesions on more severely affected rubbery-textured tubers. When cut, internal affected tissue developed a greyish appearance after several minutes. Lens-shaped cavities were apparent in two of the tubers, indicating an advanced infection. A sour-milk smell accompanied the sample. To isolate the pathogen, pieces of tuber tissue approximately 5 mm in diameter were collected from the margins of symptomatic areas and surface-sanitized in sodium hypochlorite (2%) for two minutes, rinsed twice in sterile water and plated onto tap water agar amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After three days at 21°C, colonies having distinct creamy white mycelia, a sweet, juniper-like odor, and hyaline hyphae were consistently associated with diseased tissue. Cylindrical to oval-shaped arthroconidia ranged in size from 6.6-11.0 × 3.2-5.9 µm (mean = 8.4 × 4.7, n=21), within dimensions as reported by Carmichael (1957). No other pathogens including species of Pythium and Phytophthora were recovered from the sample. Pure cultures were obtained by transferring hyphal tips to potato dextrose agar plates. Species identity was confirmed via rDNA ITS sequencing using primers ITS5/4 (White et al., 1990). DNA extraction and PCR conditions were as previously described (Woodhall et al., 2013). Resulting sequences (NCBI accession numbers MT893312 and MT893315) shared 99.4% identity with G. candidum Accession KY103453.1 on GenBank. To confirm pathogenicity, ten tubers (cv. Ciklamen) were inoculated by placing a 10mm2 plug of fully colonized PDA of G. candidum on the tuber surface, and ten tubers were mock inoculated with sterile PDA plugs. After 27 days at 21C in a dew chamber, tubers were examined for symptoms. Eight of the 10 inoculated tubers exhibited a rubbery texture and fluid leaking from tubers when cut, with two tubers also exhibiting a grey internal discoloration and the distinctive smell. Control tubers did not exhibit any symptoms. Isolations were attempted from four symptomatic tubers and G. candidum was successfully recovered from three tubers. The disease has been reported sporadically in the United Kingdom (Humphreys-Jones 1969) and Korea (Kim et al., 2011) and the pathogen occurs worldwide (Carmichael 1957). Though the fungus causes a tomato rot in the United States (US) (Pritchard and Porte, 1923; Bourret et al., 2013), and potatoes with rubbery rot originating from Australia were intercepted at a US port (Farr et al., 2020), the disease has not to our knowledge been documented on potato grown in the US. Because symptoms may be confused with pink rot and Pythium leak, it is critical for producers to obtain a correct diagnosis to facilitate appropriate management strategies.

Rhizoctonia solani AG 2-2 IIIB causing root rot of onion in Idaho.

In September of 2018, onion plants (Allium cepa cv. Joaquin) grown in one field in southwest Idaho were observed to have roots with brown discoloration over 10-20% of the total root surface area. Approximately 10% of plants over a 1 ha area were affected and these plants were about visually 50% smaller than the typical bulb size present in the field. To determine the causal agent, 3 mm pieces of symptomatic roots from four plants were placed in sodium hypochlorite (2%) for one minute, followed by two rinses in sterile water and plated on to water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After 3 days at 21°C, fungal colonies with septate hyphae with right-angled branching resembling Rhizoctonia solani were observed in over half of the 16 isolations attempted. Species identity was confirmed through rDNA ITS sequencing, as described previously (Woodhall et al., 2013), with DNA obtained from a single representative hyphal tip culture grown on Potato Dextrose Agar (PDA) which was designated isolate ON3. The resulting sequence (MT672318), was 100% identical (678/678bp) to a sequence previously identified as R. solani AG 2-2 IIIB on GenBank (FJ492137). Pathogenicity of the culture was determined by inoculating ten 20-day-old plants (cv. Joaquin) grown in premium potting compost (Scotts) with a single, fully colonized 10 mm2 plug taken from a 2-week-old PDA culture of isolate ON3. A further nine plants were inoculated with sterile PDA plugs as controls. Plants were grown in the greenhouse at 21C in a 16-hour light regime. After 24 days, each plant was assessed for root rot disease as described previously (Misawa et al. 2017). Root rot was observed on nine of the inoculated plants. Mean diseased root area was 32% of the total root surface, with a minimum of 5% and a maximum of 100% diseased root area and a standard deviation equal to 39.6. No root browning was observed on any of the control plants. Isolations were attempted from nine symptomatic plants and R. solani was successfully isolated from seven plant samples onto water agar. Sequencing was used to confirm identity as AG2-2IIIB. To our knowledge, this is the first report of R. solani AG 2-2 IIIB affecting onions in Idaho. Previous work in the Pacific Northwest recovered R. solani AG2-1, 3, 4 and 8 and also BNR AG A from stunted onions (Patzek et al., 2013). In Japan, Misawa et al. (2017) found AG 2-2 IIIB to be pathogenic to Welsh onion (Allium fistulosum). In Idaho, R. solani AG 2-2 IIIB has was previously reported causing disease in sugar beets (Strausbaugh et al. 2011) and potatoes (Woodhall et al. 2012). Growers should consider crop rotation strategies or soil treatments if R. solani AG2-2IIIB is causing disease in their crops.

First report of Fusarium proliferatum causing necrotic leaf lesions and bulb rot on storage onion (Allium cepa) in southwestern Idaho.

In September 2014, a high rate of bulb rot (5-15% depending on producer) was reported across all cultivars developing early in the storage season in the onion producing region of southwestern Idaho. Spanish yellow onion bulbs cv. Vaquero displaying tan to light brown necrotic rot were obtained. The bulb rot originated in the neck and spread to successive scales (Figure 1). In August 2015, onion cv. Redwing and Vaquero were observed to have wet necrotic lesions developing on leaves in the field (Figure 2). Margins of necrotic tissue, 1-2 cm3, were excised, surface sterilized, plated on water agar medium and incubated at 24°C. Hyphal growth was sub-cultured from eight strains (A- D in 2014; E-H in 2015) to fresh potato dextrose agar to obtain pure cultures. Cultures were characteristic of Fusarium species as described by Nelson et al. (1983) with the presence of microconidia formed on polyphialides with macroconidia present. Primers ITS4-A1 and ITS5 primers (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 1998); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999) were used to amplify regions of the ITS, elongation factor 1-α and the second largest subunit of DNA-directed RNA polymerase II. Amplicons were sequenced and analyzed using BLAST (https://www.ncbi.nlm.nih.gov/) and in combination using Pairwise DNA Alignment and Polyphasic Identification (http://www.westerdijkinstitute.nl/Fusarium/DefaultInfo.aspx?Page=Home) as described by O'Donnell et al. 2015. Analysis indicated that these strains are Fusarium proliferatum, which is part of the F. fujikuroi species complex (O'Donnell et al. 1998). Similarity (99.5%) was observed in pairwise analyses and the polyphasic identification clustering to representative F. proliferatum strain NRRL 22944 and others. Sequences were submitted to Genbank and registered accession numbers are found in Table 1. To complete Koch's postulates, cv. Vaquero onion bulbs were surface sterilized and injected with 3 × 105 microconidia into the shoulder of each bulb. Five bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 30°C in the dark. Five bulbs injected with sterile water and five non-inoculated bulbs served as controls. After 14 days, each bulb was sliced vertically down the center and inspected for rot. All eight strains induced tan to light brown necrotic rot symptoms in each inoculated bulb. No symptoms were observed for the water inoculated and the non-inoculated onion bulbs. A fungus was isolated from the necrotic tissue and confirmed to be F. proliferatum as described above. Ten µl aliquots containing 1 × 105 microconidia of F. proliferatum strains (C, E-H) were applied to leaves in triplicate of 12-week-old onion plants (cv. Vaquero) wounded with a 21-gauge needle. Water controls were included. Within three days lesions, with light chlorosis, began to form and quickly spread on the leaves. A fungus was isolated and confirmed to be F. proliferatum as described above. This is the first extensive description and identification of F. proliferatum causing bulb rot in storage in Idaho (Mohan et al. 1997). In addition, this is the first report of the fungus causing leaf infection in the field. These findings confirm F. proliferatum as the causal agent of the high incidence of bulb rot observed in 2014 and 2015. This bulb rot continues to occur in southwestern Idaho and since the pathogen can cause leaf infections growers are encouraged to be vigilant for both leaf lesions during the growing season and bulb rot in storage.

Genomics Evolutionary History and Diagnostics of the Alternaria alternata Species Group Including Apple and Asian Pear Pathotypes.

The Alternaria section alternaria (Alternaria alternata species group) represents a diverse group of saprotroph, human allergens, and plant pathogens. Alternaria taxonomy has benefited from recent phylogenetic revision but the basis of differentiation between major phylogenetic clades within the group is not yet understood. Furthermore, genomic resources have been limited for the study of host-specific pathotypes. We report near complete genomes of the apple and Asian pear pathotypes as well as draft assemblies for a further 10 isolates representing Alternaria tenuissima and Alternaria arborescens lineages. These assemblies provide the first insights into differentiation of these taxa as well as allowing the description of effector and non-effector profiles of apple and pear conditionally dispensable chromosomes (CDCs). We define the phylogenetic relationship between the isolates sequenced in this study and a further 23 Alternaria spp. based on available genomes. We determine which of these genomes represent MAT1-1-1 or MAT1-2-1 idiomorphs and designate host-specific pathotypes. We show for the first time that the apple pathotype is polyphyletic, present in both the A. arborescens and A. tenuissima lineages. Furthermore, we profile a wider set of 89 isolates for both mating type idiomorphs and toxin gene markers. Mating-type distribution indicated that gene flow has occurred since the formation of A. tenuissima and A. arborescens lineages. We also developed primers designed to AMT14, a gene from the apple pathotype toxin gene cluster with homologs in all tested pathotypes. These primers allow identification and differentiation of apple, pear, and strawberry pathotypes, providing new tools for pathogen diagnostics.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Relative Contribution of Seed Tuber- and Soilborne Inoculum to Potato Disease Development and Changes in the Population Genetic Structure of Rhizoctonia solani AG 3-PT under Field Conditions in South Africa.

Understanding the contribution of seed tuber- and soilborne inocula of Rhizoctonia solani AG 3-PT in causing potato disease epidemics is an important step in implementing effective management strategies for the pathogen. A 2-year study was conducted to evaluate the contribution of each source of inoculum using an integrative experimental approach combining field trials and molecular techniques. Two distinct sets of genetically marked isolates were used as seed tuberborne and soilborne inocula in a mark-release-recapture experiment. Disease assessments were done during tuber initiation and at tuber harvest. Both inoculum sources were found to be equally important in causing black scurf disease, whereas soilborne inocula appeared to be more important for root and stolon infection, and seedborne inocula contributed more to stem canker. However, seed tuber-transmitted genotypes accounted for 60% of the total recovered isolates when genotyped using three polymerase chain reaction restriction fragment length polymorphism markers. The changes in population structure of the experimental R. solani population over the course of the growing season and across two growing seasons were investigated using eight microsatellite markers. The populations at different sampling times were somewhat genetically differentiated, as indicated by Nei's gene diversity (0.24 to 0.27) and the fixation index (FST). The proportion of isolates with genotypes that differed from the inoculants ranged from 13 to 16% in 2013 and 2014, respectively, suggesting the possibility of emergence of new genotypes in the field. Because both soilborne and tuberborne inocula are critical, it is important to ensure the use of pathogen-free seed tubers to eliminate seed tuberborne inoculum and the introduction of new genotypes of R. solani for sustainable potato production in South Africa.

Population genetic structure of Rhizoctonia solani AG 3-PT from potatoes in South Africa.

Rhizoctonia solani AG 3-PT is an important potato pathogen causing significant yield and quality losses in potato production. However, little is known about the levels of genetic diversity and structure of this pathogen in South Africa. A total of 114 R. solani AG 3-PT isolates collected from four geographic regions were analysed for genetic diversity and structure using eight microsatellite loci. Microsatellite analysis found high intra-population genetic diversity, population differentiation and evidence of recombination. A total of 78 multilocus genotypes were identified with few shared among populations. Low levels of clonality (13-39 %) and high levels of population differentiation were observed among populations. Most of the loci were in Hardy-Weinberg equilibrium and all four populations showed evidence of a mixed reproductive mode of both clonality and recombination. The PCoA clustering method revealed genetically distinct geographic populations of R. solani AG 3-PT in South Africa. This study showed that populations of R. solani AG 3-PT in South Africa are genetically differentiated and disease management strategies should be applied accordingly. This is the first study of the population genetics of R. solani AG 3-PT in South Africa and results may help to develop knowledge-based disease management strategies.

Discrete lineages within Alternaria alternata species group: Identification using new highly variable loci and support from morphological characters.

The Alternaria alternata species group is ubiquitous in the environment acting as saprotrophs, human allergens, and plant pathogens. Many morphological species have been described within the group and it is unclear whether these represent re-descriptions of the same species or discrete evolutionary taxa. Sequencing of five loci identified three major lineages within the A. alternata species group. These loci included three new phylogenetic loci (TMA22, PGS1, and REV3) identified as highly variable based on publically available genome sequence data for Dothideomycete species. Lineages were identified as A. alternata ssp. arborescens, A. alternata ssp. tenuissima, and A. alternata ssp. gaisen in accordance with the placement of reference isolates. The phylogenetic results were supported by morphological analysis, which differentiated strains in A. alternata ssp. arborescens and A. alternata ssp. tenuissima and also aligned with previous morphological species descriptions for A. arborescens and A. tenuissima. However, phylogenetic analysis placed the morphologically described species A. alternata and A. mali within the A. alternata ssp. tenuissima and did not support them as discrete taxa. As A. alternata are of phytosanitary importance, the molecular loci used in this study offer new opportunities for molecular identification of isolates by national plant protection organizations.

Effects of damping-off caused by Rhizoctonia solani anastomosis group 2-1 on roots of wheat and oil seed rape quantified using X-ray Computed Tomography and real-time PCR.

Rhizoctonia solani is a plant pathogenic fungus that causes significant establishment and yield losses to several important food crops globally. This is the first application of high resolution X-ray micro Computed Tomography (X-ray µCT) and real-time PCR to study host-pathogen interactions in situ and elucidate the mechanism of Rhizoctonia damping-off disease over a 6-day period caused by R. solani, anastomosis group (AG) 2-1 in wheat (Triticum aestivum cv. Gallant) and oil seed rape (OSR, Brassica napus cv. Marinka). Temporal, non-destructive analysis of root system architectures was performed using RooTrak and validated by the destructive method of root washing. Disease was assessed visually and related to pathogen DNA quantification in soil using real-time PCR. R. solani AG2-1 at similar initial DNA concentrations in soil was capable of causing significant damage to the developing root systems of both wheat and OSR. Disease caused reductions in primary root number, root volume, root surface area, and convex hull which were affected less in the monocotyledonous host. Wheat was more tolerant to the pathogen, exhibited fewer symptoms and developed more complex root systems. In contrast, R. solani caused earlier damage and maceration of the taproot of the dicot, OSR. Disease severity was related to pathogen DNA accumulation in soil only for OSR, however, reductions in root traits were significantly associated with both disease and pathogen DNA. The method offers the first steps in advancing current understanding of soil-borne pathogen behavior in situ at the pore scale, which may lead to the development of mitigation measures to combat disease influence in the field.

Do particle size and surface functionality affect uptake and depuration of gold nanoparticles by aquatic invertebrates?

Because of the widespread use of engineered nanoparticles (ENPs) in consumer and industrial products, it is inevitable that these materials will enter the environment. It is often stated that the uptake of ENPs into organisms in the environment is related to the particle size and surface functionality. To test this assumption, the present study investigated the uptake and depuration of gold nanoparticle (Au NPs) coated with either citrate (Au-citrate NPs), mercaptoundecanoic acid (Au-MUDA NPs), amino polyethylene glycol (PEG) thiol (Au-NH2 NPs), or PEG (Au-PEG NP) by the aquatic invertebrate Gammarus pulex. The studies were performed using a range of standard ecotoxicity media and natural waters, resulting in varying degrees of aggregation of the different NPs. Uptake of gold by G. pulex varied depending on the surface coatings, with Au-MUDA and Au-citrate NPs being taken up to a greater extent than Au-NH2 and Au-PEG NPs in all test media and natural waters. In all test media evaluated, higher amounts of amino and PEG-coated ENPs were eliminated compared with MUDA- and citrate-coated ENPs. No obvious relationships were seen between the aggregation state of the different Au NPs in treatment and uptake, suggesting that the widely accepted assumption that Au NP uptake is related to particle size does not hold for the range of aggregation states studied (67.1-178.8 nm). Positive correlations between particle number concentration in the media and uptake were observed, indicating that this factor might partly explain the differences in uptake of a particle from different media types.

Regulatory ecotoxicity testing of engineered nanoparticles: are the results relevant to the natural environment?

Engineered nanoparticles (ENPs) will be released to the environment during use or following the disposal of ENP-containing products and concerns have been raised over the risks of ENPs to the environment. Many studies have explored the toxicity of ENPs to aquatic organisms but these studies have usually been performed with little understanding of the ENPs' behaviour in the test media and the relationship between behaviour in the media to behaviour in natural waters. This study evaluated and compared the aggregation behaviour of four model gold nanoparticle (NP) types (coated with neutral, negative, positive and amphoteric cappings) in standard ecotoxicity test media and natural waters. The effects of humic acid (HA) and test organisms on aggregation were also investigated. In standard media, positive and neutral NPs were stable, whereas amphoteric and negative NPs generally showed substantial aggregation. In natural waters, amphoteric NPs were generally found to be stable, neutral and positive NPs showed substantial aggregation while negative NPs were stable in some waters and unstable in others. HA addition stabilised the amphoteric NPs, destabilised the positive NPs and had no effect on stability of negative NPs. The presence of invertebrates generally lowered the degree of particle aggregation while macrophytes had no effect. Given the dramatically different behaviours of ENPs in various standard media and natural waters, current regulatory testing may either under- or overestimate the toxicity of nanomaterials to aquatic organisms. Therefore, there is a pressing need to employ ecotoxicity media which better represent the behaviour of ENPs in natural system.

Molecular techniques for pathogen identification and fungus detection in the environment.

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.