Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically important plant pathogens.

Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using the ITS6-ITS7 primer set that targets the ITS1 region. While this primer set shows a perfect match to most oomycete taxa, ITS7 contains 3 mismatches to the corresponding binding site of plant pathogens within the genus Aphanomyces. Polymerase chain reaction (PCR) efficiency differs for taxa with uneven primer matching characteristics, which may explain why previous studies have detected this genus at low abundance. To overcome the impact of these mismatches on PCR sensitivity, the mismatched nucleotides were replaced with degenerate nucleotides. Oomycete communities from 35 soil samples collected from asymptomatic and root rot diseased sites in pea fields across Alberta were analyzed simultaneously using ITS6-ITS7 and ITS6-ITS7-a.e. (modified version of ITS7) primer sets on 1 Illumina MiSeq run. The number of high-quality reads obtained by ITS6-ITS7-a.e. was more than twice that of ITS6-ITS7. The relative abundance of Pythium spp. was reduced and Aphanomyces spp. increased. Aphanomyces cf. cladogamus and Aphanomyces euteiches were the second and third most abundant species, respectively, in the pea rhizosphere using the ITS7-a.e. primer, but were rare using the ITS7 primer. These results indicate that use of ITS7-a.e. provides a more accurate picture of oomycete communities than ITS7 by enhancing PCR sensitivity to Aphanomyces.

Metagenomic analysis of oomycete communities from the rhizosphere of field pea on the Canadian prairies.

Oomycetes are a diverse group of microorganisms; however, little is known about their composition and biodiversity in agroecosystems. Illumina MiSeq was used to determine the type and abundance of oomycetes associated with pea root rot in the Canadian prairies. Additional objectives of the study were to identify differences in oomycete communities associated with pea root health and compare oomycete communities among the 3 prairie provinces, where field peas are commonly cultivated. Samples of soil from the rhizosphere of field pea (Pisum sativum L.) were collected from patches of asymptomatic or diseased plants from 26 commercial fields in 2013 and 2014. Oomycete communities were characterized using metagenomic analysis of the ITS1 region on Illumina MiSeq. From 105 identified operational taxonomic units (OTUs), 45 and 16 oomycete OTUs were identified at species and genus levels, respectively. Pythium was the most prevalent genus and Pythium heterothallicum the most prevalent species in all 3 provinces in both 2013 and 2014. Aphanomyces euteiches, a very important pea root rot pathogen in regions of the prairies, was detected in 57% of sites but at very low abundance (<0.2%). Multivariate analysis revealed differences in the relative abundance of species in oomycete communities between asymptomatic and diseased sites, and among years and provinces. This study demonstrated that deep amplicon sequencing can provide information on the composition and diversity of oomycete communities in agricultural soils.

Temporal Dynamics of Botrytis cinerea and Sclerotinia sclerotiorum in Seed Alfalfa Fields of Southern Alberta, Canada.

Blossom blight of seed alfalfa (Medicago sativa L.), caused by the fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum, is a potentially devastating disease on the Canadian Prairies in moist growing seasons. Monitoring the airborne spore concentrations of these pathogens could facilitate disease forecasting in the region. Nineteen seed alfalfa fields in southern Alberta, Canada were assessed throughout the growing seasons of 2014 and 2015. Trace levels of blossom blight symptoms were found in both years; however, plated floret and pod samples indicated that, overall, B. cinerea increased over the growing season whereas S. sclerotiorum decreased. In both seasons, Burkard 7-day volumetric spore samplers collected daily aerosol samples, and weather stations recorded environmental variables in three fields. Conidia and ascospores collected were quantified each day with a real-time polymerase chain reaction assay. Spore quantification indicated that both B. cinerea and S. sclerotiorum numbers remained low in July and increased in August. Both species had multiple days with high spore discharge, with seasonal maxima of 21,137 conidia and 2,265 ascospores. Exploratory model development indicated that spore discharge of both fungi is associated with environmental stressors such as large changes in relative humidity or high temperatures on preceding days.

A multiplex PCR assay for the detection and quantification of Sclerotinia sclerotiorum and Botrytis cinerea.

UNLABELLED: Traditional culture methods for identifying the plant fungal pathogens Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow and laborious. The goal of this study was to develop a multiplex real-time PCR (qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the assay increased its specificity: when the primer/probe set was tested against 21 fungal species (35 strains), amplification was detected from all S. sclerotiorum strains and no other species. For qPCR, the SsIGS_5 primer and probe set exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2)  = 0·99). SsIGS_5 was then multiplexed with a previously published primer/probe set for B. cinerea to develop a high-throughput method for the detection and quantification of DNA from both pathogens. When multiplexed, the sensitivity and specificity of both assays were not different from individual qPCR reactions. The multiplex assay is currently being used to detect and quantify S. sclerotiorum and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa fields. SIGNIFICANCE AND IMPACT OF THE STUDY: A primer and probe set for the quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The probe-based nature of this assay signifies an improvement over previous assays for this species by allowing multiplex reactions while maintaining high sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid analysis of environmental samples. In crops susceptible to both pathogens, this multiplex assay can be used to quickly quantify the presence of each pathogen.

First Report of Root Rot of Field Pea Caused by Aphanomyces euteiches in Alberta, Canada.

In recent years, root rots have severely impacted yields of field pea (Pisum sativum L.) in the Canadian province of Alberta. Above-normal precipitation levels in the springs of 2011 to 2013 led to the hypothesis that Aphanomyces euteiches Drechsler may play a role in root rot in water-saturated pea fields. To determine causal agent(s) of root rot, 145 pea fields were surveyed at flowering in July 2013 (1). Symptoms of root rot were abundant; the most prominent included red vascular streaking and dark brown rot of the tap root, indicative of Fusarium spp., but brown discoloration and cortical decay of lateral roots, indicative of A. euteiches, was also observed. Total genomic DNA was extracted from diseased root samples from each field, using the Qiagen DNeasy Plant kit, and amplified with species-specific primers for A. euteiches (2). Fusarium spp. were present in all fields, but seven fields located within a 200-km radius yielded a positive reaction for A. euteiches. Five fields were re-visited in May 2014 to collect soil for a bait test (3). Tests were performed using surface-sterilized pea seeds (cv. CDC Meadow) treated with Allegiance FL (Bayer, a.i. metalaxyl) at a rate of 110 ml/kg of seed. Five seeds per pot were planted into field soils in 10-cm pots with 12 replicate pots per field. Soils were irrigated as needed until the second-node stage and then kept at saturation for 14 days. Thirty day-old pea roots were evaluated for root rot symptoms; plated onto cornmeal agar amended with metalaxyl, benomyl, and vancomycin (MBV) without surface sterilization; and visualized microscopically for presence of oospores in the roots. Roots from three out of the five field soils showed symptoms typical of A. euteiches infection, including honey-brown discoloration, degradation of the root cortex, and presence of oospores. Root rot symptoms from the remaining fields were characteristic of Fusarium root rot, and oospores were not observed in roots. Fungal cultures with fast-growing, white, aerial mycelia characteristic of A. euteiches on MBV, were recovered from roots with Aphanomyces root rot symptoms, and transferred to PDA. To confirm pathogen identity, total DNA was extracted from 7-day-old cultures growing on PDA using the Qiagen DNeasy Plant Kit. The ribosomal DNA internal transcribed spacer (ITS) region was amplified using the primer pair ITS1 and ITS4 and sequenced (4). The sequences, deposited in GenBank with accession numbers KM486065, KM486066, and KM486067, were 100% identical to the ITS rDNA sequence of several isolates of A. euteiches using a BLASTn query. Fusarium spp. were also recovered from all root samples in the soil bait test. Total DNA extracted from roots was used in PCR assays with A. euteiches-specific primers as described above. PCR amplification of root DNA was successful only from the same three fields that showed Aphanomcyces root rot symptoms, further verifying presence of A. euteiches. The inability to detect or recover A. euteiches from two fields that had tested positive in the survey was likely due to patchy distribution of this pathogen and emphasizes the importance of rigorous soil collection methods to accurately detect pathogens. Although this is the first record of A. euteiches on field pea in Alberta, the distribution of A. euteiches within a 200-km radius in southern Alberta indicates that it has likely been present in soils for several years. The interaction between A. euteiches and Fusarium spp. infection in the root rot complex of field pea and their impact on field pea production in Alberta is currently being investigated. References: (1) S. Chatterton et al. Can. Plant Dis. Surv. 94:189, 2014. (2) C. Gangneux et al. Phytopathology 104:1138, 2014. (3) D. Malvick et al. Plant Dis. 78:361, 1994. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Net Carbon Gain and Growth of Bell Peppers, Capsicum annuum 'Cubico', Following Root Infection by Pythium aphanidermatum.

ABSTRACT The first characterization of alterations in whole-plant photosynthetic rate and carbon assimilation of bell peppers associated with infection by Pythium aphanidermatum is described. Relationships of root disease caused by P. aphanidermatum to whole-plant net carbon exchange rate (NCER), total carbon accumulation, dark respiration rates, water loss, and destructive growth parameters were quantified in vegetative, hydroponically grown pepper plants (Capsicum annuum 'Cubico'). Inoculated plants displayed lower whole-plant NCER. This translated into a loss of 28% in cumulative C gain during 7 days after inoculation and occurred before visible shoot symptoms developed. Leaf area and dry weight of shoots and roots were significantly decreased and the shoot/root ratio was higher in inoculated plants than in noninoculated plants. We propose that reduced NCER in inoculated plants was mainly due to restricted development of leaf area, because no differences in NCER and evapotranspiration were observed between control and inoculated plants when expressed based on leaf area and root dry mass, respectively. These findings indicate that Pythium infection did not affect the photosynthetic apparatus directly and that the reductions in photosynthesis and growth were not caused by inefficient water transport by diseased roots. These results enlarge on the understanding of physiological responses of host plants to early stages of root disease.

Communication skills workshops in learning disability nursing.

This article investigates the effects of a combination of direct speech, language therapy and staff training on the communication skills of nursing and support staff working with people who have a severe learning disability and challenging behaviour. The approach aimed to help staff choose, prioritize and implement changes in communication. The input was effective in increasing the knowledge of the staff group with regard to challenging behaviour and communication breakdown, and in changing the communicative behaviour of staff. The changes measured were those of longer interactions occurring between staff and service users, and the use of non-verbal communication to support interactions. The implementation of the communication strategies is being audited.

Intestinal bacteria in patients with tropical sprue.

Ten samples of jejunal fluid from patients with tropical sprue and 11 from controls without this disease were examined bacteriologically, using techniques designed to obtain the optimal yield of aerobic and anaerobic bacteria. Total colony counts did not differ significantly in the two groups studied. The organisms isolated comprised a wide range of aerobic and anaerobic bacteria. With the exception of Haemophilus species, no bacterial group was isolated in significantly greater numbers in sprue patients than in controls. The pathogenesis of tropical sprue in Natal is discussed in the light of these findings.

Characteristics and management of chronic destructive pneumonia.

In 12 years 627 patients presented to Wentworth Hospital, Natal with chronic destructive pneumonia (CDP). Common symptoms were haemoptysis, the production of foul-smelling sputum, and chest pain. The disease pursued a chronic course with acute exacerbations which may be lethal. The majority of patients were African men aged between 20 and 50 years who were free from other significant disease apart from dental infection. Radiographically and pathologically CDP had the characteristics of a necrotising pneumonia, and microbiological investigation showed mixed aerobic and anaerobic flora in the lower respiratory tract. Gram-positive aerobic cocci and Bacteroides species were the predominant organisms. In 120 patients treatment regimens were based on chloramphenicol, in 429 cephalosporins, and in 78 on combination therapy with cephalosporins, penicillin, and metronidazole. One hundred and seventy patients also required operative management in an attempt to control progress of the disease. The overall inpatient mortality rate from CDP was 7.8%. In the group of patients treated with combination therapy the mortality rate was 1.3%.

Tinidazole in the prophylaxis and treatment of anaerobic infection.

The influence of prophylactic tinidazole therapy on vaginal carriage rates of anaerobes and the development of post-operative anaerobic infection was studied in 100 women undergoing abdominal hysterectomy. Tinidazole prophylaxis (50 patients) led to a decrease of anaerobe vaginal carriage rate from 56% pre-operatively to 10 and 30% on the third and seventh post-operative days, respectively. In the control group (50 patients), no significant decrease in anaerobe yield was noted, corresponding percentages being 72, 64 and 74. Post-operative infection occurred in 34 cases (28 controls, 6 tinidazole prophylaxis). Wound swabs from patients in the latter group did not yield anaerobes on culture, and infections either resolved spontaneously (2 cases) or responded to tinidazole therapy, with or without addition of ampicillin and kanamycin (4 cases). In the control group, 21 cases of post-operative wound infection and 4 of vault infection were seen. Wound swabs from 6 of the former group yielded aerobes only and 10 mixed growth of aerobes/anaerobes. Post-operative wound/vault infections in control patients cleared spontaneously (18 cases) or responded to imidazole therapy, with or without ampicillin and kanamycin (7 cases). These data suggest that tinidazole may be a useful adjunct in imidazole prophylaxis and treatment of anaerobic infection.

Metronidazole in the prophylaxis and treatment of anaerobic infection.

The influence of prophylactic metronidazole on vaginal carriage rates of anaerobes and the development of postoperative anaerobic infection was studied in 104 women who underwent abdominal hysterectomy. Metronidazole prophylaxis in 54 patients led to a decrease in the anaerobe vaginal carriage rate from 65% pre-operatively to 17% and 28% on the 3rd and 7th postoperative days respectively. In the control group (50 patients) no significant decrease in anaerobe yield was noted, corresponding percentages being 72%, 64%, and 74%. Postoperative infection occurred in 36 patients (28 controls; 8 on prophylactic metronidazole). Wound swabs from all 8 patients in the latter group yielded aerobes, and in 1 patient mixed infection (aerobes/anaerobes) occurred. In 7 of these patients (including the patient with mixed infection), the infection resolved spontaneously, while the 8th patient responded to therapy with metronidazole, kanamycin and ampicillin. In the control patients, 21 cases of postoperative wound infection and 4 of vault infection were seen; wound swabs from patients in the former group yielded aerobes in only 6 cases, and mixed growth of aerobes/anaerobes in 10 cases. Postoperative wound/vault infections in control patients cleared spontaneously in 18 cases and responded to imidazole therapy, with or without ampicillin and kanamycin, in 7 cases.

Susceptibility of anaerobic bacteria to ten antimicrobial agents.

The susceptibility pattern of 265 anaerobic bacteria from clinical isolates to 10 antimicrobial agents was investigated by the agar dilution technique. Penicillin G, in a concentration of 16 mug/ml, was active against most organisms, important exceptions being 12% of Bacteroides melaninogenicus and 24% of B. fragilis strains. The susceptibility of strains to ampicillin was similar to their susceptibility to penicillin G. Carbenicillin, at 16 mug of imidazole per ml may have therapeutic implications.

The bacteriology of chronic destructive pneumonia.

Thirty-four patients with chronic destructive pneumonia (CDP) were investigated bacteriologically and mycologically; specimens were obtained by transtracheal aspiration, percutaneous lung puncture, and open lung biopsy. Anaerobes were isolated in the absence of aerobes in 1 case while specimens from 9 patients yielded aerobes only. In 16 patients both groups of organisms were cultured. Streptococcus viridans was isolated in 12 cases and fungi in 2 cases. Specimens from 8 patients were sterile on culture. All patients had been treated with various antibiotic combinations before specimens were taken. Aerobes, particularly Gram-positive cocci, and anaerobes probably play an important synergistic role in the pathogenesis of CDP.