Identifying the core bacterial and fungal communities within four agricultural biobeds used for the treatment of pesticide rinsates.

AIMS: This study evaluated the variability of bacterial and fungal communities within unique pesticide remediation biobeds. METHODS AND RESULTS: Four biobeds receiving different applied pesticide rinsates, were sampled throughout an operational season to determine pesticide removal efficacy and microbial communities. Biomixture samples collected from different biobed depths, were subjected to Illumina sequencing of the 16S rRNA (bacteria) and ITS2 (fungi) genes. Pesticide removal rates for all biobeds averaged 99%, with microbial community analysis revealing biobeds shared 60-70% of the most abundant bacterial and fungal orders, respectively. Though biobed depth did not greatly impact microbial community profile or diversity, bacterial and fungal taxa profiles between biobeds notably diverge at levels of genera and OTU. Biobed bacterial communities exhibited greater diversity than fungal communities between and within all biobeds. CONCLUSIONS: Biobeds receiving variable pesticide rinsates share a 'core' microbial community, exhibiting greater bacterial diversity relative to fungal diversity. Pesticide exposure increased bacterial diversity throughout the biobeds, while fungal diversity was variable, meriting further understanding of fungicide application to biobed fungal community stability. SIGNIFICANCE AND IMPACT OF THE STUDY: Biobeds achieve high treatment efficacy of unique pesticide rinsates, regardless of differentiation of specific genera in response to specific compounds; supporting biobeds as a robust engineered system for pesticide rinsates bioremediation.

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.

Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance.

This study investigated how the timing of application of the biofungicide Serenade (Bacillus subtilis QST713) or it components (product filtrate and bacterial cell suspension) influenced infection of canola by Plasmodiophora brassicae under controlled conditions. The biofungicide and its components were applied as a soil drench at 5% concentration (vol/vol or equivalent CFU) to a planting mix infested with P. brassicae at seeding or at transplanting 7 or 14 days after seeding (DAS) to target primary and secondary zoospores of P. brassicae. Quantitative polymerase chain reaction (qPCR) was used to assess root colonization by B. subtilis as well as P. brassicae. The biofungicide was consistently more effective than the individual components in reducing infection by P. brassicae. Two applications were more effective than one, with the biofungicide suppressing infection completely and the individual components reducing clubroot severity by 62 to 83%. The biofungicide also reduced genomic DNA of P. brassicae in canola roots by 26 to 99% at 7 and 14 DAS, and the qPCR results were strongly correlated with root hair infection (%) assessed at the same time (r = 0.84 to 0.95). qPCR was also used to quantify the transcript activity of nine host-defense-related genes in inoculated plants treated with Serenade at 14 DAS for potential induced resistance. Genes encoding the jasmonic acid (BnOPR2), ethylene (BnACO), and phenylpropanoid (BnOPCL and BnCCR) pathways were upregulated by 2.2- to 23-fold in plants treated with the biofungicide relative to control plants. This induced defense response was translocated to the foliage (determined based on the inhibition of infection by Leptosphaeria maculans). It is possible that antibiosis and induced resistance are involved in clubroot suppression by Serenade. Activity against the infection from both primary and secondary zoospores of P. brassicae may be required for maximum efficacy against clubroot.

First Report of Rhizoctonia solani AG-4 and AG-2-2 on Lupinus angustifolius in Canada.

Narrow-leaved lupine (Lupinus angustifolius L.) is grown as a grain legume crop in Australia and Europe where it is used as feedstock in the livestock and aquaculture industries. During July 2003, a stem rot disease was observed in narrow-leaved lupine (cv. Arabella) plants in a research plot at the Crop Diversification Centre North (CDCN), Edmonton, Alberta, Canada. The disease was also found on cv. Rose at the CDCN and cv. Arabella in experimental fields near Devon, Ellerslie, and Westlock, Alberta during the late spring of 2004. Diseased plants showed dark brown-to-black stems with sunken and constricted lesions at the soil level. Young leaves became shrunken and twisted and seedlings collapsed. Rhizoctonia solani was consistently isolated from lesions on taproots and basal stems of diseased plants. Colonies of cream-colored mycelia grew close to the surface of potato dextrose agar (PDA). Most sclerotia formed inside the medium. Agar disks (1 cm in diameter) of isolates LP-11Bb, LP-24C, and LP-25C were attached to the opposite sides of basal stems (180° apart) of 1-month-old lupine seedlings (cv. Arabella). Inoculated plants were incubated for 2 days in black plastic bags under a greenhouse bench at approximately 20°C. All isolates caused brown lesions on the lower stems (extending up to 7 cm above ground level), girdling, and root rot. Plants wilted within 7 to 10 days after inoculation, and aerial mycelia appeared on the basal stems. R. solani was reisolated from the infected crown tissues using PDA to complete Koch's postulates. The isolates were paired with AG tester strains of R. solani. Isolates LP-11Bb and LP-24C were identified as AG-4 while isolate LP-25C was identified as AG-2-2. In another trial, eight isolates of R. solani (unknown AG types) were tested for virulence on L. angustifolius cv. Arabella using the inoculation method described above. All isolates were pathogenic, and disease severity that was based on a 0 to 4 scale ranged from 2.7 to 3.2. The most virulent strain was LP-24C, which caused a 77% loss in fresh weight compared with the noninoculated control plants. R. solani AG-8 is associated with Rhizoctonia disease of lupine in Australia (1) and also causes bare patch disease of wheat. To our knowledge, this is the first report of R. solani on lupine in Canada. This disease could have a significant impact on the commercial production of lupine in Alberta. Reference: (1) M. W. Sweetingham et al. Pages 466-486 in: Advances in Lupin Disease Management in Australia. Proc. Int. Lupin Conf., 8th. G. D. Hill, ed. International Lupin Association, Canterbury, New Zealand, 1999.

Identification of Lentil Germ Plasm Resistant to Colletotrichum truncatum and Characterization of Two Pathogen Races.

ABSTRACT A total of 1,771 lentil accessions from the U.S. lentil collection (U.S. Department of Agriculture-Agricultural Research Service, Pullman, WA) and the Institut für Pflanzengenetik und Kulturpflanzenforschung (Gatersleben, Germany) were screened for resistance to Colletotrichum truncatum, the cause of anthracnose. About 95% of the accessions were susceptible when inoculated with a single isolate in the field. Retesting, under controlled conditions, of accessions rated as resistant or moderately resistant in the field resulted in identification of anthracnose resistance in four accessions from the U.S. collection (PI 320937, PI 320952 [cv. Indianhead], PI 345629, and 468901), and 12 accessions from the German collection (Lens 3, 102, 104, 106, 107, 119, 122, 134, 135, 177, 195, and 209). Seven of the accessions were used as host differentials to characterize pathogenic variability of 50 single-spore isolates collected in Manitoba and Saskatchewan, Canada. The presence of two distinct races was demonstrated. Isolates of C. truncatum avirulent on cv. Indianhead, PI 320937, PI 345629, PI 468901, Lens 102, Lens 104, and Lens 195 were designated race Ct1. Isolates that were virulent on these seven entries were designated race Ct0, indicating their lack of avirulence genes. Race Ct0 was isolated more frequently from commercial seed samples than race Ct1, but the two races were isolated with similar frequency from plants in commercial fields planted to susceptible cultivars. Race Ct0, to which no resistance has yet been identified, presents a high risk to lentil production in Canada and potentially worldwide.

Genetic Diversity of Ascochyta rabiei in Canada.

Assessment of variability of Ascochyta rabiei (teleomorph: Didymella rabiei) was based on virulence tests of 40 isolates and on random amplified polymorphic DNA (RAPD) analysis of 39 isolates from Canada. In addition, isolates of A. rabiei from other countries were assessed in the virulence (18 isolates) and RAPD (20 isolates) analyses. Seven isolates of A. lentis (teleo-morph: Didymella lentis) and two of A. pinodes (teleomorph: Mycosphaerella pinodes) also were included in the RAPD analysis. Significant line-isolate interactions in the virulence tests indicated that certain isolates were virulent only on certain lines. Canadian isolates were grouped into 14 pathotypes using eight chickpea differentials. These groupings also encompassed 17 of the 18 isolates from other countries. RAPD analysis of all 68 isolates using 8 primers produced 112 fragments, of which 96% were polymorphic. Similarities among A. rabiei isolates from Canada ranged from 20 to 100%. In the RAPD dendrogram, all five A. rabiei isolates from Australia, three of six from Syria, three of five from the United States, and one of two from India clustered within the major groups of Canadian isolates. There was a weak association between RAPD and pathotype groups. A. rabiei was 45% similar to A. lentis and only 14% similar to A. pinodes. The levels of DNA variability and virulence among isolates show that the population of A. rabiei in Canada is highly diverse.

First Report of Resistance to Benomyl Fungicide in Sclerotinia sclerotiorum.

Benomyl fungicide (Benlate) is used worldwide to control ascomycete pathogens, but resistance has developed in several pathogen populations (1). On the Canadian prairies, benomyl is used to reduce injury caused by Sclerotinia sclerotiorum (Lib.) de Bary on canola (Brassica napus, B. rapa) and alfalfa (Medicago sativa) seed crops. To determine if populations are resistant to benomyl, isolates of S. sclerotiorum collected from 15 fields (12 alfalfa and 3 canola, one isolate per field) in 2000 were grown on potato dextrose agar amended with benomyl at 0, 0.05, 0.5, 5, 50, and 500 mg/liter. Plugs of mycelium from the margin of an actively growing colony were placed in the center of a 10-cm-diameter petri dish containing 15 ml of test medium and incubated on a laboratory bench. Linear growth (mean of maximum width and right angle) of each colony (three replicates each) was measured after 5 to 6 days. The growth of isolates from 13 fields was inhibited by low concentrations of benomyl (EC50 < 8 mg/liter), but two isolates were very resistant (EC50 > 200 mg/liter). Resistant cultures were isolated from infected canola plants in the only two fields in the study in which reduced efficacy of benomyl was suspected. The distribution and importance of benomyl-resistant populations of S. sclerotiorum in the region remains to be determined. Reference: (1) T. R. Pettitt et al. Mycol. Res. 97:1172, 1993.

First Report of Dollar Spot, Caused by Sclerotinia homoeocarpa, on Poa pratensis in Saskatchewan, Canada.

Dollar spot disease affects many species of grasses in North America but has not been previously reported from Saskatchewan. In July 2000, symptoms were observed on golf course fairways in Saskatoon. No dollar spot disease was observed on adjacent putting greens or tees composed of Agrostis palustris (creeping bentgrass), perhaps because the tees and greens were grown under a higher nitrogen fertility regime. Fungicide treatments are usually not required for turf disease control during the warm, dry summer growing season in Saskatoon, and no fungicides had been applied at this location. Daytime temperatures near 25°C and heavy dew at night preceded the disease outbreak, which affected about 5% of the turf across large areas of several fairways. The fairways were originally seeded to Festuca rubra subsp. rubra (common creeping red fescue) and Poa pratensis (Kentucky bluegrass) but also contained annual bluegrass (P. annua). The disease was observed on leaf blades of all three species. In addition to 5-cm-diameter circular patches of brown grass, sharply delimited individual leaf lesions and cobweb-like aerial mycelia on the grass were observed. Fungal isolates were obtained by plating infected P. pratensis leaf blades on potato dextrose agar and then transferring to oat agar. On oat agar, isolates produced white, fluffy aerial mycelium, columnar when mature, and usually with a cinnamon base and dark brown stromata or sclerotial flakes on and in the agar. DNA was extracted from an isolate and amplified using the primers ITS1 and ITS4 (1). A 500-bp fragment presumed to contain the internal transcribed spacer region of the ribosomal DNA (ITS) was purified and sequenced (1), and it showed 96% identity with the ITS of a Sclerotinia homoeocarpa isolate, Genbank accession AF067640. To test Koch's postulates, P. pratensis cv. Loft 1757 was grown from seed in 15 ml of sand at 20°C under constant fluorescent light. Two-week-old turf was inoculated with 5-mm-diameter mycelial plugs of the fungus from 1-week-old cultures on potato dextrose agar by placing inoculum plugs on the sand. Inoculated turf was incubated in a loosely-lidded clear plastic container at 20°C under constant fluorescent lighting. After 1 week, lesions and white aerial hyphae were observed on the leaf blades, while no disease was observed on the uninoculated controls. The fungus was reisolated from foliar lesions to complete Koch's postulates. In addition to P. pratensis, both P. annua and A. palustris cv. Penncross were also inoculated and showed disease symptoms, with greater disease severity on P. annua. Reference: (1) T. Hsiang and C. Wu. Mycol. Res. 104:16, 2000.

Stem Smut (Ustilago hypodytes) on Intermediate Wheatgrass in Canada.

Intermediate wheatgrass (Thinopyrum intermedium [Host] Barkworth & D.R. Dewey) (syn. Agropyron intermedium [Host] Beauv.) is becoming an important forage grass species in Alberta, Canada. Severe losses in seed yield due to stem smut (Ustilago hypodytes [Schlecht.] Fr.) were noted in a 70-acre field near Warner, AB, in 1999. The crop had been established in 1993 and harvested for seed each year. Smut symptoms (5% incidence) were noted initially in 1997. Incidence, determined by counting the number of symptomatic stems, increased to 10% in 1998 and 50% in 1999. The symptoms usually appeared in the first week of June. Brown sori developed on infected stems, especially between the uppermost node and the leaf below the flag leaf, and gradually became black during the period of seed filling, which is characteristic of stem smut (1). Teliospores were smooth, spherical to oval, light to dark brown, and 4.5 to 5.0 × 5.0 to 6.8 µm in dimension, which is also consistent with previous descriptions of U. hypodytes. Infected stems occasionally flowered, but did not set seed, so seed yield losses were proportional to disease incidence. Plants infected with stem smut were often stunted. Tissues in the smutty stem often became sunken and stems became twisted and thinner than normal due to the propagation of sori in the stem over time. Stem smut has been reported on crested wheatgrass and slender wheatgrass in other parts of Canada (2) and on T. intermedium in the United States (3). This is the first report of stem smut affecting commercial grass seed production in Alberta, Canada. This disease could also have a significant impact on the seed production of intermediate wheatgrass elsewhere. References: (1) G. W. Fischer. 1953. Manual of the North American Smut Fungi. Ronald Press, New York. (2) B. D. Gossen and D. Regnier. Can. Plant Dis. Surv. 71:88-89, 1991. (3) J. F. Karn and J. M. Krupinsky. Phytopathology 73:1152-1155, 1983.