Fluopyram Suppresses Population Densities of Heterodera glycines in Field and Greenhouse Studies in Michigan.

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, causes significant damage to soybean production annually. Fluopyram is a fungicide commonly used in soybean seed treatments intended to control soilborne fungal pathogens; however, recent studies have also suggested inhibitory effects on SCN. We examined the effects of a fluopyram seed treatment, ILeVO, on SCN reproduction, sudden death syndrome (SDS) development, and yield in a 3-year field study. Overall, fluopyram had a significant effect on yield (P = 0.046) and end-of-season SCN eggs and second-stage juveniles (Pf, P = 0.033) but no significant effect on SCN reproduction (Rf) or SDS disease index (P > 0.05). Post hoc tests indicated that fluopyram increased yield and suppressed SCN quantities. However, Rf was consistently greater than 1 whether or not the seed was treated with fluopyram, indicating that SCN populations were still increasing in the presence of fluopyram. A follow-up greenhouse study indicated that fluopyram reduced SCN relative to nontreated controls, as observed in the field, but only reduced SCN DNA within roots of a susceptible cultivar. These results indicate that fluopyram can suppress SCN quantities relative to nontreated seed but may not successfully reduce nematode populations without the use of additional management strategies.

Tar Spot: An Understudied Disease Threatening Corn Production in the Americas.

Tar spot of corn has been a major foliar disease in several Latin American countries since 1904. In 2015, tar spot was first documented in the United States and has led to significant yield losses of approximately 4.5 million t. Tar spot is caused by an obligate pathogen, Phyllachora maydis, and thus requires a living host to grow and reproduce. Due to its obligate nature, biological and epidemiological studies are limited and impact of disease in corn production has been understudied. Here we present the current literature and gaps in knowledge of tar spot of corn in the Americas, its etiology, distribution, impact and known management strategies as a resource for understanding the pathosystem. This will in tern guide current and future research and aid in the development of effective management strategies for this disease.

Integrated Effects of Genetic Resistance and Prothioconazole + Tebuconazole Application Timing on Fusarium Head Blight in Wheat.

Integrated Fusarium head blight (FHB) management programs consisting of different combinations of cultivar resistance class and an application of the fungicide prothioconazole + tebuconazole at or after 50% early anthesis were evaluated for efficacy against FHB incidence (INC; percentage of diseased spikes), index (IND; percentage of diseased spikelets per spike), Fusarium damaged kernel (FDK), deoxynivalenol (DON) toxin contamination, grain yield, and test weight (TW) in inoculated field trials conducted in 11 U.S. states in 2014 and 2015. Mean log response ratios and corresponding percent control values for INC, IND, FDK, and DON, and mean differences in yield and TW relative to a nontreated, inoculated susceptible check (S_CK), were estimated through network meta-analyses as measures of efficacy. Results from the analyses were then used to estimate the economic benefit of each management program for a range of grain prices and fungicide applications costs. Management programs consisting of a moderately resistant (MR) cultivar treated with the fungicide were the most efficacious, reducing INC by 60 to 69%, IND by 71 to 76%, FDK by 66 to 72%, and DON by 60 to 64% relative to S_CK, compared with 56 to 62% for INC, 68 to 72% for IND, 66 to 68% for FDK, and 58 to 61% for DON for programs with a moderately susceptible (MS) cultivar. The least efficacious programs were those with a fungicide application to a susceptible (S) cultivar, with less than a 45% reduction of INC, IND, FDK, or DON. All programs were more efficacious under conditions favorable for FHB compared with less favorable conditions, with applications made at 50% early anthesis being of comparable efficacy to those made 2 to 7 days later. Programs with an MS cultivar resulted in the highest mean yield increases relative to S_CK (541 to 753 kg/ha), followed by programs with an S cultivar (386 to 498 kg/ha) and programs with an MR cultivar (250 to 337 kg/ha). Integrated management programs with an MS or MR cultivar treated with the fungicide at or after 50% early anthesis were the most likely to result in a 50 or 75% control of IND, FDK, or DON in a future trial. At a fixed fungicide application cost, these programs were $4 to $319/MT more economically beneficial than corresponding fungicide-only programs, depending on the cultivar and grain price. These findings demonstrate the benefits of combining genetic resistance with a prothioconazole + tebuconazole treatment to manage FHB, even if that treatment is applied a few days after 50% early anthesis.

Pathogenicity and Virulence of Soilborne Oomycetes on Phaseolus vulgaris.

Dry bean (Phaseolus vulgaris L.) is a globally important leguminous food crop. Yields can be reduced by high incidence of soilborne oomycetes that cause seedling disease. Breeders have attempted to develop Pythium root rot-resistant bean varieties; however, relationships between dry bean and most soilborne oomycete species remain uncharacterized. Oomycete species (n = 28), including Pythium spp. and Phytopythium spp., were tested in a growth chamber seedling assay at 20°C and an in vitro seed assay at 20°C and 26°C to evaluate their pathogenicity and virulence on 'Red Hawk' dark red kidney bean and 'Zorro' black bean. Root size or disease severity was significantly impacted by 14 oomycete species, though results varied by bean variety, temperature, and assay. Of these 14 pathogenic oomycete species, 11 species exhibited significant differences in DSI due to temperature on at least one bean variety. Pythium aphanidermatum, P. myriotylum, P. ultimum, P. ultimum var. sporangiiferium, and P. ultimum var. ultimum were the most virulent species in both assays, causing seed rot and pre-emergence damping-off of dry bean. Oomycete species were clustered into three groups based on symptom development: seed rot pathogens, root rot pathogens, or nonpathogens. Intraspecific variability in virulence was observed for eight of the 14 pathogenic oomycete species. Improved understanding of Pythium and Phytopythium interactions with dry bean may enable breeders and pathologists to more effectively evaluate strategies for oomycete seedling disease management.

First Report of Aster Yellows Phytoplasma in Soybean in Michigan.

During the summer of 2012, soybean plants in a commercial field in Clinton County, Michigan, exhibited symptoms characteristic of phytoplasmal diseases (1,2). Symptoms included extensive top dieback, stunting, purple stem surfaces, internal necrosis, leaf vein discoloration, and bud proliferation. Approximately 80% of plants in a half hectare along the southern edge of the field were symptomatic, although the majority of plants in the 4-ha field appeared symptomless. Total genomic DNA was extracted from one symptomatic and one asymptomatic leaf sample using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to manufacturer's instructions. The DNA was used as template in direct PCR primed by the phytoplasma-universal primers P1/P7 followed by nested PCR primed by P1/AYint (3). Reactions containing template DNA from the symptomatic plant yielded ribosomal RNA gene amplicons of 1.8 kbp (P1/P7-primed) and 1.6 kbp (P1/AYint-primed), respectively. Reactions containing template DNA from the asymptomatic plant or water did not yield amplicons. The products of PCRs primed by P1/P7 were purified using PureLink PCR Purification kit (Life Technologies, Carlsbad, CA) and cloned in a pGem T-Easy vector system (Promega, Madison, WI). Three separate clones were sequenced using the sequencing primers M13For, M13Rev, SAYF nt 755, (5'-AAAGCGTGGGGAGCAAACAG), and SAYR nt 1159, (5'-TTTGACGTCGTCCCCACCTT). The sequences of all three clones were identical. A consensus (Sequencher 4.1, Gene Codes Corporation, Ann Arbor, MI) nucleotide sequence of 1,830 bp was deposited in GenBank under the accession number KF528320. A BLASTn similarity search revealed that the sequence shared 100% identity to rDNA of aster yellows phytoplasma (AF222063). Additionally, analysis of the 16Sr group/subgroup classification, based on in silico RFLP analyses using iPhyClassifier (4), indicated that the soybean phytoplasma is a member of subgroup 16SrI-B aster yellows phytoplasma subgroup. In a phylogenic tree deduced using the neighbor joining algorithm, the phytoplasma consensus sequence obtained from soybean in Michigan clustered with other group 16SrI (aster yellows phytoplasma) strains. While aster yellows phytoplasma has been previously reported in soybean in Wisconsin (2), to our knowledge, this is the first report of aster yellows in soybean in Michigan. References: (1) C. R. Grau et al. Compendium of Soybean Diseases, 4th ed. G. L. Hartman et al., eds. American Phytopathological Society, St. Paul, MN, 1999. (2) M. E. Lee et al. Can. J. Plant Pathol. 24:125, 2002. (3) C. D. Smart et al. Appl. Env. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

A Coordinated Effort to Manage Soybean Rust in North America: A Success Story in Soybean Disease Monitoring.

Existing crop monitoring programs determine the incidence and distribution of plant diseases and pathogens and assess the damage caused within a crop production region. These programs have traditionally used observed or predicted disease and pathogen data and environmental information to prescribe management practices that minimize crop loss. Monitoring programs are especially important for crops with broad geographic distribution or for diseases that can cause rapid and great economic losses. Successful monitoring programs have been developed for several plant diseases, including downy mildew of cucurbits, Fusarium head blight of wheat, potato late blight, and rusts of cereal crops. A recent example of a successful disease-monitoring program for an economically important crop is the soybean rust (SBR) monitoring effort within North America. SBR, caused by the fungus Phakopsora pachyrhizi, was first identified in the continental United States in November 2004. SBR causes moderate to severe yield losses globally. The fungus produces foliar lesions on soybean (Glycine max) and other legume hosts. P. pachyrhizi diverts nutrients from the host to its own growth and reproduction. The lesions also reduce photosynthetic area. Uredinia rupture the host epidermis and diminish stomatal regulation of transpiration to cause tissue desiccation and premature defoliation. Severe soybean yield losses can occur if plants defoliate during the mid-reproductive growth stages. The rapid response to the threat of SBR in North America resulted in an unprecedented amount of information dissemination and the development of a real-time, publicly available monitoring and prediction system known as the Soybean Rust-Pest Information Platform for Extension and Education (SBR-PIPE). The objectives of this article are (i) to highlight the successful response effort to SBR in North America, and (ii) to introduce researchers to the quantity and type of data generated by SBR-PIPE. Data from this system may now be used to answer questions about the biology, ecology, and epidemiology of an important pathogen and disease of soybean.

First Report of Soybean vein necrosis virus on Soybeans in Michigan.

Soybean vein necrosis virus (SVNV) is associated with an emerging disease in soybean producing regions of the United States. Soybean leaves with necrotic vein symptoms were initially noted in 2008 or 2009 in fields across Arkansas, Kansas, Missouri, Illinois, Mississippi, Tennessee, and Kentucky and SVNV was determined to be the causal agent (2). In 2012, widespread reports of SVNV were made across most soybean (Glycine max) producing states including the recent confirmation of SVNV in Iowa and Wisconsin (1). Foliar symptoms similar to those reported for SVNV were observed at approximately 1 to 10% incidence in soybean fields across Michigan in late August and September of 2012, including fields located in Cass, Ingham, Midland, Saginaw, and Van Buren counties. Symptoms included chlorosis and necrosis which initiated on the veins with subsequent spread across the leaf. An initial sample collected from the East Lansing Agricultural Research Station was confirmed to have SVNV with a polyclonal antibody using double antibody sandwich (DAS)-ELISA at AC Diagnostics, Inc. (Fayetteville, AR) and with reverse transcription PCR by Ioannis Tzanetakis, University of Arkansas, Fayetteville. Additional samples from five fields were subsequently collected from Cass, Ingham, and Van Buren counties. Duplicate leaf tissue samples were tested with DAS-ELISA using the SVNV test kit (AC Diagnostics). All symptomatic leaf samples exhibited a strong positive reaction based on the optical density reading at 405 nm. Absorbance reading that exceeded the healthy soybean tissue by a standard deviation of +3× were considered positive. Total RNA was also extracted from each sample using the RNeasy Plant Mini Kit (Qiagen, Germantown, MD). Complementary DNA (cDNA) was generated using virus-specific LdetR and SdetR primers (2) with the High Capacity RT cDNA kit (Life Technologies; Carlsbad, CA). The cDNA was used as template for PCR with the SVNV-specific primers that amplify regions of the L (LdetF/LdetR) and the S (SdetF/SdetR) RNAs (1). Amplification products of the expected 297 and 861 bp size, respectively, were detected in all symptomatic samples while no amplification products were generated from healthy soybean plant tissues grown under greenhouse conditions. Significantly, this is the first documentation and confirmation of the widespread prevalence of SVNV across the state of Michigan in 2012. References: (1) D. L. Smith et al. Plant Dis. http://dx.doi.org/10.1094/PDIS-11-12-1096-PDN . (2) J. Zhou et al. Virus Genes 43:289, 2011.

Long-term respiratory outcomes following solid organ transplantation in children: A retrospective cohort study.

BACKGROUND: Solid-organ transplantation (SOT) has become commonly used in children and is associated with excellent survival rates into adulthood. Data regarding long-term respiratory outcomes following pediatric transplantation are lacking. We aimed to describe the prevalence and nature of respiratory pathology following pediatric heart, kidney, and liver transplant, and identify potential risk factors for respiratory complications. METHODS: Retrospective review involving all children under active follow-up at the provincial transplant service in British Columbia, Canada, following SOT. RESULTS: Of 118 children, 33% experienced respiratory complications, increasing to 54% in heart transplant recipients. Chronic or recurrent cough with persistent chest x-ray changes was the most common clinical picture, and most infections were with nonopportunistic organisms typically found in otherwise healthy children. A history of respiratory illness before transplant was significantly associated with risk of posttransplant respiratory complications. Eight percentage8% were diagnosed with bronchiectasis, which was more common in recipients of heart and kidney transplant. Bronchiectasis was associated with recurrent hospital admissions with lower respiratory tract infections, treatment of acute rejection episodes, and treatment with sirolimus. INTERPRETATION: Respiratory morbidity is common after pediatric SOT, and bronchiectasis rates were disproportionately high in this patient group. We hypothesize that this relates to recurrent infections resulting from iatrogenic immunosuppression. Direct pulmonary toxicity from immunosuppression drugs may also be contributory. A high index of suspicion for respiratory complications is needed following childhood SOT, particularly in those with a history of respiratory disease before transplant, experiencing recurrent or severe respiratory tract infections, or exposed to intensified immunosuppression.

First Report and Confirmed Distribution of Soybean Sudden Death Syndrome Caused by Fusarium virguliforme in Southern Michigan.

Leaf lesions and root rot symptoms typical of soybean sudden death syndrome (SDS) caused by Fusarium virguliforme O'Donnell & T. Aoki were observed in commercial soybeans (Glycine max (L.) Merr.) in southern Michigan. Leaf symptoms ranged from chlorotic spots to severe interveinal chlorosis and necrosis, no foliar pathogens were noted. In 2008, isolates were collected from Berrien and St. Joseph counties. In 2009, isolates were collected from Cass, St. Joseph, Van Buren, Allegan, and Monroe counties. Pieces of roots with root rot symptoms were washed prior to surface disinfestation with 70% ethanol for 30 s and 0.5% NaOCl for 1 min and incubated on water agar (WA) in petri plates amended with 50 µg/ml of chloramphenicol for the production of sporodochia. Alternatively, spores were collected directly from nondisinfested roots expressing blue sporodochia. Single-spore cultures were derived by streaking macroconidia with a bacterial loop onto 3% WA + chloramphenicol and incubated overnight. With a dissecting microscope, single germinated macroconidia were collected with a sterile 0.2-mm-diameter insect pin and transferred to potato dextrose agar (PDA). Cultures on PDA grew slowly and developed blue-to-purple masses of sporodochia typical of F. virguliforme descriptions and similar to a representative isolate, Mont-1, grown alongside (1,2). Size of macroconidia from the six representative isolates, one from each county (including isolates derived from surface-disinfested and nondisinfested roots), and Mont-1 were determined to be within the range for F. virguliforme (42 to 56 × 5 to 6 µm), with an average of four septa per macroconidia. Identity of the representative isolates was confirmed by partial DNA sequencing of both strands of the internal transcribed spacer (ITS) region of the ribosomal RNA gene, translation elongation factor 1-α, and ß-tubulin loci. All six representative isolates were identical in each of the three loci and matched with 100% similarity F. virguliforme accessions in GenBank and Fusarium-ID database searches, except for the ß-tubulin locus in which a single nucleotide insertion was noted (Accession Nos. HM453328-HM453330). Sequences were 98 to 99% similar to other SDS Fusarium spp. not yet recorded in the United States. Koch's postulates were performed in the greenhouse according to Malvick and Bussey (3). Infested sorghum seed (~20 g) was placed 2 cm below soybean seed of susceptible cv. Williams 82 in plastic pots. Noninfested sorghum seed was used as a negative control and sorghum infested with Mont-1 as a positive control. Chlorotic spots developed 2 weeks after establishing the trial, and 3 to 4 weeks postinoculation, severe SDS symptoms of foliar interveinal chlorosis and necrosis and severe root rot developed. Koch's postulates were completed by reisolating F. virguliforme from a subset of infected plants. In addition, an isolate of F. virguliforme collected in 2008 was used to inoculate a 2009 field trial in East Lansing, MI with no history of SDS. Typical SDS symptoms developed in the field trial and F. virguliforme was isolated from a symptomatic plant that was identified as described above. Despite being reported across the majority of soybean-producing states, to our knowledge, this is the first confirmation and distribution report for SDS in Michigan. References: (1) T. Aoki et al. Mycoscience 46:162, 2005. (2) G. L. Hartman et al. Plant Dis. 81:515, 1997. (3) D. K. Malvick and K. E. Bussey. Can. J. Plant Pathol. 30:467, 2008.

First Report of Chickpea Wilt Caused by Clonostachys rhizophaga in Syria.

In 2007 and 2008, disease symptoms were observed on four cultivars of chickpea (Cicer arietinum L.), including two of the most popular cultivars grown in Syria (Ghab 3 and Ghab 4), in a replicated on-farm trial conducted in the fertile Al Ghab Plains. Affected plants exhibited chlorosis of the foliage, vascular discoloration, and death. In both years, plant mortality reached 100% in plots of cvs. ICC 12004, Ghab 3, and Ghab 4, but only 60% in plots of cv. ILC 97-706. Five monosporic isolates obtained from surface-disinfested stems and roots were identified morphologically. All micromorphological characteristics indicated that the isolated fungi fit the description of Clonostachys rhizophaga Schroers (1). Wilting of chickpea was widespread in the area, and fungal isolations from a random sample of diseased plants in neighboring farmers' fields revealed the presence of C. rhizophaga. In culture, isolates formed dimorphic, Verticillium-like (primary) or penicillate (secondary) conidiophores and ovoidal to elongate, slightly curved or asymmetrical, 5 to 9 µm long and 2.5 to 3.5 µm wide conidia showing a slightly laterally displaced hilum. The identification of the five isolates as C. rhizophaga was supported by sequencing approximately 600 bp of the ß-tubulin gene (tub2). Two representative sequences have been deposited under GenBank, Accession No. FJ593882 for strain CBS 124507 and No. FJ593883 for CBS 124511. Both were 100% similar to the sequence of C. rhizophaga strain CBS 361.77 (GenBank Accession No. AF358158) but differed by a deletion of 2 nucleotides relative to the ex-type strain of C. rhizophaga, CBS 202.37 (GenBank Accession No. AF358156). Two methods were used to inoculate plants and complete Koch's postulates. Method 1 used a 10-mm-diameter mycelial plug to inoculate healthy 3-day-old seedlings grown on 40 ml of Hoagland nutrient agar medium in a glass tube (one seedling per tube). The plug was placed mycelial-side down on the surface of the medium, and the fungus subsequently colonized the medium and penetrated the plant roots. Method 2 involved mixing autoclaved seed that had been colonized by each isolate with sterilized soil (1:12 vol/vol) prior to transplanting healthy seedlings into the soil mix. Thirty plants of each cultivar were tested per isolate per method, and controls received sterile agar plugs or autoclaved chickpea seed only. Irrespective of inoculation method, all five isolates caused wilt and plant death of all cultivars within 15 days (method 1) or 2 months (method 2) postinoculation. Symptoms were similar to those originally observed in the field and controls remained healthy. C. rhizophaga was recovered from all affected plants. To our knowledge, this is the first report of C. rhizophaga as a pathogen of chickpea. In an earlier report, C. rhizophaga (as Verticillium rhizophagum Tehon & Jacobs, nom. invalid.) was identified as the causal agent of a disastrous disease of Ulmus americana in Ohio (2). C. rhizophaga has been reported from Chile, Ecuador, the United States, and Switzerland (1). References: (1) H.-J. Schroers. Stud. Mycol. 46:85, 2001. (2) L.-R. Tehon and H. L. Jacobs. Bull. Davey Tree Expert Company, Kent, OH. 6:3, 1936.

Identification of Fungal Communities Within the Tar Spot Complex of Corn in Michigan via Next-Generation Sequencing.

Tar spot is a fungal disease complex of corn that has been destructive and yield limiting in Central and South America for nearly 50 years. Phyllachora maydis, the causal agent of tar spot, is an emerging corn pathogen in the United States, first reported in 2015 from major corn producing regions of the country. The tar spot disease complex putatively includes Monographella maydis (syn. Microdochium maydis), which increases disease damage through the development of necrotic halos surrounding tar spot lesions. These necrotic halos, termed "fish-eye" symptoms, have been identified in the United States, though Monographella maydis has not yet been confirmed. A recent surge in disease severity and loss of yield attributed to tar spot in the United States has led to increased attention and expanded efforts to understand the disease complex and how to manage it. In this study, next-generation sequencing of the internal transcribed spacer-1 (ITS1) ribosomal DNA was used to identify fungal taxa that distinguish tar spot infections with or without fish-eye symptoms. Fungal communities within tar spot only lesions were significantly different from communities having fish-eye symptoms. Two low abundance OTUs were identified as Microdochium sp., however, neither were associated with fish-eye symptom development. Interestingly, a single OTU was found to be significantly more abundant in fish-eye lesions compared to tar spot lesions and had a 91% ITS1 identity to Neottiosporina paspali. In addition, the occurrence of this OTU was positively associated with Phyllachora maydis fish-eye symptom networks, but not in tar spot symptom networks. Neottiosporina paspali has been reported to cause necrotic lesions on various monocot grasses. Whether the related fungus we detected is part of the tar-spot complex of corn and responsible for fish-eye lesions remains to be tested. Alternatively, many OTUs identified as Phyllachora maydis, suggesting that different isolate genotypes may be capable of causing both tar spot and fish-eye symptoms, independent of other fungi. We conclude that Monographella maydis is not required for fish-eye symptoms in tar spot of corn.

First Report of Spring Black Stem and Leaf Spot of Alfalfa in Washington State Caused by Phoma medicaginis.

Lesions were observed on leaves and stems of alfalfa (Medicago sativa L.) growing as weeds in Pullman, Washington in June of 2001. Lesions appeared similar to those described for spring black stem and leaf spot caused by Phoma medicaginis Malbr. & Roum. in Roum. var. medicaginis Boerema (synonyms Phoma herbarum Westend. var. medicaginis Fckl. and Ascochyta imperfecta Peck). Sporulation was induced by placing surface-disinfested pieces of infected tissue on 3% water agar (WA) for 24 h under fluorescent light with a 12-h photoperiod. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia after 18 h. Isolates had cultural and conidial morphology similar to descriptions of P. medicaginis and isolate ATCC52798 when grown on V8 agar and PDA at room temperature (3). Distinction between P. medicaginis var. medicaginis and P. medicaginis var. macrospora was not attempted. Conidial suspensions (1 × 106 conidia/ml) of isolates AS1, AS2, AS3, and AS4 were spray inoculated to runoff onto 3-week-old plants. PI lines 536535 and 536534 of M. sativa subsp. sativa (4-trifolate stage) and PI lines 442896 and 577609 of M. truncatula (5- to 7-trifolate stage) from the USDA Western Region Plant Introduction Station, Pullman, Washington were inoculated, with at least two replicate plants inoculated per isolate. Plants were incubated in a dew chamber at 20°C in the dark for 24 h to promote infection and then transferred to a growth chamber at 18°C with a 12-h photoperiod. Lesions were apparent on M. sativa subsp. sativa plants 4 days postinoculation (dpi) and 7 dpi on M. truncatula plants. At 12 dpi, many dark brown lesions with chlorotic halos were noted on leaves of M. sativa subsp. sativa, occasionally killing the entire trifoliate leaf and progressing approximately 1 cm down the stem. According to the previously published 1-to-5 visual rating scale for this disease (4), disease scores on both genotypes of M. sativa subsp. sativa were 4 (susceptible), while disease ratings on M. truncatula were 1-2 (resistant) with a few dark brown lesions noted on leaves and stems generally restricted to less than 2 mm in diameter. DNA was extracted from isolates AS1 and AS4, and PCR was performed using gpd-1 and gpd-2 primers for the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) (1), and EF1-728F and EF1-986R primers for the translation elongation factor 1-alpha gene (EF) (2), resulting in amplification of an approximately 600-bp fragment from each primer set. Amplicons were direct-sequenced on both strands, and BLAST searches of the NCBI nucleotide database were conducted with consensus G3PD and EF sequences of both isolates AS1 and AS4. Closest matches obtained for the G3PD and EF sequences were P. medicaginis isolate ATCC52798 (Accession No. DQ525740) and P. medicaginis var. medicaginis CBS316.90 (Accession No. AY831548), respectively. The G3PD and EF sequences for these isolates have been deposited in GenBank database (Accession Nos. EU394712-EU394715). To our knowledge, this is the first confirmed report of spring black stem and leaf spot of alfalfa in Washington State supported by Koch's postulates, cultural morphology, and multigene sequencing. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (3) G. C. Kinsey. No. 1503 in: IMI Descriptions of Fungi and Bacteria. CABI Bioscience, Surrey, UK, 2002. (4) R. M. Salter and K. L. Leath. Spring blackstem and leafspot resistance. Online publication. North American Alfalfa Improvement Conference, Beltsville, MD, 1992.

First Report of Ascochyta Blight of Pisum elatius (Wild Pea) in the Republic of Georgia Caused by Ascochyta pisi.

Characteristic Ascochyta blight lesions were observed on leaves and pods of wild pea (Pisum elatius Steven. ex M. Bieb.) growing at three sites in the Republic of Georgia during June and July of 2004. Site characteristics were 41°36.11'N, 44°31.34'E (elevation 919 m), 41°54.221'N, 44°05.667'E (elevation 744 m), and 41°44.907'N, 43°12.263'E (elevation 884 m). Lesions appeared similar to those induced by Ascochyta pisi Lib. on cultivated pea (P. sativum L.). Fungi were isolated by surface disinfesting small pieces of infected tissue in 95% EtOH for 10 s, 1% NaOCl for 1 min, and then in deionized sterile H20 for 1 min. Tissue pieces were placed on 3% water agar (WA) for 24 h under fluorescent lights with a 12-h photoperiod to induce sporulation. Single-conidial isolations were made by streaking conidia on 3% WA and picking germinated conidia 18 h later. Three fungi (isolates Georgia-6, -7, and -12) had colony morphology similar to that of A. pisi on V8 juice agar. Conidial suspensions (1 × 105 conidia/ml) of each isolate above were spray inoculated to runoff on three genotypes of 2-week-old P. elatius plants. Plants inoculated included PI lines 560055 and 513252 and W6 line 15006 from the USDA Western Region Plant Introduction Station, Pullman, WA with 11 replicate plants inoculated per isolate. Plants were incubated in a growth chamber for 48 h at 18°C and covered with a plastic cup to maintain high humidity. Characteristic Ascochyta blight lesions were apparent 7 days after inoculation. DNA was extracted from each isolate and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD), 364 bp of chitin synthase 1, and 330 bp of the translation elongation factor 1-alpha gene were amplified with gpd-1 and gpd-2 primers (1), CHS-79 and CHS-354 primers (2), and EF1-728F and EF1-986R primers (2), respectively. Amplicons were direct sequenced on both strands, and BLAST searches of the NCBI nucleotide database with consensus G3PD, CHS, and EF sequences of isolates Georgia-6, -7, and -12 were performed. The closest match obtained for the G3PD sequences was A. pisi isolate ATCC 201617 (Accession No. DQ383963). G3PD sequences for Georgia-6, -7, and -12 were deposited in GenBank (Accession Nos. DQ383966 [Georgia-6 and -7] and DQ383963 [A. pisi isolate AP1 and Georgia-12]). Closest matches to CHS and EF sequences were A. pisi isolate ATCC 201618 (EF Accession No. DQ386494) and Didymella fabae isolate ATCC 96418 (CHS Accession No. DQ386481, EFAccession No. DQ386492), respectively. CHS sequences for Georgia-6, -7, and -12 were identical to each other and to A. fabae isolate AF1 and were deposited in GenBank (Accession No. DQ386481. EF sequences for Georgia-6, -7, and -12 were deposited in GenBank (Accession Nos. DQ386494 [Georgia-6 and A. pisi isolate AP2], DQ386495, and DQ386496, respectively. These results, coupled with the morphological identification and inoculation results, confirm the identity of the fungus as A. pisi. To our knowledge, this is the first report of Ascochyta blight of P. elatius in the Republic of Georgia. References: (1) M. L. Berbee et al. Mycologia 91:964. 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999.

Effects of maltreatment and parental schizophrenia spectrum disorders on early childhood social-emotional functioning: a population record linkage study.

AIMS: Childhood maltreatment and a family history of a schizophrenia spectrum disorder (SSD) are each associated with social-emotional dysfunction in childhood. Both are also strong risk factors for adult SSDs, and social-emotional dysfunction in childhood may be an antecedent of these disorders. We used data from a large Australian population cohort to determine the independent and moderating effects of maltreatment and parental SSDs on early childhood social-emotional functioning. METHODS: The New South Wales Child Development Study combines intergenerational multi-agency data using record linkage methods. Multiple measures of social-emotional functioning (social competency, prosocial/helping behaviour, anxious/fearful behaviour; aggressive behaviour, and hyperactivity/inattention) on 69 116 kindergarten children (age ~5 years) were linked with government records of child maltreatment and parental presentations to health services for SSD. Multivariable analyses investigated the association between maltreatment and social-emotional functioning, adjusting for demographic variables and parental SSD history, in the population sample and in sub-cohorts exposed and not exposed to parental SSD history. We also examined the association of parental SSD history and social-emotional functioning, adjusting for demographic variables and maltreatment. RESULTS: Medium-sized associations were identified between maltreatment and poor social competency, aggressive behaviour and hyperactivity/inattention; small associations were revealed between maltreatment and poor prosocial/helping and anxious/fearful behaviours. These associations did not differ greatly when adjusted for parental SSD, and were greater in magnitude among children with no history of parental SSD. Small associations between parental SSD and poor social-emotional functioning remained after adjusting for demographic variables and maltreatment. CONCLUSIONS: Childhood maltreatment and history of parental SSD are associated independently with poor early childhood social-emotional functioning, with the impact of exposure to maltreatment on social-emotional functioning in early childhood of greater magnitude than that observed for parental SSDs. The impact of maltreatment was reduced in the context of parental SSDs. The influence of parental SSDs on later outcomes of maltreated children may become more apparent during adolescence and young adulthood when overt symptoms of SSD are likely to emerge. Early intervention to strengthen childhood social-emotional functioning might mitigate the impact of maltreatment, and potentially also avert future psychopathology.

Host Specificity of Ascochyta spp. Infecting Legumes of the Viciae and Cicerae Tribes and Pathogenicity of an Interspecific Hybrid.

ABSTRACT Ascochyta spp. (teleomorphs: Didymella spp.) infect a number of legumes, including many economically important species, and the diseases they cause represent serious limitations of legume production worldwide. Ascochyta rabiei, A. fabae, A. pisi, A. lentis, and A. viciae-villosae are pathogens of chickpea (Cicer arietinum), faba bean (Vicia faba), pea (Pisum sativum), lentil (Lens culinaris), and hairy vetch (V. villosa), respectively. Inoculations in the greenhouse and in growth chambers demonstrated that A. fabae, A. lentis, A. pisi, A. rabiei, and A. viciae-villosae were host specific. Isolates caused no visible disease symptoms on "nonhost" plants (plants other than the hosts they were originally isolated from) but were recovered consistently from inoculated, surface-disinfested, nonhost tissues. Interspecific crosses of A. pisi x A. fabae and A. viciae-villosae x A. lentis produced pseudothecia with viable ascospores, and the hybrid status of the ascospore progeny was verified by the segregation of mating type and amplified fragment length polymorphism (AFLP) markers. Interspecific progeny were morphologically normal in culture but exhibited more phenotypic variation compared with progeny from intraspecific crosses. Mating type and the majority of AFLP markers segregated in Mendelian 1:1 ratios in both intraspecific and interspecific crosses. A total of 11 and 7% of AFLP markers showed segregation distortion among progeny from interspecific crosses and intraspecific crosses, respectively; however, this difference was not significant (P = 0.90). Only 30 of 114 progeny isolates from the A. fabae x A. pisi cross inoculated in the greenhouse caused lesions on pea and only 4 caused disease on faba bean. In all, 15 of 110 progeny isolates were pathogenic to pea and none were pathogenic to faba bean under growth chamber conditions. Although no obvious postzygotic, intrinsic isolating barriers were identified in any of the interspecific crosses, it appears that host specialization may act as both a prezygotic, ecological isolating barrier and a postzygotic, extrinsic, ecological isolating barrier in these fungi. Host specificity, coupled with low pathogenic fitness of hybrids, may be an important speciation mechanism contributing to the maintenance of hostspecific, phylogenetic lineages of these fungi.

First Report of Ascochyta Blight of Vicia hirsuta (Hairy Tare) in the Republic of Georgia Caused by Ascochyta sp.

Tan lesions with dark margins containing concentric rings of black pycnidia were observed on leaves and pods of hairy tare (Vicia hirsuta L.) growing near Ateni, GA (41°54.631'N, 44°05.586'E, elev. 730 m) on 1 July 2004. Lesions were reminiscent of those induced by Ascochyta rabiei (Pass.) Labrousse on chickpea (Cicer arietinum L.). At the time of collection, necrotic lesions were observed on the stems, leaflets, and pods of several plants. The fungus was isolated by surface-disinfecting small pieces of infected tissue in 95% EtOH for 10 s, 1% NaOCl for 1 min, and then deionized H20 for 1 min. Tissue pieces were placed on 3% water agar (WA) for 24 h under fluorescent lights with a 12-h photoperiod to induce sporulation. Single-conidial isolations were made by streaking cirrhi on 3% WA and picking germinated single conidia. After 14 days of growth, the isolated fungus had colony morphology similar to that of A. rabiei on V8 juice agar. A conidial suspension of the fungus (1 × 105 conidia/ml) was spray-inoculated onto 2-week-old plants including PI lines 628303, 628304, 420171, and 422499 of V. hirsuta and C. arietinum cv. Burpee. Plants were obtained from the USDA Western Region Plant Introduction Station, Pullman, WA, and 20 replicate plants of each genotype were inoculated. Inoculated plants were covered with a plastic cup to maintain high humidity and incubated in a growth chamber for 48 h at 18°C. Following removal of the cups, characteristic Ascochyta blight lesions were apparent 14 days after inoculation on both plant species. DNA was extracted from the isolate and 610 bp of the glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD), 364 bp of the chitin synthase 1 gene, and 330 bp of the translation elongation factor 1-alpha gene were amplified with gpd-1 and gpd-2 primers (1), CHS-79 and CHS-354 primers (2), and EF1-728F and EF1-986R primers (2), respectively. Amplicons were direct sequenced on both strands and a BLAST search of the NCBI nucleotide database with consensus G3PD, CHS, and EF sequences revealed the chickpea pathogen Didymella rabiei (anamorph Ascochyta rabiei) accessions DQ383958, DQ386480, and DQ386488 as the closest matches in the databases with 95, 95, and 88% sequence similarity, respectively. These results, coupled with the morphological identification and the inoculation results, confirm the identity of the fungus as Ascochyta sp. Further research needs to be performed to determine if this represents a new species of Ascochyta. The identification of this fungus is part of a larger project to develop a phylogeny for Ascochyta spp. infecting cultivated legumes and their wild relatives that will provide a framework for the study of the evolution of host specificity and speciation of plant-pathogenic fungi. This is the second report of an Ascochyta species on V. hirsuta, and to our knowledge, the first report of Ascochyta blight of this host in the Republic of Georgia. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999.

Local mucociliary defence mechanisms.

The lung is continually at risk of exposure to noxious environmental agents and respiratory pathogens. An elaborate series of defence mechanisms have been developed to protect the airways from these insults. The lower respiratory tract is protected by local mucociliary mechanisms that involve the integration of the ciliated epithelium, periciliary fluid and mucus. Mucus acts as a physical and chemical barrier onto which particles and organisms adhere. Cilia lining the respiratory tract propel the overlying mucus to the oropharynx where it is either swallowed or expectorated. Regulation of periciliary fluid is thought essential to maintenance of both mucociliary clearance and to produce an environment in which airway antimicrobial peptides and defensins are effective. Disruption of mucociliary clearance may be caused by diseases such as cystic fibrosis, primary ciliary dyskinesia and asthma or may be secondary to pollutant exposure and viral or bacterial infections.

Analysis of ciliary beat pattern and beat frequency using digital high speed imaging: comparison with the photomultiplier and photodiode methods.

BACKGROUND: The aim of this study was to determine the relationship of the power and recovery stroke of respiratory cilia using digital high speed video imaging. Beat frequency measurements made using digital high speed video were also compared with those obtained using the photomultiplier and modified photodiode techniques. METHOD: Ciliated epithelium was obtained by brushing the inferior nasal turbinate of 20 healthy subjects. Ciliated edges were observed by microscopy and the deviation of cilia during their recovery stroke relative to the path travelled during their power stroke was measured. Beat frequency measurements made by digital high speed video analysis were compared with those obtained using the photomultiplier and modified photodiode. RESULTS: Cilia were found to beat with a forward power stroke and a backward recovery stroke within the same plane. The mean angular deviation of the cilia during the recovery stroke from the plane of the forward power stroke was only 3.6 degrees (95% CI 3.1 to 4.1). There was a significant difference in beat frequency measurement between the digital high speed video (13.2 Hz (95% CI 11.8 to 14.6)) and both photomultiplier (12.0 Hz (95% CI 10.8 to 13.1), p = 0.01) and photodiode (11.2 Hz (95% CI 9.9 to 12.5), p<0.001) techniques. The Bland-Altman limits of agreement for the digital high speed video were -2.75 to 5.15 Hz with the photomultiplier and -2.30 to 6.06 Hz with the photodiode. CONCLUSION: Respiratory cilia beat forwards and backwards within the same plane without a classical sideways recovery sweep. Digital high speed video imaging allows both ciliary beat frequency and beat pattern to be evaluated.