First Report of Target Spot of Broadleaf Tobacco Caused by Rhizoctonia solani (AG-3) in Connecticut.

In June 2011, 15 transplant beds of broadleaf cigar wrapper tobacco (Nicotiana tabacum L., cv. C9) plants in Hartford County, Connecticut, were observed with almost every plant diseased. Leaf lesion symptoms ranged from small (2 to 3 mm) water-soaked spots to larger (2 to 3 cm) lesions. Disease was subsequently observed, also at nearly 100% incidence in a 10-hectare field on that farm and at additional broadleaf tobacco farms from two other towns in Hartford County and one town in Tolland County. Lesions exhibited a pattern of concentric rings, necrotic centers and tears in the centers, and margins that often resulted in a shot-hole appearance. Some lesions had chlorotic halos. Rhizoctonia solani Kuhn (Thanatephorus cucumeris A. B. Frank) was isolated from the margins of lesions that had been surface sterilized in 0.5% NaOCl for 30 s and then rinsed in sterile distilled water and placed on the surface of half-strength potato dextrose agar (PDA). Multiple isolations were made and the pathogen was identified on the basis of mycelial characteristics including multinucleate cells, septate hyphae wider than 7 µm, and hyphal branches occurring at approximately right angles, constricted at the base (4). Eight-week-old potted tobacco plants were each inoculated by spraying with a mycelial suspension (1 × 105 CFU) of an isolate of R. solani recovered from tobacco onto leaves, or with water alone (five plants each). The plants were placed in plastic bags in a 24°C growth chamber and misted. After 2 days, the bags were removed and the potted plants placed in trays filled to a depth of 1 cm with water in the growth chamber. After 8 days, the pathogen was reisolated from all inoculated plants exhibiting water-soaked spots as disease symptoms. Leaves inoculated with water or half-strength PDA plugs alone were asymptomatic. DNA was liberated from hyphae of the R. solani isolate by bead beating in STE buffer using 0.15 mm zirconium beads. Two microliters of the eluate was used to amplify the ITS region. Amplified DNA was purified in a Qiagen QIAquick PCR purification kit and submitted to the Yale science hill genomic facility for standard Sanger dideoxy sequencing. The sequence was exactly the same as an isolate from Massachusetts that we sequenced in 2010 (GenBank Accession No. HQ241274). The ITS sequence confirmed our identification of this new isolate as R. solani anastomosis group (AG) 3. This disease has been previously reported on tobacco from South America, South Africa, and the southern United States (1), Canada (3), and Massachusetts (2). Conditions were very conducive for disease because 2011 was a very wet year in Connecticut. To our knowledge, this is the first report of this disease in broadleaf cigar wrapper tobacco in Connecticut. The sequence data suggested that it may have been introduced to Connecticut from Massachusetts. We have found the target spot pathogen distributed across the tobacco producing area of Connecticut. This constitutes a serious threat as there are no systemic fungicides currently registered for control of this disease in broadleaf tobacco. References: (1) J. S. Johnk et al. Phytopathology 83:854, 1993. (2) J. A. LaMondia and C. R. Vossbrinck, Plant Dis. 95:496, 2010. (3) R. D. Reeleder et al., Plant Dis., 80:712. (4) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St. Paul, MN, 1991.

First Report of Target Spot of Tobacco Caused by Rhizoctonia solani (AG-3) in Massachusetts.

In June 2010, shade-grown cigar wrapper tobacco (Nicotiana tabacum L.) plants in Hampshire County, Massachusetts were observed with leaf lesion symptoms that ranged from small (2 to 3 mm) water-soaked spots to larger (2 to 3 cm) lesions. Lesions had a pattern of concentric rings, necrotic centers and tears in the centers, and margins that often resulted in a shot-hole appearance. Some lesions had chlorotic halos. Rhizoctonia solani Kuhn (Thanatephorus cucumeris A.B. Frank) was isolated from lesions and identified on the basis of mycelial characteristics including multinucleate cells, septate hyphae wider than 7 µm and hyphal branches occurring at approximately right angles, and constricted at the base (3). Eight-week-old, potted tobacco plants were each inoculated either by spraying with a mycelial suspension (1 × 105 CFU) (five plants) or by placing colonized half-strength potato dextrose agar (PDA) plugs (0.2 cm) of an isolate of R. solani recovered from tobacco onto leaves (five plants) or with water or half-strength PDA plugs alone (five plants each). The plants were placed in plastic bags in a 24°C growth chamber and misted. After 2 days, the bags were removed and the potted plants were placed in trays filled with water to a depth of 1 cm in the growth chamber. After 8 days, the pathogen was reisolated from inoculated plants exhibiting water-soaked spots as disease symptoms. Leaves inoculated with water or half-strength PDA plugs alone were not diseased. DNA was extracted from the R. solani isolate and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified and sequenced (GenBank Accession No. HQ241274). The ITS sequence confirmed our identification of this new isolate as R. solani anastomosis group (AG) 3. This disease had been previously reported on tobacco from South America, South Africa, the southern United States (1), and Canada (2). To our knowledge, this is the first report of this disease in cigar wrapper tobacco in New England. The humid environmental conditions under which shade tobacco is grown make this new disease a significant threat for the Massachusetts and Connecticut growing area. References: (1) J. S. Johnk et al. Phytopathology 83:854, 1993 (2) R. D. Reeleder et al. Plant Dis. 80:712, 1996. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St. Paul, MN, 1991.

First Report of Blight of Common Bean Caused by Phytophthora capsici in Connecticut.

Phytophthora capsici Leonion was first identified on pepper (Capsicum annuum L.) and is widespread on solanaceous and cucurbitaceous crops. It was first documented on Phaseolus lunatus L. in Delaware in 2002 (1), followed by reports on snap beans (Phaseolus vulgaris L.) in Michigan in 2003 (2), and on Long Island, NY in 2008 ( http://vegetablemdonline.ppath.cornell.edu/ NewsArticles/Bean_phytoJune09.html ). In 2009, we observed snap and wax beans in commercial production with water-soaked lesions on foliage, stems, and pods. Twelve to sixteen hectares were affected in the flood plain of the Connecticut River in central Connecticut. Weather conditions had been warm and very wet. Lesions displayed white mycelia and sporangia. P. capsici was isolated from surface-sterilized tissue on potato dextrose agar (PDA) and malt extract agar. Hyphal tips were subcultured onto V8 media for further analysis. To confirm Koch's postulates, two isolates were tested for pathogenicity against bean (cv. Valentino) and pepper (cv. Cayenne) by placing colonized PDA plugs or PDA alone next to the crown or in stem branches. Symptoms similar to those observed in the field on bean and pepper developed on inoculated plants and the pathogen was reisolated. Controls did not develop disease. Sporangia of P. capsici growing on V8 medium were ellipsoid, ovoid, pyriform, but occasionally irregular, papillate, and 54.0 ± 5.7 × 31.1 ± 4.7 µm (n = 31) with a length/width (L/W) ratio of 1.8 ± 0.3. The papillae were 5.4 ± 0.9 µm (n = 31) and the pedicels were 24.5 ± 12.6 × 3.0 ± 1.0 µm. Sporangia collected from bean plants were smaller with longer pedicels; the sporangia were 44.9 ± 9.1 × 26.0 ± 2.8 µm with a L/W ratio of 1.7 ± 0.2; papillae were 4.6 ± 1.0 µm; and the pedicels were 49 ± 20.0 × 2.8 ± 0.9 µm (n = 20). To confirm the identity of our isolate genetically, DNA was extracted from one P. capsici isolate and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified and sequenced (GenBank Accession No. GU011684). The ITS sequence was identical to sequences of P. capsisci in GenBank and confirmed our identification of this new isolate as P. capsici. To our knowledge, this is the first report of P. capsici infecting Phaseolus vulgaris in Connecticut and New England. References: (1) C. R. Davidson et al. Plant Dis. 85:886, 2002. (2) A. J. Gevens et al. Plant Dis. 92:201, 2008.

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg.

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.

A phylogenetic approach to following West Nile virus in Connecticut.

The 1999 outbreak of West Nile (WN) virus in the northeastern United States was the first known natural occurrence of this flavivirus in the Western Hemisphere. In 1999 and 2000, 82 independent Connecticut WN virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. Nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with WN-NY99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ending at 1125. This region encodes portions of the nucleocapsid and envelope proteins and includes the entire coding regions for the premembrane and membrane proteins. Amino acid changes occurred at seven loci in six isolates relative to the WN-NY99 strain. Although 34 of the isolates showed sequences identical to the WN-NY99 isolate, we were able to show geographical-based clusters of mutations. In particular, 26 isolates were characterized by mutation of C to T at position 858. This group apparently originated in Stamford, CT and disseminated to sites located as far as 54 miles from Stamford. Sequences of WN virus isolated from both brain and heart tissues from the same avian host were identical in all 14 tested individual birds, suggesting that the mutations we have documented are real and not caused by culture, RNA extraction, or PCR procedures. We conclude that this portion of the viral genome will enable us to follow the geographical and temporal movement of variant WN virus strains as they adapt to North America.

Mosquito surveillance for West Nile virus in Connecticut, 2000: isolation from Culex pipiens, Cx. restuans, Cx. salinarius, and Culiseta melanura.

Fourteen isolations of West Nile (WN) virus were obtained from four mosquito species (Culex pipiens [5], Cx. restuans [4], Cx. salinarius [2], and Culiseta melanura [3]) in statewide surveillance conducted from June through October 2000. Most isolates were obtained from mosquitoes collected in densely populated residential locales in Fairfield and New Haven counties, where the highest rates of dead crow sightings were reported and where WN virus was detected in 1999. Minimum field infection rates per 1,000 mosquitoes ranged from 0.5 to 1.8 (county based) and from 1.3 to 76.9 (site specific). Cx. restuans appears to be important in initiating WN virus transmission among birds in early summer; Cx. pipiens appears to play a greater role in amplifying virus later in the season. Cs. melanura could be important in the circulation of WN virus among birds in sylvan environments; Cx. salinarius is a suspected vector of WN virus to humans and horses.

Impact of a North American isolate of the microsporidium Nosema carpocapsae on a laboratory population of the codling moth, Cydia pomonella.

Nosema carpocapsae is a microsporidian pathogen of the codling moth, Cydia pomonella. We report the occurrence of this pathogen in a colony originating from collections made in the United States. This is the first record of N. carpocapsae infecting North American codling moths. This North American isolate of N. carpocapsae was indistinguishable from isolates received from New Zealand and Bulgaria, based on small subunit ribosomal RNA sequencing, but was more virulent than the previously described New Zealand isolate. In the laboratory, infected larvae and pupae had increased mortality compared to their uninfected counterparts and developmental time increased by 1 week. There was no effect on female fecundity. Within a cohort of eggs laid by infected females, neonates that emerged first were more likely to be uninfected. We established an uninfected colony by interrupting horizontal transmission and only utilizing the larvae that emerged from the first-laid eggs.

Isolation of West Nile virus from mosquitoes, crows, and a Cooper's hawk in Connecticut.

West Nile (WN) virus, a mosquito-transmitted virus native to Africa, Asia, and Europe, was isolated from two species of mosquitoes, Culex pipiens and Aedes vexans, and from brain tissues of 28 American crows, Corvus brachyrhynchos, and one Cooper's hawk, Accipiter cooperii, in Connecticut. A portion of the genome of virus isolates from four different hosts was sequenced and analyzed by comparative phylogenetic analysis. Our isolates from Connecticut were similar to one another and most closely related to two WN isolates from Romania (2.8 and 3.6 percent difference). If established in North America, WN virus will likely have severe effects on human health and on the health of populations of birds.

A new Microsporidium sp. (microsporidia) from the musculature of the mountain whitefish Prosopium williamsoni from British Columbia: morphology and phylogeny.

Few microsporidia have been reported from whitefish species (subfamily Coregoninae). For the most part, these microsporidia have been incompletely described. In a survey of parasites of mountain whitefish Prosopium williamsoni collected from Kootenay Lake, British Columbia, we encountered an unusual microsporidium infecting the endomysium of the skeletal musculature. Spores were uninucleate, ovoid to pyriform, and were 5.6 (5-7) microm x 3.2 (3-4) microm with 13-16 coils in the polar filament. We describe here this organism as a new species based on its site of development and its relationship among fish microsporidia based on small subunit ribosomal DNA sequence data, i.e., our analysis showed that it is not closely related to other microsporidia for which ribosomal DNA sequence is available thus far.

Verification of intermediate hosts in the life cycles of microsporidia by small subunit rDNA sequencing.

Small subunit rDNA sequences were obtained from field-collected Amblyospora connecticus (Microsporida: Amblyosporidae) spores isolated from the mosquito, Aedes cantator (Diptera: Culicidae), and from field collected spores isolated from the putative intermediate host, Acanthocyclops vernalis (Copepoda: Cyclopidae). The ribosomal DNA sequences of the spores isolated from the two hosts were identical. These findings corroborate previous laboratory transmission studies and validate the intermediary role of A. vernalis in the life cycle of this microsporidium. These data represent the first comparative sequence analysis of a microsporidium isolated from its definitive and intermediate hosts. The results demonstrate the effectiveness of using rDNA sequence data for screening potential intermediate hosts. Unlike laboratory transmission tests, which can take months or years to complete, this technique can be completed in days and can be performed on a single infected organism.

Phylogeny of amblyospora (Microsporida: amblyosporidae) and related genera based on small subunit ribosomal DNA data: A possible example of host parasite cospeciation.

Small subunit ribosomal RNA (SSU rRNA) gene sequences were analyzed for six species and four genera of microsporidia from mosquito hosts; Amblyospora stimuli (Aedes stimulans), Amblyospora californica (Culex tarsalis), Amblyospora sp. (Culex salinarius), Edhazardia aedis (Aedes aegypti), Culicosporella lunata (Culex pilosus), and Parathelohania anophelis (Anopheles quadrimaculatus). Comparison of these sequences to those of other microsporidia show that these sequences are longer with the SSU rRNA gene of E. aedis being the longest microsporidia sequenced to date (1447 base pairs). Parsimony, maximum likelihood, and distance methods produced identical trees, suggesting that the above microsporidian taxa, contrary to current classification schemes, form a monophyletic group. Relationships within this group are further supported by high bootstrap and decay analysis values. Based on the molecular analysis, P. anophelis is the most divergent species in this group of mosquito parasites. Amblyospora is paraphyletic with A. californica and Amblyospora sp., forming a sister taxon to a clade composed of E. aedis and A. stimuli. Culicosporella lunata comprises a sister taxon to the Amblyospora/Edhazardia clade. The pattern of host relationships on the tree provides preliminary evidence that the branching pattern seen here may indicate that host-parasite cospeciation is an important mechanism of evolution in this group.

Microsporidiosis: molecular and diagnostic aspects.

The term 'microsporidia' is a nontaxonomic designation which is used to refer to a group of intracellular parasites belonging to the phylum Microspora. These eukaryotic obligate intracellular protozoans have been described infecting every major animal group, especially insects, fish and mammals. They are important agricultural parasites in commercially important insects, fish, laboratory rodents, rabbits, fur-bearing animals, and primates. There is now an increasing recognition of microsporidia as important opportunistic pathogens in persons infected with the human immunodeficiency virus (HIV). Microsporidia possess ribosomes with features resembling prokaryotes. Phylogenetic analysis of the rRNA sequence from several of the microsporidia suggests that these organisms were early branches in the eukaryotic evolutionary line. The data on these molecular phylogenetic relationships are reviewed in this paper. Inroads have recently been made into the molecular biology of these organisms and these data are also presented. Diagnosis of microsporidia infection from stool examination is possible and has replaced biopsy as the initial diagnostic procedure in many laboratories. These staining techniques can be difficult, however, due to the small size of the spores. The specific identification of microsporidian species has classically depended on ultrastructural examination. With the cloning of the rRNA genes from the human pathogenic microsporidia it has been possible to apply polymerase chain reaction (PCR) techniques for the diagnosis of microsporidial infection at the species level. Both staining and PCR techniques for the diagnosis of microsporidia are reviewed.

Phylogenetic position of Amblyospora Hazard & Oldacre (Microspora:Amblyosporidae) based on small subunit rRNA data and its implication for the evolution of the microsporidia.

Sequences of the small subunit rRNA genes of Amblvospora california and an Amblyospora sp. from Culex salinarius were determined. These sequences were compared phylogenetically with 16 other microsporidia. The results suggest Amblyospora forms a sister taxon to the rest of the microsporidia examined. The basal position of Amblyospora is discussed with respect to the evolution of microsporidian life cycles. These sequences represent the longest microsporidian small subunit rRNA genes sequenced to date, 1,359 and 1,358 bp. respectively. Structural features and GC content (49% for both) are comparable to those of other microsporidia which have been sequenced.

A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria.

Microsporidia are small (1-20 micron) obligate intracellular parasites of a variety of eukaryotes, and they are serious opportunistic pathogens of immunocompromised patients [1]. Microsporidia are often assigned to the first branch in gene trees of eukaryotes [2,3], and are reported to lack mitochondria [2,4]. Like diplomonads and trichomonads, microsporidia are hypothesised to have diverged from the main eukaryotic stock prior to the event that led to the mitochondrion endosymbiosis [2,4]. They have thus assumed importance as putative relics of premitochondrion eukaryote evolution. Recent data have now revealed that diplomonads and trichomonads contain genes that probably originated from the mitochondrion endosymbiont [5-9], leaving microsporidia as chief candidates for an extant primitively amitochondriate eukaryote group. We have now identified a gene in the microsporidium Vairimorpha necatrix that appears to be orthologous to the eukaryotic (symbiont-derived) Hsp70 gene, the protein product of which normally functions in mitochondria. The simplest interpretation of our data is that microporidia have lost mitochondria while retaining genetic evidence of their past presence. This strongly suggests that microsporidia are not primitively amitochondriate and makes feasible an evolutionary scenario whereby all extant eukaryotes share a common ancestor which contained mitochondria.

A microsporidian isolated from an AIDS patient corresponds to Encephalitozoon cuniculi III, originally isolated from domestic dogs.

The ribosomal DNA internal transcribed spacer (ITS) region of a recently cultured human Encephalitozoon cuniculi isolate was analyzed by gene amplification and DNA sequencing. Restriction endonuclease digestion (FokI) and double-stranded DNA heteroduplex mobility shift analysis were performed to determine their utility for strain differentiation. The human E. cuniculi isolate was identical to E. cuniculi III, which had been isolated only from domestic dogs until now. The patient providing the isolate owned a pet dog, but no microsporidia were detected in the pet's urine.

Restriction analysis of the ribosomal DNA internal transcribed spacer region of Culex restuans and mosquitoes in the Culex pipiens complex.

Members of the Culex pipiens Linn. complex in the eastern, southern, and central United States are the primary vectors of St. Louis encephalitis virus. Although species and subspecies in the complex can be identified as 4th-instar larvae and by characters on the male genitalia, adult females cannot be identified accurately. In this study a ribosomal DNA (rDNA) segment that includes the internal transcribed spacer region (ITS) was amplified from Culex pipiens pipiens Linn., Culex quinquefasciatus Say, and Culex restuans Theobald. The DNA was amplified from single abdomens or single legs. The amplified rDNA segment from Cx. restuans is 90 base pairs smaller than those from members of the Cx. pipiens complex. Ribosomal DNA was amplified separately from 3 individuals for each population of Cx. pipiens and analyzed by restriction digestion. Intrapopulation variation is seen, because for each population, bands are present that are common to all 3 individuals within the population, but are also unique to that population. These results indicate that this method may provide a means for distinguishing among the mosquitoes in the Cx. pipiens complex.

Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix.

An RNA homologous to U2 RNA and a single copy gene encoding the RNA homolog have been characterized in the microsporidian, Vairimorpha necatrix. The RNA which is 165 nucleotides in length possesses significant similarity to U2 RNA, particularly in the 5' half of the molecule. The U2 homolog contains the highly conserved GUAGUA branch point binding sequence seen in all U2 RNAs except those of the trypanosomes. A U2 RNA sequence element implicated in a U2:U6 RNA intermolecular pairing is also present in the U2 homolog. The V. necatrix U2 RNA homolog differs at positions previously found to be invariant in U2 RNAs and appears to lack an Sm binding site sequence. The RNA can be folded into a secondary structure possessing three of the four principal stem-loops proposed for the consensus U2 RNA structure. A cis-diol containing cap structure is present at the 5' end of the U2 homolog. Unlike the cap structures seen in U-snRNAs and mRNAs it is neither 2,2,7-trimethylguanosine, gamma-monomethyl phosphate, nor 7-methylguanosine.