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Hydrogels are attractive soilless media for plant cultivation with strong water and nutrient retention. However, pristine hydrogels contain mostly ultra-micro pores and lack air-filled porosity for root zone aeration. Herein we report a porous hydrogel composite comprising an agarose network and porous growing mix particle (GMP) fillers. The agarose backbone allowed the composite to sustain a 12-d growth cycle for red cabbage microgreens without the need for watering or crew interaction. Moreover, the GMP induced greater total pore volume and increased the prevalence of pores30 μm by 8-fold. Further investigation suggested that the nutrients from GMP accounted for a 54 % increase in microgreen yield over pristine hydrogel, while the porous structure introduced by GMP improved the yield by another 44 %. Increased air-filled porosity accelerated the water transport and loss of hydrogel but maintained favorable water potential levels for plant extraction. Finally, the hydrogel composite supported microgreen growth satisfyingly under simulated microgravity despite some morphological changes. Results of this study reveal a novel growth substrate that is lightweight, convenient, and water-efficient, while effectively sustaining plant growth for multiple applications including indoor farming and space farming.

Streptomyces strain NRRL B-2795T (DSM 112329T=NRRL B-2795T) is described as the type strain of Streptomyces griseiscabiei sp. nov. using whole-genome average nucleotide identity and multilocus sequence analyses in addition to phenotypic characterization of carbon source utilization, spore chain morphology, melanin production, salt tolerance, pH tolerance, plant pathogenicity and antibiotic resistance. This strain was previously classified as Streptomyces scabiei but suggested as a potential novel species. A second Streptomyces strain, NRRL B-16521, previously named Streptomyces scabiei , and also previously suggested as a potential novel species, is assigned to Streptomyces acidiscabies based on whole-genome average nucleotide identity. Morphological and biochemical characterizations also support this designation for NRRL B-16521. Both Streptomyces sp. strain NRRL B-2795T and NRRL B-16521 cause common scab on multiple cultivars of potato.

Varroa destructor is a cosmopolitan pest and leading cause of colony loss of the European honey bee. Historically described as a competent vector of honey bee viruses, this arthropod vector is cause for the global pandemic of Deformed wing virus, now endemic in honeybee populations. Our work shows viral spread is driven by Varroa actively switching from one adult bee to another as they feed. Assays using fluorescent microspheres were used to show the movement of fluids in both directions between host and vector when Varroa feed. Therefore, Varroa could be in either an infectious or naïve state dependent upon the disease status of their host. We tested this and confirm that the relative risk of a Varroa feeding was dependent on the infectiousness of their previous host. Varroa exhibit remarkable heterogeneity in their host switching behavior, with some Varroa switching infrequently while others switch at least daily. As a result, relatively few of the most active Varroa parasitize the majority of bees. This multiple feeding behavior has analogs in vectorial capacity models of other systems, where promiscuous feeding by individual vectors is a leading driver of vectorial capacity. We propose that the honeybee-Varroa relationship offers a unique opportunity to apply principles of vectorial capacity to a social organism, as virus transmission is both vectored and occurs through multiple host-to-host routes common to a crowded society.

Two isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from a small pool in marshland near the mouth of the Nanticoke River in Maryland, USA. The isolates IIBBL 257-1T and IIBBL 257-2 had identical 16S rRNA gene sequences as determined by PCR, and highly similar fatty acid and biochemical profiles. The 16S rRNA gene sequences indicated the isolates belonged to the genus Chromobacterium . Genomic sequencing of IIBBL 257-1T revealed a genome of 4.27 Mb, with a G+C content of 63.6 %. Whole genome comparisons with other members of the Chromobacterium using JSpecies and the genome blast distance phylogeny approach indicated that among described species, IIBBL 257-1T was most closely related to C. amazonense and C. phragmitis . Comparison of the IIBBL 257-1T genome with those of type strains of these species resulted in ANIb and dDDH values of ca. 85 and 30 %, respectively, for both. These results demonstrate that IIBBL 257-1T and IIBBL 257–2 represent a new taxon within the genus Chromobacterium . We propose the name Chromobacterium paludis sp. nov. for this taxon; the type strain is IIBBL 257-1T (=NRRL B-65555T=JCM 33770T).

Summary Punctodera mulveyi n. sp. is described and illustrated from turf grass (Poa annua) in golf course greens with other fescues in Bandon, Coos County, Oregon, USA. Females and cysts are characterised by a saccate, globose to ovoid or pear-shaped body with a protruding neck. The cuticle has a lace-like pattern of ridges and heavy punctations on the subsurface. Cysts have distinctive vulval and anal circumfenestral patterns with heavy bullae scattered around the fenestral area, these being absent in young cysts. Second-stage juveniles (J2) vermiform, tapering to a long and cylindrical tail with a bluntly rounded to occasionally clavate tail terminus. Morphologically the new species resembles all known species of Punctodera using both light microscopy and scanning electron microscopy observations, but differs from the other species either by the J2 body and stylet length, shape of head, tail and tail terminus, female and male stylet or spicule length, and in having distinctive vulval and anal circumfenestral patterns in the cysts. Molecular analysis with sequence alignments and phylogenetic trees of ITS rDNA, nuclear heat shock protein 90 and mitochondrial COI sequences separated P. mulveyi n. sp. from P. matadorensis, P. punctata, P. stonei and P. chalcoensis, but 18S and 28S were relatively conserved with a few bp differences and there were insufficient Punctodera species sequences to give strong support to a new species designation. A morphologically most closely related species, P. stonei from Canada, further supported the status of P. mulveyi n. sp. An identification key to all five nominal species of Punctodera is given.

This study examined the ovipositional behavior of Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae) by evaluating how parasitoid density and access to nutrition influenced percent parasitism on egg masses of different sizes in laboratory tests. When three parasitoids were exposed to A. tristis egg masses with only three to five eggs, 72.7% of parasitoids became trapped in the eggs and failed to emerge successfully. These results suggest that competition between larvae within the egg may have reduced the fitness of the surviving parasitoid. Continual access to honey water did not significantly influence parasitism rates on A. armigera egg masses and only increased parasitism on A. tristis egg masses with 20–25 eggs. Overall, parasitism rates were higher on A. armigera egg masses than on A. tristis egg masses, and parasitoids were more likely to emerge successfully from A. armigera eggs than from A. tristis eggs. Parasitoids spent the same amount of time probing eggs of the two species, but they spent significantly more time drilling into A. tristis eggs than A. armigera eggs. Measurements taken using transmission electron microscopy determined that the average combined width of the epicuticle and exocuticle of the egg chorion was significantly greater for A. tristis eggs than for A. armigera eggs. This difference may account for the lower rates of parasitism and parasitoid emergence and for the increased time spent drilling into A. tristis eggs compared with A. armigera eggs.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Gamasellodes garybauchani sp. nov. Rueda-Ramírez & Santos is described based on specimens collected at the Beltsville Agricultural Research Center (BARC) (Maryland, USA). Twenty-one specimens were used for the description, of which five were subjected to low temperature scanning electron microscopy (LTSEM), followed by DNA extraction for genetic analysis and subsequent slide mounting. The remaining 16 specimens were subjected to only DNA extraction for genetic analysis and subsequent slide mounting. Morphological characters of the new species observed both in the images obtained in LTSEM and in the observation of the mounted specimens are detailed. Molecular information available on the Barcode of Life Datasysetm (BOLD) is presented. An update to the recent key to the Gamasellodes species is provided.

Current and future legislation requiring the reduction of pesticides use, coupled with global initiatives for the promotion of soil health and conservation of soil biodiversity are creating opportunities for studies aimed at highlighting ecosystem services provided by functioning soil food webs in agricultural systems, including soil predatory mites. However, the key personnel for performing such studies are expert taxonomists, who are already spread very thin. To meet this demand, we propose an integrative approach where scientists (without expertise in taxonomy) play a significant role in supporting expert taxonomists. Soil samples were collected at the USDA ARS Farming Systems Project (FSP) at the Beltsville Agricultural Research Center, either incubated or not, followed by extraction of mites and nematodes. Improved modified Berlese funnels and an extraction protocol were utilized to improve sequencing success. Incubation dramatically enhanced the number of extracted individuals per sample whilst the daily freezing of extracted mites substantially improved the sequencing success rate compared to previous studies. Taken together, this led to the addition of records for eight Mesostigmata and ten Oribatida BINs to BOLD (Barcode of Life Datasystem). Fifteen species of Mesostigmata species were found, with three dominant species, Cycetogamasus diviortus (Athias-Henriot, 1967) (Parasitidae), Lasioseius youcefi Athias-Henriot 1959 (Blattisociidae) and a new species of Gamasellodes (Ascidae). LTSEM imaging followed by molecular identification contributed further details to the published descriptions of C. diviortus and L. youcefi. In line with our general aim, collecting, extracting, identification to morpho-species, sample preparation for DNA barcoding and uploading relevant information to BOLD was performed by trained personnel, but without taxonomic expertise. Whereas our skilled taxonomists focused on the morphological identifications using light microscopy, expanding on existing descriptions using LTSEM images and in a subsequent manuscript the description of a new species. We believe this division of tasks and labor will set the stage for further collaborative integrated studies between ecologists, biocontrol specialists and expert taxonomists for the identification, evaluation, and description of known and novel soil acarine biological control agents (BCAs).

Eriophyoid mites, also known as bud, gall, or rust mites, impact nearly all crops of economic importance, including food crops, timber, and ornamental plants. Currently, they are divided into three families (Phytoptidae, Eriophyidae and Diptilomiopidae) with an estimated one million species, of which just over 5600 have been described.

A study was conducted to investigate epidemiological aspects of papaya virus E (PpVE), a cytorhabdovirus commonly found in papaya (Carica papaya L.) plantings in Ecuador. Besides papaya, PpVE was found in three Fabaceae weeds, including Rhynchosia minima, Centrosema plumieri, and Macroptilium lathyroides, the latter being the species with the highest virus prevalence. Greenhouse experiments showed that in M. lathyroides, single infections of PpVE induce only mild leaf mosaic, whereas in mixed infections with cowpea severe mosaic virus, PpVE contributes to severe mosaic. In papaya, PpVE did not induce noticeable symptoms in single or mixed infections with papaya ringspot virus. Transmission experiments confirmed that whiteflies (Bemisia tabaci) transmit PpVE in a semipersistent, nonpropagative manner.

The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George’s County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 μm, stylet from 70 to 75 μm long with anchor shaped knobs, vulva located at 70–73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17–20 µm and gubernaculum = 5.0–5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

Barbary sheep (Ammotragus lervia) is a North African native wild Caprinae, introduced in the 1970s in new territories such as Spain, the USA, and Mexico. Here, we describe Sarcocystis species in Barbary sheep. Sarcocysts were found in 19 out of 56 adult A. lervia in Southern Spain and characterized morphologically and molecularly. By light microscopy, sarcocysts had thin ( 2 μm) walls. By transmission electron microscopy, sarcocysts with thick walls had Type 14 villar protrusions corresponding to S. tenella/S. capracanis of domestic sheep (Ovis aries) or goats (Capra hircus). Sarcocysts with thin walls had Type 7b villar protrusions that corresponded to S. arieticanis/S. hircicanis of domestic sheep or goats. Molecular analyses allowed the identification of only thick-walled Sarcocystis species. Six sarcocysts were assigned to S. tenella (99.2–100% and 95.6–100% sequence similarity within 18S rRNA and COI, respectively) and 19 sarcocysts were assigned to S. capracanis (98.5–99.8% and 97.9–99.0% sequence similarity within 18S rRNA and COI, respectively). Further studies are needed for taxonomic identification of sarcocysts in Barbary sheep because Sarcocystis species in sheep and goats are not cross transmissible despite morphological similarities.

Thirteen isolates of Gram-stain-negative, motile, violet-pigmented bacteria were isolated from marshes along tidal portions of the Potomac and James rivers in Maryland and Virginia, USA, respectively. 16S rRNA gene sequences and fatty acid analysis revealed a high degree of relatedness among the isolates, and genomic sequencing of two isolates, IIBBL 112-1T and IIBBL 274-1 (from the Potomac and James rivers, respectively), revealed highly similar genomic sequences, with a blast-based average nucleotide identity (ANIb) of ca. 98.7 %. Phylogenetic analysis of 16S rRNA gene sequences suggested that the species most highly related to IIBBL 112-1T were Chromobacterium amazonense , Chromobacterium subtsugae and Chromobacterium sphagni . However, deletion of a 25-nucleotide sequence that may have been horizontally acquired by both IIBBL 112-1T and C. amazonense resulted in a substantially different analysis; in the latter case, the species nearest IIBBL 112-1T were Chromobacterium violaceum , Chromobacterium vaccinii and Chromobacterium piscinae . Whole-genome alignments between either IIBBL 112-1T or IIBBL 274-1 and the type strains of C. vaccinii or C. violaceum resulted in ANIb values in the range of ca. 87 %, while alignment with C. amazonense CBMAI 310T resulted in an ANIb of ca. 83 %. Collectively, these data demonstrate that IIBBL 112-1T and IIBBL 274-1 represent a new taxon within the genus Chromobacterium . We propose the name Chromobacterium phragmitis sp. nov. for this taxon; the type strain is IIBBL 112-1T (=NRRL B-67132T=JCM 31884T).

Heliothine eggs are commonly found on agricultural commodities and ornamental plants transported through domestic and international commerce. Tobacco budworm [Chloridea (Heliothis) virescens (F.)], Chloridea subflexa (Guenée), and the corn earworm [Helicoverpa zea (Boddie)] are indigenous pests of the American continent. Interceptions of the Old World bollworm [Helicoverpa armigera (Hübner)] at various ports of entry into the United States have been detected due to the invasion of this pest in South America, adding to the complexity and importance of decision making at plant inspection stations. The ability to distinguish the eggs of C. virescens from C. subflexa and Helicoverpa species is a critical component for conducting risk assessments by quarantine authorities, taxonomists, and crop consultants. We developed a simple, rapid (~60 min), inexpensive ($0.06 per sample), and accurate (100% reliability) technique to distinguish C. virescens eggs from the possibility of being H. armigera based on the presence, number, and/or size of aeropyle holes on the primary ribs of eggs, near the micropylar rosette. In this location, aeropyles were easily visible at 40× magnification in 213 fresh and ethanol-preserved C. virescens eggs once treated with Hoyer’s solution. A small number of C. subflexa had one or two aeropyles on a few of the primary ribs, whereas no aeropyles were found on 411 H. zea and 269 H. armigera eggs analyzed using optical, electron, and confocal microscopy. We conclude that in most cases multiple aeropyle holes positively identify H. virescens eggs from H. zea or H. armigera, and C. subflexa. This technique potentially will reduce the number of specimens that need to be sent for molecular identification thereby saving program time and resources.

Significance Varroa destructor causes considerable damage to honey bees and subsequently the field of apiculture through just one process: feeding. For five decades, we have believed that these mites consume hemolymph like a tick consumes blood, and that Varroa cause harm primarily by vectoring viruses. Our work shows that they cause damage more directly. Varroa externally digest and consume fat body tissue rather than blood. These findings explain the failure of some previous attempts at developing effectively targeted treatment strategies for Varroa control. Furthermore, it provides some explanation for the diverse array of debilitating pathologies associated with Varroa that were unexplained by hemolymph removal alone. Our work provides a path forward for the development of novel treatment strategies for Varroa., The parasitic mite Varroa destructor is the greatest single driver of the global honey bee health decline. Better understanding of the association of this parasite and its host is critical to developing sustainable management practices. Our work shows that this parasite is not consuming hemolymph, as has been the accepted view, but damages host bees by consuming fat body, a tissue roughly analogous to the mammalian liver. Both hemolymph and fat body in honey bees were marked with fluorescent biostains. The fluorescence profile in the guts of mites allowed to feed on these bees was very different from that of the hemolymph of the host bee but consistently matched the fluorescence profile unique to the fat body. Via transmission electron microscopy, we observed externally digested fat body tissue in the wounds of parasitized bees. Mites in their reproductive phase were then fed a diet composed of one or both tissues. Mites fed hemolymph showed fitness metrics no different from the starved control. Mites fed fat body survived longer and produced more eggs than those fed hemolymph, suggesting that fat body is integral to their diet when feeding on brood as well. Collectively, these findings strongly suggest that Varroa are exploiting the fat body as their primary source of sustenance: a tissue integral to proper immune function, pesticide detoxification, overwinter survival, and several other essential processes in healthy bees. These findings underscore a need to revisit our understanding of this parasite and its impacts, both direct and indirect, on honey bee health.

The Mythimna unipuncta nucleopolyhedrovirus isolate KY310 (MyunNPV-KY310) is an alphabaculovirus isolated from a true armyworm (Mythimna unipuncta) population in Kentucky, USA. Occlusion bodies of this virus were examined by electron microscopy and the genome sequence was determined by 454 pyrosequencing. MyunNPV-KY310 occlusion bodies consisted of irregular polyhedra measuring 0.8–1.8 µm in diameter and containing multiple virions, with one to six nucleocapsids per virion. The genome sequence was determined to be 156,647 bp with a nucleotide distribution of 43.9% G+C. 152 ORFs and six homologous repeat (hr) regions were annotated for the sequence, including the 38 core genes of family Baculoviridae and an additional group of 26 conserved alphabaculovirus genes. BLAST queries and phylogenetic inference confirmed that MyunNPV-KY310 is most closely related to the alphabaculovirus Leucania separata nucleopolyhedrovirus isolate AH1, which infects Mythimna separata. In contrast, MyunNPV-KY310 did not exhibit a close relationship with Mythimna unipuncta nucleopolyhedrovirus isolate #7, an alphabaculovirus from the same host species. MyunNPV-KY310 lacks the gp64 envelope glycoprotein, which is a characteristic of group II alphabaculoviruses. However, this virus and five other alphabaculoviruses lacking gp64 are placed outside the group I and group II clades in core gene phylogenies, further demonstrating that viruses of genus Alphabaculovirus do not occur in two monophyletic clades. Potential instances of MyunNPV-KY310 ORFs arising by horizontal transfer were detected. Although there are now genome sequences of four different baculoviruses from M. unipuncta, comparison of their genome sequences provides little insight into the genetic basis for their host specificity.

Rhizobia colonize legumes and reduce N2 to NH3 in root nodules. The current model is that symbiotic rhizobia bacteroids avoid assimilating this NH3. Instead, host legume cells form glutamine from NH3, and the nitrogen is returned to the bacteroid as dicarboxylates, peptides, and amino acids. In soybean cells surrounding bacteroids, glutamine also is converted to ureides. One problem for soybean cultivation is inefficiency in symbiotic N2 fixation, the biochemical basis of which is unknown. Here, the proteomes of bacteroids of Bradyrhizobium elkanii USDA76 isolated from N2 fixation-efficient Peking and -inefficient Williams 82 soybean nodules were analyzed by mass spectrometry. Nearly half of the encoded bacterial proteins were quantified. Efficient bacteroids produced greater amounts of enzymes to form Nod factors and had increased amounts of signaling proteins, transporters, and enzymes needed to generate ATP to power nitrogenase and to acquire resources. Parallel investigation of nodule proteins revealed that Peking had no significantly greater accumulation of enzymes needed to assimilate NH3 than Williams 82. Instead, efficient bacteroids had increased amounts of enzymes to produce amino acids, including glutamine, and to form ureide precursors. These results support a model for efficient symbiotic N2 fixation in soybean where the bacteroid assimilates NH3 for itself.

Rodents are intermediate hosts for many species of Sarcocystis. Little is known of Sarcocystis cymruensis that uses the Brown rat (Rattus norvegicus) as intermediate hosts and the domestic cat (Felis catus) as experimental definitive host. Here, we identified and described Sarcocystis cymruensis in naturally infected R. norvegicus from Grenada, West Indies. Rats (n = 167) were trapped in various locations in two parishes (St. George and St. David). Microscopic, thin (

A facile and template-free solvothermal method was developed as a bottom-up approach to synthesize mesoporous/macroporous MOF nanosheets in a simple and scalable way. It was found that starting coordination complexes of different copper(ii)-ligand compounds mediated the controlled growth and morphology of MOF crystals. By controlling the size and shape of the MOF crystals, the possibility to adjust and tailor the structure and performances of the assemblies was demonstrated. This work provides a bottom-up approach to synthesize MOF films and nanosheets in a simple and scalable way, which may have potential in energy and biomedical applications.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

The protozoan parasite Sarcocystis neurona is an important cause of disease in horses (equine protozoal myeloencephalitis, EPM) and marine mammals. Isolated reports of clinical EPM-like disease have been documented in a zebra, raccoon, domestic cat, domestic dog, ferret, skunk, mink, lynx, red panda and fisher. The predominant disease is encephalomyelitis associated with schizonts in neural tissues. Here, we report highly disseminated sarcocystosis, in many tissues of a captive White-nosed coati (Nasua narica molaris). The 14year old, neutered male coati was euthanized due to progressive weakness, lethargy, and inappetence. Schizonts, including free and intracellular merozoites were detected in many cell types, and differed morphologically from S. neurona schizonts in horses. Only a few sarcocysts were seen in skeletal muscle and the myocardium. Immunohistochemically, the protozoa reacted positively to S. neurona but not to Toxoplasma gondii antibodies. Severe inflammtory disease detected in the stomach, intestine, adrenal and thyroid glands, ciliary body of eye, and urinary bladder associated with schizonts in the coati has not been reported earlier in any host with EPM. Although, a few schizonts were found in the brain, encephalitis was minimal and not the cause of clinical signs. Multilocus PCR-DNA sequencing using DNA derived from the coati lung tissue identified an S. neurona infection using the 18S, 28S and ITS-1 markers, and a novel genotype using primer pairs against antigenic surface proteins (SnSAG3, SnSAG4, SnSAG1-5-6) and microsatellite markers (MS, SN7, SN9). Although the genotype was similar to the widely distributed Type VI strain, it possessed a novel allele at SnSAG5, and a different MS combination of repeats at SN7 and SN9. Whether this severe parasitism was related to the host or the parasite needs further investigation.

Here, we report a new species, Sarcocystis pantherophisi n. sp., with the Eastern rat snake (Pantherophis alleghaniensis) as natural definitive host and the interferon gamma gene knockout (KO) mouse as the experimental intermediate host. Sporocysts (n = 15) from intestinal contents of the snake were 10.838.9 lm. Sporocysts were orally infective to KO mice but not to laboratory-raised albino outbred house mice (Mus musculus). The interferon gamma KO mice developed schizont-associated neurological signs, and schizonts were cultivated in vitro from the brain. Mature sarcocysts were found in skeletal muscles of KO mice examined 41 days postinoculation (PI). Sarcocysts were slender, up to 70 lm wide and up to 3.5 mm long. By light microscopy, sarcocysts appeared thin-walled (, 1 lm) without projections. By transmission electron microscopy, the sarcocyst wall was a variant of "type 1'' (type 1i, new designation). The parasitophorous vacuolar membrane (pvm) had approximately 100-nm-wide3100-nm-long bleb-like evaginations interspersed with 100-nm-wide 3 650-nm-long elongated protrusions at irregular distances, and invaginations into the ground substance layer (gs) for a very short distance (6 nm). The gs was smooth, up to 500 nm thick, without tubules, and contained a few vesicles. Longitudinally cut bradyzoites at 54 days PI were bananashaped, 7.832.2 lm (n = 5). Molecular characterization using 18S rRNA, 28S rRNA, ITS-1, and cox1 genes indicated a close relationship with other Sarcocystis parasites that have snake-rodent life cycles. The parasite in the present study was molecularly and biologically similar to a previously reported isolate (designated earlier as Sarcocystis sp. ex Pantherophis alleghaniensis) from P. alleghaniensis, and it was structurally different from other Sarcocystis species so far described. Public domain – authored by a U.S. government employee

Soil predatory mites feed on a diverse diet making them excellent candidates for conservation biocontrol. Free-living nematodes (FLN) are commonly found in soils and serve as prey for many acarine predators. Our goal was to determine whether conservation biological control of plant-parasitic nematodes by predators could be enhanced by provisioning FLN with their culture medium (FLNCM) under semi-field conditions. We conducted two experiments on dwarf tomato plants, the first until the beginning of flowering and the second until harvest. The treatments evaluated were with and without: 1) the root-knot nematode Meloidogyne incognita, 2) the predator Stratiolaelaps scimitus, and 3) the FLN Rhabditella axei in its culture medium. In both experiments, gall abundance was lowest in the combined treatment of FLN and predators. Similar reduction in gall abundance occurred when only predators or only FLNCM was added to the soil mix. Additionally, in the FLNCM treatment, foliar macronutrients N and K were significantly higher than the negative control. Our original aim was to use FLN as a supplementary food source for predators in conservation IPM. Based on the significant reduction in gall numbers, and the increase in foliar macronutrients, it is clear that the FLNCM treatment played additional roles. Finally, for demonstration, the predation of M. incognita was visualized in high resolution imaging using a low-temperature-scanning electron microscope. Accordingly, we recommend that future research focus on identifying soil amendments that will foster the establishment of beneficial microbiota, FLN and soil predators for the conservation biological control of soil pests.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Escherichia coli O157:H7 and Salmonella enterica are foodborne pathogens with major public health concern in the U.S. These pathogens utilize several virulence factors to initiate infections in humans. The antimicrobial effect of seven glucosinolate hydrolysis compounds against Salmonella and E. coli O157:H7 was investigated by the disc diffusion assay. Among the tested compounds, benzyl isothiocyanate (BIT), which exerted the highest antimicrobial activity, was evaluated for its anti-virulence properties against these pathogens. The effect of BIT on motility of Salmonella and E. coli O157:H7 and Shiga toxin production by E. coli O157:H7 was determined by the motility assay and ELISA procedure, respectively. Confocal and transmission electron microscopy (TEM) procedures were used to determine bacterial damage at the cellular level. Results revealed that sub-inhibitory concentrations (SICs) of BIT significantly inhibited the motility of both bacteria (P 0.05). Shiga toxin production by E. coli O157:H7 was decreased by ~32% in the presence of BIT at SICs. TEM results showed the disruption of outer membrane, release of cytoplasmic contents, and cell lysis following BIT treatment. Results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by reducing the virulence factors including bacterial motility and Shiga toxin production.

The complete genome of a new rhabdovirus infecting papaya (Carica papayaL.) was sequenced and characterized. The genome consists of 13,469 nucleotides with six canonical open reading frames (ORFs) predicted from the antigenomic strand. In addition, two overlapping short ORFs were predicted between ORFs 3 and 4. Phylogenetic analyses using amino acid sequences from the nucleocapsid, glycoprotein and polymerase, grouped the virus with members of the genusCytorhabdovirus, with rice stripe mosaic virus, yerba mate chlorosis-associated virus and Colocasia bobone disease-associated virus as closest relatives. The 3’ leader and 5’ trailer sequences were 144 and 167 nt long, respectively. Each end contains complementary sequences prone to form panhandle structures. The motif 3’-AUUCUUUUUG-5’, conserved across rhabdoviruses, was identified in all but one intergenic regions; whereas the motif 3’-ACAAAAACACA-5’ was found in three intergenic junctions. This is the first complete genome of a cytorhabdovirus infecting papaya. The virus was prevalent in commercial plantings of Los Ríos, the most important papaya producing province of Ecuador. During the final stage of this manuscript preparation, the genome of a bean-associated cytorhabdovirus became available. Nucleotide identity (97%) between both genomes indicated that the two viruses are strains of the same species, for which we propose the name papaya cytorhabdovirus E.

Feeding behaviors and biomechanics of female Varroa destructor mites are revealed from AC-DC electropenetrography (EPG) recordings of mites feeding from Apis mellifera honey bee pupae and histology of mite internal ingestion apparatus. EPG signals characteristic of arthropod suction feeding (ingestion) were identified for mites that fed on pupae during overnight recordings. Ingestion by these mites was confirmed afterwards by observing internally fluorescent microbeads previously injected into their hosts. Micrographs of internal ingestion apparatus illustrate the connection between a gnathosomal tube and a pharyngeal lumen, which is surrounded by alternating dilator and constrictor muscles. Inspection of EPG signals showed the muscularized mite pharyngeal pump operates at a mean repetition rate of 4.5 cycles/s to ingest host fluids. Separate feeding events observed for mites numbered between 23 and 33 over approximately 16 h of recording, with each event lasting ~10 s. Feeding events were each separated by ~2 min. Consecutive feeding events separated by either locomotion or prolonged periods of quiescence were grouped into feeding bouts, which ranged in number from one to six. Statistical analyses of EPG data revealed that feeding events were prolonged for mites having lower pharyngeal pump frequencies, and mites having prolonged feeding events went unfed for significantly more time between feeding events. These results suggest that mites may adjust behaviors to meet limitations of their feeding apparatus to acquire similar amounts of food. Data reported here help to provide a more robust view of Varroa mite feeding than those previously reported and are both reminiscent of, as well as distinct from, some other acarines and fluid-feeding insects.

An analysis of transcriptomes from the antennae of the three South American stink bugs (Euschistus heros, Chinavia ubica, and Dichelops melacanthus) revealed the presence of picorna-like virus genome-length RNAs with high sequence identity to the genome of Halyomorpha halys virus (HhV), originally discovered in the transcriptome of the brown marmorated stink bug, Halyomorpha halys. Features of the genome, phylogenetic relationships to other viruses, and the appearances of virus-like particles isolated from host stink bugs all confirm that these viruses are iflaviruses and isolates of an undescribed species. Iflavirus RNAs were present at high levels (40%-90% of transcriptome reads) in the stink bug antennal transcriptomes. In whole-insect transcriptomes of H. halys, HhV reads were >500-fold more abundant in adults than in nymphs. We identified from field population a subject of species E. heros infected by this iflavirus. The results of the analysis suggest that these iflaviruses are able to produce large quantities of their RNAs without causing any obvious pathology to their hosts.

Spodoptera exempta nucleopolyhedrovirus (SpexNPV) is a viral pathogen of the African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), a significant agricultural pest of cereal crops in Africa. SpexNPV has been evaluated as a potential insecticide for control of this pest and has served as the subject of research on baculovirus pathology and transmission. Occlusion bodies (OBs) of SpexNPV isolate 244.1 were examined, and the nucleotide sequence of the genome was determined and characterized. SpexNPV-244.1 OBs consisted of irregular polyhedra with a size and appearance typical for alphabaculoviruses. Virions within the polyhedra contained 1-8 nucleocapsids per unit envelope. The SpexNPV-244.1 genome was comprised of a 129,528 bp circular sequence, in which 139 ORFs were annotated. Five homologous regions (hrs) consisting of a variable number of 28-bp imperfect palindromes were identified in the genome. The genome sequence contained the 38 core genes of family Baculoviridae, as well as three ORFs unique to the SpexNPV sequence and one ORF that was apparently acquired by horizontal gene transfer with a betabaculovirus ancestor. Phylogenetic inference with core gene amino acid sequence alignments placed SpexNPV-244.1 in a lineage containing alphabaculoviruses of Spodoptera frugiperda and Spodopotera exigua which in turn is part of a larger group of alphabaculoviruses from the subfamily Noctuinae in the lepidopteran family Noctuidae. Kimura-2-parameter pairwise nucleotide distances indicated that SpexNPV-244.1 represented a different and previously unlisted species in the genus Alphabaculovirus. Gene parity plots indicated that the gene order of SpexNPV-244.l was extensively collinear with that of Spodoptera exigua NPV (SeMNPV). These plots also revealed a group of 17 core genes whose order was conserved in other alpha- and betabaculoviruses.

A mild isolate of Papaya ringspot virus type-P, abbreviated as PRSV-mild, from Ecuador was sequenced and characterized. The most distinguishing symptom induced by PRSV-mild was gray powder-like leaf spots radiating from secondary veins. In greenhouse experiments, PRSV-mild did not confer durable protection against a severe isolate of the virus (PRSV-sev), obtained from the same field. Furthermore, isolate specific detection in cross-protected plants showed that PRSV-sev becomes dominant in infections, rendering PRSV-mild undetectable at 90 - 120 days post superinfection. Virus testing using isolate-specific primers detected PRSV-mild in two out of five surveyed provinces, with 10% and 48% of incidence in Santo Domingo and Los Ríos, respectively. Comparative genomics showed that PRSV-mild lacks two amino acids from the coat protein region, whereas amino acid determinants for asymptomatic phenotypes were not identified. Recombination events were not predicted in the genomes of the Ecuadorean isolates. Phylogenetic analyses placed both PRSV-mild and PRSV-sev in a clade that includes an additional PRSV isolate from Ecuador and others from South America.

Biofilm formation is an important strategy for foodborne bacterial pathogens to survive in stressful environments such as fresh produce processing facilities. Bacterial cell aggregation strongly promotes the initiation of microcolonies and the formation of biofilms on abiological surfaces. We previously showed that Ralstonia insidiosa, an environmental bacterial species frequently isolated from fresh produce facilities, may serve as a “bridge bacterium” that strongly enhanced the incorporation of several foodborne bacterial pathogens into dual species biofilms. While the R. insidiosa strain exhibited moderate cell aggregation in liquid culture, co-culturing Listeria monocytogenes with R. insidiosa resulted in significant augmentation of cell aggregation. Electron microscopy indicated that L. monocytogenes cells were initially attracted to the R. insidiosa aggregates and formed large dual species aggregates that were predominately composed of L. monocytogenes cells. The predominant presence of L. monocytogenes in the dual species aggregates was also confirmed by differential plating. These findings suggest that bridge bacteria such as R. insidiosa play critical roles in the survival of foodborne bacterial pathogens, such as L. monocytogenes and Escherichia coli, by promoting multispecies biofilm formation. The implications of such bridge bacteria on food safety need to be further evaluated.

Controlled ripening of climacteric fruits, such as bananas and avocados, is a critical step to provide consumers with high-quality products while reducing postharvest losses. Prior to ripening, these fruits can be stored for an extended period of time but are usually not suitable for consumption. However, once ripening is initiated, they undergo irreversible changes that lead to rapid quality loss and decay if not consumed within a short window of time. Therefore, technologies to slow the ripening process after its onset or to stimulate ripening immediately before consumption are in high demand. In this study, we developed a solid porous metal-organic framework (MOF) to encapsulate gaseous ethylene for subsequent release. We evaluated the feasibility of this technology for on-demand stimulated ripening of bananas and avocados. Copper terephthalate (CuTPA) MOF was synthesized via a solvothermal method and loaded with ethylene gas. Its crystalline structure and chemical composition were characterized by X-ray diffraction crystallography, porosity by N2 and ethylene isotherms, and morphology by electron microscopy. The MOF loaded with ethylene (MOF-ethylene) was placed inside sealed containers with preclimacteric bananas and avocados and stored at 16 °C. The headspace gas composition and fruit color and texture were monitored periodically. Results showed that this CuTPA MOF is highly porous, with a total pore volume of 0.39 cm(3)/g. A 50 mg portion of MOF-ethylene can absorb and release up to 654 μL/L of ethylene in a 4 L container. MOF-ethylene significantly accelerated the ripening-related color and firmness changes of treated bananas and avocados. This result suggests that MOF-ethylene technology could be used for postharvest application to stimulate ripening just before the point of consumption.

Paraffin-embedded blocks of brain of a nine months old bull calf that died of neurological signs in 1982 in Germany were restudied. Numerous schizonts and merozoites were found associated with extensive but focal necrosis and severe meningoencephalitis. Developing stages of schizonts as well as free merozoites were identified. The schizonts were primarily in perivascular areas. Ultrastructurally, schizonts were seen both in capillaries and in extravascular space. Merozoites were often concentrated in adventitial layers of capillaries. Schizonts divided by endopolygeny, the nucleus became multi-lobed, and at the terminal stage nuclear lobes were incorporated into budding merozoites. Individual merozoites were seen in neurons, astrocytes, oligodendrocytes, leukocytes, and vascular endothelial cells. Occasionally merozoites were present in the nucleus of mononuclear cells. Individual merozoites were ovoid, 3-5×2-3μm in size, and contained a prominent nucleus, numerous micronemes, a conoid, but no rhoptries. Schizonts and merozoites did not react to polyclonal rabbit Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona antibodies but did react to Sarcocystis cruzi antibodies. Because of morphological characteristics and the type of lesions, the parasite was likely due to an unidentified Sarcocystis species, different from S. cruzi.

Root-lesion nematodes (Pratylenchus spp.) of the genus Pratylenchus Filipjev, 1936, are among the most important nematode pests on soybean (Glycine max (L.) Merr.), along with soybean cyst and root-knot nematodes. In May 2015 and 2016, a total of six soil samples were collected from a soybean field in Walcott, Richland County, ND and submitted to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), USDA, ARS, MD for analysis. Later, in 2019, additional nematodes recovered from a greenhouse culture on soybean originally from the same field were submitted for further analysis. Males, females, and juveniles of Pratylenchus sp. were recovered from soil and root samples and were examined morphologically and molecularly. DNA from single nematodes were extracted, and the nucleotides feature of three genomic regions targeting on the D2&ndash, D3 region of 28S rDNA and ITS rDNA and mitochondrial cytochrome oxidase subunit I (COX1) gene were characterized. Phylogeny trees were constructed to ascertain the relationships with other Pratylenchus spp., and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to provide a rapid and reliable differentiation from other common Pratylenchus spp. Molecular features indicated that it is a new, unnamed Pratylenchus sp. that is different from morphologically closely related Pratylenchus spp., including P. convallariae, P. pratensis, P. fallax, and P. flakkensis. In conclusion, both morphological and molecular observations indicate that the North Dakota isolate on soybean represents a new root-lesion nematode species which is named and described herein as Pratylenchus dakotaensis n. sp.

Chemical sanitizers are commonly used for fresh produce washing to reduce potential presence of foodborne pathogens and spoilage bacteria, and to prevent cross-contamination. In this study, we evaluated the susceptibility of foodborne pathogens, including Shiga-toxigenic Escherichia coli, Salmonella enterica, and Listeria monocytogenes, to free chlorine (FC) and peracetic acid (PAA) in spinach and romaine lettuce rinsates as simulated leafy green wash water. All inoculated pathogens exhibited notably enhanced tolerance (over 2 log of increased recovery) to FC, but not to PAA, when treated in fresh produce rinsates in comparison to that in sterile water. Both FC and PAA effectively inactivated the inoculated foodborne pathogens, rendering them undetectable after 30 s exposure to 10 mg/L FC or 30 mg/L PAA, by selective plating or non-selective enrichment. However, potentially viable cells for all the inoculated foodborne pathogens were detected by PMA-qPCR, especially in wash water samples treated with PAA at concentrations up to 50 mg/L. While laser confocal microscopy after differential staining suggested significant presence of viable but non-culturable (VBNC) populations in PAA treated wash water samples, resuscitation in vitro of the potential VBNC cells was not achieved. This study provides scientific data to better understand the risks of potential VBNC foodborne pathogens during fresh produce washing.