Effects of Field Fumigation and Inoculation With the Pecan Truffle (Tuber lyonii) on the Fungal Community of Pecan (Carya illinoinensis) Seedlings Over 5 Years

Truffle fungi are esteemed for their aromatic qualities and are among the most widely cultivated edible ectomycorrhizal fungi. Here we document a successful method for establishing Tuber lyonii, the pecan truffle, on pecan (Carya illinoinensis) seedlings in a field setting. We assessed the impacts of soil fumigation and varying concentrations of truffle spore inoculum on the ectomycorrhizal fungal and the complete fungal communities as well as the colonization of T. lyonii on pecan roots at three nurseries in Georgia, United States. To identify fungal communities on pecan seedlings, we performed high-throughput amplicon sequencing of the fungal ITS1 rDNA region. Our 5-year long field experiment demonstrates that fumigation and inoculation together resulted in the highest persistence of T. lyonii on pecan roots. While fungal OTU numbers fluctuated over the years of our experiments, there was no statistical support to demonstrate diversification of communities when Shannon diversity metrics were used. However, we did find that older seedlings were less likely to be dominated by T. lyonii compared to younger ones, suggesting successional changes in the fungal community over time. This suggests that transplanting inoculated seedlings after 2 or 3 years post-inoculation is optimal for future truffle propagation efforts. Our results demonstrate that T. lyonii can be established in situ with methods that are compatible with current pecan nursery industry practices and that fungal communities on pecan seedlings vary depending on the experimental treatments used during planting. While the pecan truffle is not yet widely cultivated, our results provide insights for future large-scale cultivation of this and perhaps other Tuber species.

Phylogenetic prospecting for cryptic species of the genus Merluccius (Actinopterygii: Merlucciidae)

Hakes of the genus Merluccius include 11 valid species as well a number of rare morphotypes suspected to be “cryptic species”. Concatenated nucDNA ITS1-rDNA and mtDNA cyt b sequences plus nested ITS1Nes sequences allowed to ascribe 14 specimens of nine rare morphotypes from the South Pacific and the South Atlantic to the phylogenetic backbone of this genus. Bayesian analyses pointed to M. bilinearis and M. albidus as the oldest species of the genus and the New World cluster, respectively. The phylogenetic status of M. angustimanus from the upper Gulf of California suggests its hybrid origin between M. gayi and M. productus from about 0.25 MYA, although an ever since confinement of a subset of those species cannot be ruled out. The molecular phylodiagnostic test suggests a common origin of all rare morphotypes and the absence of cryptic hake species in the Southern Cone. The molecular background of the morphotypes distributed between the Western Pacific South of New Zealand and the western Atlantic South of Argentina is compatible with their hybrid origin between M. gayi and both, M. australis or M. hubbsi, respectively.

Updates on Geographical Dispersion of Leishmania Parasites Causing Cutaneous Affections in Algeria

Leishmaniases are neglected tropical diseases of public health concern in Algeria. To update the geographical distribution of Leishmania spp. causing cutaneous affection, we examined a set of Giemsa-stained smears prepared from skin lesions of the patients suspected to have cutaneous leishmaniasis (CL) in various geographical areas in Algeria. The identification of Leishmania parasites was performed using microscopy, conventional PCR, and PCR–RFLP (PCR-Restriction Fragment Length Polymorphism) targeting ITS1-rDNA. Among 32 smears provided from 27 suspected patients with cutaneous lesions, no trace of parasites was observed in the smear of three patients using microscopy and molecular approaches. Furthermore, four patients presented at least two lesions. PCR–RFLP confirmed the presence of Leishmania in 29 smears prepared from 24 patients. Two biopsies, negative after microscopic examination, were found positive by PCR. Of these 29 PCR positive smears (24 patients), 20 were identified using RFLP–PCR as L. major, two as L. tropica, and two as L. infantum. We found L. major infected patients from Ain skhouna, Biskra, El M’hir, Ghardaïa, M’Sila, and Saida, in agreement with previously reported cases. Furthermore, we highlighted for the first time, the identification of L. major in the patients from Bourkika, Bou Kremissa, Bou Saada Clef, Hajout, Maghnia, Médéa, Menaceur, Messad, Mostaghanem, Nador, Oran, and Sidi Okba. A phylogenetic reconstruction performed with sequences collected from the PCR products confirmed these identifications. Our data provide additional information on the geographical extension of CL caused by L. tropica and L. infantum in Algeria.

AcanR3990 qPCR: a novel, highly sensitive, bioinformatically-informed assay to detect Angiostrongylus cantonensis infections

Background Angiostrongylus cantonensis (Ac), or the rat lungworm, is a major cause of eosinophilic meningitis. Humans are infected by ingesting the 3 rd stage larvae from primary hosts, snails and slugs, or paratenic hosts. The currently used molecular test is a qPCR assay targeting the ITS1 rDNA region (ITS1) of Ac. Methods In silico design of a more sensitive qPCR assay was performed based on tandem repeats predicted to be the most abundant by the RepeatExplorer algorithm. Genomic DNA (gDNA) of Ac were used to determine the analytical sensitivity and specificity of the best primer/probe combination. This assay was then applied to clinical and environmental samples. Results The limit of detection of the best performing assay, AcanR3990, was 1 fg (the DNA equivalent of 1/100,000 dilution of a single 3 rd stage larvae). Out of 127 CDC archived CSF samples from varied geographic locations, the AcanR3990 qPCR detected the presence of Ac in 49/49 ITS1 confirmed angiostrongyliasis patients along with 15/73 samples previously negative by ITS1 qPCR despite strong clinical suspicion for angiostrongyliasis. Intermediate hosts (gastropods) and an accidental host, a symptomatic horse, were also tested with similar improvement in detection observed. AcanR3990 qPCR did not cross-react in five CSF from patients with proven neurocysticercosis, toxocariasis, gnathostomiasis and baylisascariasis. AcanR3990 qPCR failed to amplify genomic DNA from the other related Angiostrongylus species tested except for A. mackerrasae (Am), a neurotropic species limited to Australia that would be expected to present with a clinical syndrome indistinguishable from Ac. Conclusion These results suggest AcanR3990 qPCR assay is highly sensitive and specific with potential wide applicability as a One Health detection method for Ac and Am.

Genetic data of the ITS1-5.8S rDNA sequences of small liver flukes (Opisthorchiidae) from cats in Northern Vietnam

Domestic cats and humans are favorite final hosts for some hepatic trematodes of Opisthorchiidae. The present study examined the sequence variation in the nuclear ribosomal DNA (rDNA) region, including the internal transcribed spacer (ITS1) and the 5.8S gene, of two human opisthorchiid species Clonorchis sinensis (Cobbold, 1875) Looss, 1907 and Opisthorchis viverrini (Poirier, 1886) Stiles & Hassal, 1896, in cats sampled in Northern Vietnam. The length of 30 partial ITS1‒5.8S rDNA sequences of C. sinensis from different hosts and localities varied from 761 to 766 bp. The results showed completely identity for all investigated 5.8S rDNA sequences of C. sinensis. In contrast, the variations were revealed in the complete ITS1 sequences of C. sinensis. Three polymorphic sites, indel and new sites with intragenomic polymorphism were detected. The length of the complete ITS1 rDNA region ranged from 656 to 661 bp. There are no substitutions downstream of the 5 bp insertion, and the different ratio of nucleotide in polymorphic sites with presence/absence of insertion leads to the existence of 6 genotypes in the investigated samples. The major genotype was obtained in 70% of sequences, other genotypes were presented by 1, 2 or 3 sequences. Nucleotide diversity in whole sample of C. sinensis was 0.00122 ± 0.00073. Partial sequences of ITS1‒5.8S rDNA of O. viverrini were 745 bp in length, and no difference was observed. Phylogenetic analyses by neighbor joining phylogram inferred from ITS1‒5.8S rDNA of 3 sequences of O. viverrini and 30 sequences of C. sinensis were formed two separately monophyletic clades.

Growth, fatty, and amino acid profiles of the soil alga Vischeria sp. E71.10 (Eustigmatophyceae) under different cultivation conditions

In this study, a unicellular soil alga isolated from farmland in Germany was surveyed. The investigation of the hypervariable molecular markers ITS1 rDNA and ITS2 rDNA identified strain E71.10 as conspecific with Vischeria sp. SAG 51.91 (Eustigmatophyceae). The culture was tested for biomass generation and for the yield of fatty acids and amino acids. The survey included four different culture conditions (conventional, elevated CO2, nitrogen depletion, or sodium chloride stress) at room temperature. The best yield of dry biomass was achieved applying 1% CO2, whereas nitrogen-free medium resulted into least growth. The fatty acid content peaked in nitrogen-free medium at 59% per dry mass. Eicosapentaenoic acid was the most abundant fatty acid in all treatments (except for nitrogen free), accounting for 10.44 to 16.72 g/100 g dry mass. The highest content of amino acids (20%) was achieved under conventional conditions. The results show that abiotic factors strongly influence to which extent metabolites are intracellularly stored and they confirm also for this yet undescribed strain of Vischeria that Eustigmatophyceae are promising candidates for biotechnology.

Super Infection of Cutaneous Leishmaniasis Caused by Leishmania major and L. tropica to Crithidia fasciculata in Shiraz, Iran

Background: The aims of the current study were to determined present status of CL in Shiraz City, identify the causative species of Leishmania and conduct phylogenetic evaluations in detected parasites. Methods: This study was conducted on 70 individuals with suspected CL that referred to the major health centers of Shiraz (Valfajr), Fars province, Iran, from Sep 2016 to Jul 2017. DNA was extracted from cultured Leishmania promastigotes and PCR-RFLP were performed using ITS1-rDNA gene. Results: Overall, 39 male (55.70%) and 31 (44.30%) female were found to be positive microscopically. All of direct examined positive samples were confirmed to be positive for Leishmania spp. DNA. Based upon the PCRRFLP patterns and phylogenetic analysis, 46 (65.72%), 17 (24.28%) and 7 (10%) isolates were clearly identified as L. major, L. tropica and C. fasciculata, respectively. Conclusion: The dominat detected species in Shiraz City was L. major and L. tropica, respectively. CL has high prevalence in Shiraz City; therefore, more studies on leishmaniasis in the natural vectors and also reservoirs infection in this region is exceedingly recommended. Skin leisons due to C. fasciculata, was described for the first time in Iran (Shiraz City).

New morphological observations on Neolobocriconema serratum (Khan & Siddiqi, 1963) Mehta & Raski, 1971 (Rhabditida: Criconematidae)

Summary A population of Neolobocriconema serratum was recovered from the rhizosphere of Hosta sp., northern Iran, for the first time. It was studied using morphological and molecular data and new morphological observations were made. The Iranian population is characterised by females having 37-43 body annuli, their outer margin ornamented with 12-14 small lobe-like outgrowths under light microscopy, anastomoses occur sparsely in some females and the succeeding annulus is usually anteriorly bent opposite to the vulva. The cephalic region has a single smooth wavy annulus with grooves and is separated from the first, wider body annulus by a very short neck. Four prominent discoid submedian lobes and two lateral pseudolips present, appearing two layered (i.e., one above the other) in scanning electron microscopy (SEM). Body 487-607 μm long, stylet 85-93 μm long with rounded to anchor-shaped knobs, ovary very long, vulva generally closed, but open in some specimens under SEM. Tail conoid with bluntly rounded end and males absent. The recovered population agreed well with the type population based upon the morphometric data and female morphology. In having an almost identical morphology and morphometrics, N. allantoideum is proposed as a junior synonym of N. serratum. The molecular phylogenetic analyses were done by using near-full-length sequences of the small subunit ribosomal DNA (SSU rDNA), the internal transcribed spacer 1 (ITS1 rDNA) and the mitochondrial cytochrome c oxidase subunit I (COI mtDNA). The Iranian population of this species formed a clade with three other populations of the species in the SSU rDNA, and with one population in both the ITS1 rDNA and the COI mtDNA trees using Bayesian inference.