DNA Internal Transcribed Spacer Region Research Articles

Bionomic characterization of Anopheles mosquitoes in the Ethiopian highlands and lowlands

BackgroundThe protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands—resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands.MethodsHourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1).ResultsIn the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) (n = 1970, 41.9%), followed by An. demeilloni (n = 1133, 24.1%) and An. cinereus (n = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. (n = 1190, 36.9%), An. pretoriensis (n = 899, 27.9%), and An. demeilloni (n = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations.ConclusionsThe high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations.Graphical

Pholiotina sphagnicola sp. nov. (Bolbitiaceae, Agaricales) from Mongolia

A new species Pholiotina sphagnicola sp. nov., was discovered in Mongolia. Its identification was based on phylogenetic analyses of combined nuclear ribosomal DNA internal transcribed spacer region (ITS), nuclear ribosomal DNA 28S subunit (nrLSU), and translation elongation factor 1-alpha (tef1-α), as well as the morphological analyses. The species is characterized by a pileus ranging from hemispherical to convex, with indistinct striae on the margin. The ring is located above the stipe, and the upper sides crenulate-striate. Basidia, which resemble rabbit ears, are 2(1)-spored and have sterigmata ranging from 3 to 13 μm in length. Cheilocystidia are lageniform with a long neck and lanceolate shape; some may exhibit slight swelling at the apex.

Multiple Anopheles species complicate downstream analysis and decision-making in a malaria pre-elimination area in southern Mozambique

BackgroundDifferent anopheline species (even within a species group/complex) can differ in their feeding and resting behaviours, which impact both malaria transmission patterns as well as the efficacy of vector control interventions. While morphological identification of sampled specimens is an important first step towards understanding species diversity and abundance, misidentification can result in the implementation of less effective vector control measures, and consequently smaller reductions in the number of local malaria cases. Focusing on southern Mozambique, a malaria pre-elimination area where malaria remains persistent, the aims of this preliminary study were to use molecular identification (CO1 and ITS2 barcoding) to (1) validate the results from the morphological identification (with a particular focus on Anopheles pharoensis and Anopheles squamosus), and (2) have a closer look at the Anopheles coustani group (which includes Anopheles tenebrosus and Anopheles ziemanni).MethodsFemale anopheline mosquitoes (n = 81) were identified morphologically and subsequently sequenced at the ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or cytochrome oxidase subunit 1 (CO1) loci towards species determination.ResultsOut of the 62 specimens that were identified morphologically to species, 4 (6.5%) were misidentified. Regarding the An. coustani group, morphological identification showed that several members are present in southern Mozambique, including An. coustani sensu lato (s.l.), An. ziemanni and An. tenebrosus. However, based on both ITS2 and CO1 sequences, the exact species remains unknown for the latter two members until voucher sequences are available for comparison.ConclusionThe reason(s) for morphological misidentification of anopheline mosquitoes need to be mitigated. This is usually related to both the capacity (i.e. training) of the microscopist to identify anopheline species, and the information provided in the dichotomous identification key. As the An. coustani complex contributes to (residual) malaria transmission in sub-Saharan Africa, it may play a role in the observed persistent malaria in southern Mozambique. A better baseline characterizing of the local anophelines species diversity and behaviours will allow us to improve entomological surveillance strategies, better understand the impact of vector control on each local vector species, and identify new approaches to target those vector species.

First report of Perkinsus olseni infections in blood cockles Tegillarca granosa on the south coast of Korea

The protozoan parasite Perkinsus olseni has become a focus of attention since it has been responsible for mass mortalities and economic losses in a wide range of bivalve hosts globally. The P. olseni host range along the south coast of Korea may extend beyond what was previously understood, and blood cockles in the Family Arcidae are also suggested to be potential hosts of P. olseni. In the present study, we applied histology and molecular techniques to identify Perkinsus sp. infections in the blood cockles Tegillarca granosa, which have been commercially exploited on the south coast of Korea for several decades. Histology and molecular techniques, including genus-specific immunofluorescence assay, species-specific fluorescence in situ hybridization, and phylogeny based on the ribosomal DNA internal transcribed spacer region revealed that T. granosa is infected by P. olseni, although the prevalence was low (0.5%). Histology revealed massive hemocyte infiltrations in the mantle, gill, and digestive gland connective tissues, indicating that the infection exerts negative impacts on the host cockles.

Molecular prevalence of Enterocytozoon bieneusi in stray cats of İzmir, Türkiye

The phylum Microsporidia contains obligate single celled parasites that can infect many vertebrate hosts including humans. Enterocytozoon bieneusi is considered as the most diagnosed species in humans. E. bieneusi has also been detected in many animals such as cats, dogs and cattle. Among these animals, cats are carriers of type D and IV which are the most common human pathogenic genotypes of E. bieneusi. In Türkiye, the prevalence of E. bieneusi in stray cats is not well known. Therefore, in this study, the molecular prevalence of E. bieneusi in stray cats (n = 339) was determined by Real-Time PCR targeting ribosomal DNA ITS (internal transcribed spacer) region of E. bieneusi. Initially, the analytical sensitivity of Real-Time PCR was determined by a plasmid control and then E. bieneusi DNA was investigated in fecal samples of stray cats. The results showed that the analytical sensitivity of Real-Time PCR targeting ITS region of E. bieneusi was ≤1 copy plasmid/reaction. Analysis of fecal samples revealed that the molecular prevalence of E. bieneusi was 50.15% (170/339). Overall, these results showed that the Real-Time PCR successfully detected E. bieneusi in cat’s fecal samples and stray cats can be an important source for transmission of E. bieneusi to humans and other animals.

Chemical and Genetic Variability of Istrian Foeniculum vulgare Wild Populations.

Wild Foeniculum vulgare populations from the region of Istria have been subjected to a genetic and chemical study. Headspace-gas chromatography analysis of volatile secondary metabolites and PCR-RFLP analysis of the ribosomal DNA internal transcribed spacer region has been chosen to analyze the chemical and genetic traits of single plants, respectively. Large intrapopulation and interpopulation differences have been observed in both chemical profiles and restriction patterns of PCR products. The data from chemical and genetic analyses were pooled and used to assess allele frequencies of three putative genetic loci on individual populations. The pooled allele frequencies were used to determine interpopulation distances for phenogram reconstruction. The combined use of chemical and genetic datasets for genetic variation analysis proved to be a more comprehensive approach for such a study, compared to the use of single datasets, even using such relatively simple analytical tools.

Species diversity and phylogenetic relationships within the tribe Mentheae (Lamiaceae) in Uzbekistan using ITS sequence data

Regular monitoring and comprehensive study of natural populations rare endangered species, both by classical taxonomy and molecular genetic analysis methods are of considerable importance all over the world. This work has focused at the research of species diversity flora of Uzbekistan using combinations of DNA markers. Currently, there are no universal DNA site for plant species identification. Pairwise sequence alignments and multiple sequence alignments are made using ClustalW program. DNA sequence data were used to verify the taxonomic identity and phylogenetic aspects of the studied representatives the tribe Mentheae (Lamiaceae) collected in Uzbekistan territory. The phylogenetic analyzes of this tribe was reconstructed for 44 species, representing 19 genera using nuclear ribosomal DNA internal transcribed spacer region (nrITS), containing ITS1 and ITS4 adopted as an important basis, since it can provide a high resolution of relationships. 15 consensus ITS gene sequences were obtained for studied species in this work and 29 published molecular sequence data from GenBank were used. Dendrograms were constructed using hierarchical clustering with the software Mega X. The parsimony analyses were resulted in an accurate consensus tree. Three major clades was identified within the tribe Mentheae. So, this paper demonstrates the successfully usefulness of DNA technology as a tool for genetic inventory at the species diversity assessment of rare endangered plants.

Morphological and pathogenic characterization of Corynespora cassiicola isolates reveals specific genotypic interactions in soybean

AbstractSoybean target spot, caused by the fungus Corynespora cassiicola, is an important disease in northwestern Argentina (NWA). A cultural, morphological, and virulence characterization of 24 C. cassiicola isolates from different geographic localities in NWA was performed. The isolates were classified into five different cultural phenotypes, one of which has not been previously described. The ribosomal DNA internal transcribed spacer region sequence from five representative isolates (587 bp) exhibited 100% identity with C. cassiicola. A high variability of conidial morphology was observed among isolates, but no correlation with cultural phenotype was observed. When the soybean cultivar A 8000 RG was challenged with the 24 isolates, different degrees of virulence were observed, ranging from highly virulent to nonvirulent. No association of virulence with cultural or morphological characters was observed, but a relationship with geographical origin was demonstrated. Histopathological studies were performed on a nonvirulent and a highly virulent isolate. Microscopic observations of infected tissues of the former showed low mycelium development restricted to the upper epidermis, a thickening of the cuticle and primary wall of subepidermal cells, and accumulation of callose in phloematic vessels. In tissue infected with the latter, there was abundant mycelium development accompanied by cellular disorganization in mesophyll cells. Pathological challenges of isolates on nine different cultivars indicated that the degree of virulence of isolates depends on the plant genotype, demonstrating that the C. cassiicola–soybean interaction is highly specific. Understanding the genetic basis of this interaction will provide new information for better disease management and breeding strategies.

Dissimilarity in the Nuclear Ribosomal DNA Internal Transcribed Spacer Regions of Haplophyllum spp. Founded in Ashdagh Mountain, Sangaw, Kurdistan of Iraq

Studying and understanding the changes in the molecular sequences of plants are important to better identification and classifying the species. Internal transcribed spacer (ITS) regions are consider an informative genetic sequence to find out the variation among species of the same genus. In this study, ITS regions of Haplophyllum collected in Ashdagh Mountain were amplified, analyzed, and compared with reference species gathered from gene bank. The results showed that the collected species was closely related to Haplophyllum blanchei and Haplophyllum tuberculatum. In addition, there were differences in the number of the base pairs in the ITS1 region between Haplophyllum sp. and H. blanchei and H. tuberculatum. The transition to transversion ratio between Haplophyllum sp. and H. tuberculatum was lower (=1) than with other species. The results reveals that the studied plant is could be a new species in Iraq. To the best of our knowledge, this study is the first molecular taxonomic study done to identify Haplophyllum species in Ashdagh mountain in Sangaw/Suleimani.

Molecular Phylogenetic Study of Strains Morphologically Identified as <i>Exophiala dermatitidis</i> from Clinical and Environmental Specimens in Japan

In this study, we aimed to clarify the phylogenetic distribution of Exophiala dermatitidis in Japan and describe the characteristics of genotypes. We examined 67 clinical and environmental isolates that were morphologically identified and preserved as E. dermatitidis and we confirmed the identification on the basis of the ribosomal DNA internal transcribed spacer region. Genotype sequences were aligned and compared using maximum likelihood phylogenetic tree analyses of the ITS1 region. Additionally, the strains of each genotype were tested for mycological characteristics, such as growth temperature, growth rate, and drug sensitivity. The 67 strains examined were isolated from Japan, the United States, Brazil, Venezuela, and China. In accordance with the establishment of a phylogenetic tree for the ITS1 region, 45 of the 49 Japanese strains were classified as genotype A, two as genotype B, and two as genotype D (A2 according to the method of Matos et al. (2003)). Chinese strains were divided into genotypes A and D (A2), and South American strains were classified into genotypes A, B, B2, and C2, while all strains from the U.S. belonged to genotype A. New genotype groups B2 and C2 were identified in Brazilian and Venezuelan strains, respectively. There were no specific differences among the genotypes or isolated regions in the antifungal susceptibility test for all E. dermatitidis isolates. However, genotypes B2 and D (A2) exhibited growth at higher temperatures than the other genotypes.

Occurrence of Volutella Blight Caused by Pseudonectria foliicola on Boxwood in Tennessee.

Boxwood (Buxus sp. L.) is a very popular evergreen shrub in the United States which is widely used as landscape plant and fresh greenery. Boxwood 'Green velvet' (B. sinica var. insularis x B. sempervirens) plants grown in field condition exhibiting Volutella blight symptoms were found in a commercial nursery in Warren Co., Tennessee in May 2019. Leaves appeared red, brown or tan color on affected plants. Waxy, salmon pink colored fruiting bodies (sporodochia) were observed underneath the affected leaves using a hand lens (Figure 1). Leaf drop was also observed on plants. Black lesions under the bark were observed in some of the plants. The disease severity (percentage leaf area diseased) was nearly 40% and the disease incidence was nearly 30% of 1,000 plants. Infected leaf and stem tissues collected from four symptomatic plants were surface sterilized with 70% ethanol and washed with sterile distilled water. Culturing the infected leaf and stem pieces, 5-mm in size, on potato dextrose agar (PDA) consistently yielded white fluffy aerial mycelium growth with scattered salmon-color slimy masses of conidia forming from sporodochia after 10 days incubation at 25°C in a 12-h fluorescent light and dark cycle. A total of two isolates (FBG2020_396 and FBG2020_405) were hyphal tip purified on PDA. The conidia (n = 50) were hyaline, aseptate, fusiform to ellipsoidal measuring average of 7.8 × 3.3 μm (range: 4.84 to 13.2 μm × 2.2 to 4.64 μm). To confirm the pathogen identity, total DNA was extracted using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA) directly from a 5-day old culture of isolates (FBG2020_396 and FBG2020_405) on PDA. The ribosomal DNA internal transcribed spacer region (ITS), β-tubulin (tub2) and part of 28S large ribosomal subunit (LSU) regions were amplified by PCR using the primer pairs ITS 5/ITS 4, T1/BTb2 and LR0R/LR5, respectively (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; Rehner and Samuels 1994; Vilgalys and Hester 1990; White et al. 1990). Newly generated sequences - GenBank/NCBI acc. nos. MW459251, MW465902 (ITS), MW464656, MW464657 (tub2) and MW459255, MW465903 (LSU) were 100% identical to Pseudonectria foliicola L. Lombard & Crous ex-type (CBS 123190) sequences KM231776, KM232035 and NG_058095, respectively. To complete Koch's postulates, six boxwood 'Green velvet' plants grown in 10 cm square pots (containing 40% coarse sand and 60% ground pine bark) were inoculated by spraying conidial suspension of P. foliicola [FBG2020_396 (1 × 105 conidia/mL)] obtained from 2-wk-old PDA cultures. Plants were covered with clear plastic humidity domes for 3 days and then they were maintained in a growth chamber at 25°C and 60% RH in a 12-h fluorescent light and dark cycle. Six control boxwood plants were maintained in the same environment without pathogen introduction. Pathogenicity test was conducted twice. After 10 days, typical symptoms of Volutella blight developed on the inoculated plants and microscopic examination revealed the same pathogen morphology as the original isolate. Pseudonectria foliicola was consistently re-isolated from leaves and stems. All control boxwood plants remained symptom-free and P. foliicola was not isolated from the leaves or stems. Pseudonectria foliicola causing Volutella blight has been reported on B. sempervirens in Czech Republic (Spetik et al. 2020), New Zealand (Lombard et al. 2015); Buxus sp. in Illinois, Maryland, Massachusetts, North Carolina and Washington (Salgado-Salazar et al. 2019). To our knowledge, this is the first report of Volutella blight of boxwood caused by P. foliicola in Tennessee. Pseudonectria foliicola is an opportunistic pathogen and infects weak, stressed, and injured boxwood plants/cuttings (Rivera et al. 2018). This pathogen could cause a serious economic loss to boxwood nursery growers, as it can significantly affect the ornamental value of boxwood plants and fresh greenery. Integration of sanitation practices with other disease management strategies such as biorational products and reduced-risk fungicides will be necessary for limiting the spread of pathogen and successful management of P. foliicola on boxwood in both field and postharvest conditions.

Genetic diversity of Amomum xanthioides and its related species from Southeast Asia and China.

Amomum Semen, the seed mass of Amomum xanthioides, has been imported from Southeast Asia and China and used for the treatment of gastric and intestinal disorders. A. xanthioides has been treated as a synonym of A. villosum var. xanthioides. Furthermore, A. villosum var. villosum, A. villosum var. xanthioides, or A. longiligulare have been described as the botanical origin of Amomi Fructus, which is a similar crude drug in Chinese Pharmacopoeia. Under these circumstances, the botanical origin of Amomum Semen was changed to A. villosum var. xanthioides, A. villosum var. villosum, or A. longiligulare in Supplement II to the 17th edition of the Japanese Pharmacopoeia. To develop an objective identification method for Amomum Semen and to confirm the phylogenetic relationship among Amomum taxa, the nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacer region and chloroplast DNA partial matK-trnK and trnH-psbA intergenic spacer regions were determined in specimens collected from Southeast Asia and China, including those from the type localities of each taxon. Six taxa were divided into four groups. A. xanthioides from Myanmar belonging to group 1 was discriminated from A. villosum var. xanthioides from China of group 2. A. villosum and its varieties were divided into two groups: group 2 included those from China, and group 3 consisted of A. villosum from Laos. A. longiligulare from China and Laos and A. uliginosum from Laos belonged to group 3 and group 4, respectively. These findings illustrate the phylogenetic basis for the need for taxonomical reorganization among the Amomum species.

Analysis of conidiogenesis and lifelong conidial production from single conidiophores of Podosphaera aphanis on strawberry leaves using digital microscopic and electrostatic techniques

In this study, strawberry plants (Fragaria × ananassa Duchesne ex Rozier) grown via elevated cultivation in a greenhouse were heavily infested with powdery mildew. We isolated the powdery mildew fungus from strawberry leaves and identified the isolate as Podosphaera aphanis (Wallroth) U. Braun & S. Takamatsu var. aphanis KSP-7 N, based on its morphological characteristics and sequencing of the ribosomal DNA internal transcribed spacer region. The host range of KSP-7 N was assessed by inoculating 17 plant families comprising 49 species (a total of 110 cultivars) with conidia. The fungus caused severe powdery mildew on the tested strawberry plants (commercial, wild, and false strawberries), produced scattered conidia on conidiophores, and formed completely catenated conidia within approximately 27 h from conidiophore erection to the first release of mature conidia. Six conidia were produced on each conidiophore; only those at the apex reached maturity. The cycles of conidial release were repeated 17 to 21 times at intervals of approximately 6 h, during gradual upward elongation of the conidiophores. At the final stage, conidia were released without growth or septation of the generative cells. Conidiophores produced an average of 38 conidia during a 96-h period. These results will help to clarify the modes of conidiogenesis, the lifetime of conidiophores, and the production of conidia on a conidiophore among powdery mildew pathogens isolated from strawberry leaves.

The endangered epiphytic orchid Dendrobium okinawense has a highly specific mycorrhizal association with a single Tulasnellaceae fungus

ABSTRACT Epiphytic orchids, which account for 68% of vascular epiphytes, contribute substantially to plant species diversity in forest canopies. Many orchid species are naturally rare and have limited geographical distributions. All orchids rely on mycorrhizal fungi for nutrient acquisition during seed germination and seedling establishment, and many species retain these associations throughout their lifecycle. Due to their reliance on mycorrhizal fungi, epiphytic orchid species with highly specific mycorrhizal associations may be geographically restricted and are often rare. Dendrobium okinawense is a rare epiphytic orchid endemic to the Yambaru Forest in Japan and to southern Taiwan. We hypothesized that this species exhibits high mycorrhizal specificity and tested this hypothesis via identification of the species’ mycorrhizal associates. We collected root samples of this orchid from four tree species and assessed mycorrhizal fungi in 25 root fragments harvested from 10 plants. Fungi were identified using molecular methods based on fungal isolates and DNA extracted from mycorrhizal roots. Detected fungi were assigned to 21 operational taxonomic units (OTUs) based on 97% sequence similarity of nuclear ribosomal DNA internal transcribed spacer region sequences. TU11, an OTU belonging to Tulasnellaceae (Basidiomycota), was the most frequently detected OTU, occurring in 20 root fragments from all 10 sampled individuals. Our results indicate that Dendrobium okinawense has a highly specific association with TU11. Further, this specificity may be high relative to the Dendrobium species assessed in other studies. These findings support the hypothesis that Dendrobium okinawense exhibits high mycorrhizal specificity.

Phenotypic plasticity and taxonomy of Cladophora gracilis (Griffiths) Kützing (Cladophorales, Chlorophyta) in the western Yellow Sea with the implication of its DNA barcode

Due to extensive morphological plasticity, the taxonomy of Cladophora species remains unclear. As one of the widely distributed species, C. gracilis was reported to hold many varieties, which make it difficult to identify the species. This study explored the morphology diversity of nine C. gracilis samples collected along the coast of western Yellow Sea. Some samples showed extremely varied characteristics, by which one cannot classify them correctly. Hence, 18S rDNA and nuclear ribosomal DNA internal transcribed spacer regions (ITS) sequences were employed to delimit species. For 18S rDNA, sequence similarity ranged from 99.6% to 100%. For ITS region, the similarity ranged from 98.7% to 100%. Molecular data strongly suggested that the morphologically heterogeneous samples were actually the same species. Characteristics comparison of the samples revealed that the taxonomy criteria including branching pattern and density, thallus color, height and texture varied widely, influenced by environmental conditions and age of alga. Besides, cell dimensions, as the relatively stable criterion, also exhibited intraspecific variance. Successful application of 18S rDNA and ITS sequences indicated that molecular method can be a powerful assistant as DNA barcodes to traditional morphology taxonomy.

Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia

BackgroundUnderstanding local Anopheles species compositions and bionomic traits are vital for an effective malaria vector intervention strategy. Though eight malaria vectors, including species complexes, have been documented across the island of Sulawesi, Indonesia, a comprehensive survey linking morphological and molecular species identification has not been conducted in this global hotspot of biodiversity.ResultsEighteen distinct species of Anopheles were molecularly identified in a 1 km2 area in Karama village, West Mamuju Province, Sulawesi. Known species included An. aconitus, An. karwari, An. peditaeniatus, An. vagus, An. barbirostris, An. tessellatus, An. nigerrimus, An. crawfordi, An. maculatus, An. flavirostris and An. kochi. Of the 18 distinct sequence groups identified through both ribosomal DNA internal transcribed spacer region 2, and mitochondrial DNA cytochrome c oxidase subunit 1 loci, 8 could not be identified to species through comparison to published sequences. The comparison of morphological and molecular identities determined that interpretations of local species compositions for primary and expected species in Karama (An. barbirostris and An. vagus) had the highest rate of accuracy (92.1% and 87.6%, respectively) when compared to molecular analysis. However, the remaining distinct sequences molecularly identified to species were identified correctly by morphological methods less frequently, from 0 to 83%.ConclusionsKarama, Indonesia has a high diversity of Anopheles spp. The unexpected high number of Anopheles species in a small area points to possible complex transmission dynamics and limitations with vector control based on possible varying behaviors and interactions with both humans and interventions. Morphological identification of Anopheles spp. in this study was more accurate for primary and expected species than secondary or unexpected species. Finally, the inability to identify seven sequence groups to species with consensus sequences implies that future studies employing sequencing are required to clarify species compositions in the Nigerrimus Subgroup, among others, as well as their distribution and vector status. Use of molecular methods in conjunction with morphological investigations for analysis of species composition, population dynamics and bionomic characteristics is directly implicated in understanding drivers of malaria transmission, intervention effectiveness, and the pursuit of malaria elimination.

New records of Indigofera cordifolia Heyne ex Roth. (Fabales: Fabaceae) in Saudi Arabia based on morphological and molecular evidence

Indigofera cordifolia Heyne ex Roth. so far is reported only in the Arabian Peninsula from Oman. A recent field expedition in the Farasan Archipelago, Saudi Arabia, Red Sea, resulted in documentation of this species in Dumsuk Island. Morphological examination and molecular identification based on the nuclear ribosomal DNA internal transcribed spacer region of this plant species was investigated in this study. The result show that the internal transcribed spacer had high species-level discrimination efficiency for the identification of Indigofera cordifolia that may provide help in authentic identification and management process of this rare and nationally endangered species.

A Survey of Trunk Disease Pathogens within Citrus Trees in Iran.

Citrus trees with cankers and dieback symptoms were observed in Bushehr (Bushehr province, Iran). Isolations were made from diseased cankers and branches. Recovered fungal isolates were identified using cultural and morphological characteristics, as well as comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, translation elongation factor 1α, β-tubulin, and actin gene regions. Dothiorella viticola, Lasiodiplodia theobromae, Neoscytalidium hyalinum, Phaeoacremonium (P.) parasiticum, P. italicum, P. iranianum, P. rubrigenum, P. minimum, P. croatiense, P. fraxinopensylvanicum, Phaeoacremonium sp., Cadophora luteo-olivacea, Biscogniauxia (B.) mediterranea, Colletotrichum gloeosporioides, C. boninense, Peyronellaea (Pa.) pinodella, Stilbocrea (S.) walteri, and several isolates of Phoma, Pestalotiopsis, and Fusarium species were obtained from diseased trees. The pathogenicity tests were conducted by artificial inoculation of excised shoots of healthy acid lime trees (Citrus aurantifolia) under controlled conditions. Lasiodiplodia theobromae was the most virulent and caused the longest lesions within 40 days of inoculation. According to literature reviews, this is the first report of L. theobromae and N. hyalinum on citrus in Iran. Additionally, we report several Phaeoacremonium species, S. walteri, Pa. pinodella and C. luteo-olivacea on citrus trees for the first time in the world.

Anopheles cinereus implicated as a vector of malaria transmission in the highlands of north-west Ethiopia

BackgroundTransmission of malaria in the highlands of Ethiopia is poorly understood and usually attributed to importation by mobile populations or local transmission by Anopheles arabiensis. To characterize and identify Anopheles species present in a highland area of northern Ethiopia, adult and larval collections were performed in Gondar town and the neighboring Senbet Debir village (Dembia district, > 2000 meters above sea level, masl), in addition to Bahir Dar town (capital of Amhara region) and Kumer Aftit village (Metema district, < 2000 masl).MethodsCDC-light traps were used to collect adult mosquitoes and larval collections were performed from rain pools for rearing into adults for species identification. Collections were made September-March 2016–2018. Adult mosquitoes were identified morphologically and a subset of randomly chosen specimens were identified to species by sequencing the ribosomal DNA internal transcribed spacer region 2 (ITS2) and mitochondrial DNA cytochrome c oxidase subunit 1 (cox1).ResultsThe primary species of Anopheles identified at elevations higher than 2000 masl was An. cinereus, which was confirmed molecularly by ITS2 and cox1 sequencing. Interestingly, two unknown species were also sequenced, in addition to two specimens of An. pretoriensis. The species collected at sites with elevations less than 2000 masl (Bahir Dar town and Kumer Aftit village) was An. arabiensis. Three Plasmodium falciparum-positive specimens were identified molecularly as An. cinereus.ConclusionsThe presence of Plasmodium-positive An. cinereus in areas greater than 2000 masl incriminates this species as a potential vector contributing to non-peak malaria transmission in Ethiopian highland areas.

Soybean Fungal Endophytes Alternaria and Diaporthe spp. are Differentially Impacted by Fungicide Application.

In field trials in Iowa, we investigated the association of a fungicide applied at early pod set to the diversity and composition of foliar endophytic fungi in presenescent soybeans. The main purpose of our study was to determine whether fungicides affect the microbiome of soybean plants during the pod-fill reproductive stage. In a replicated experiment focused on the impact of a fungicide application including a quinone outside inhibitor (QoI) and a pyrazole-carboxamide spanning two growing seasons, healthy stems and leaves near the tops of soybean were sampled for endophytic fungi. The survey yielded 1,791 isolates belonging to 17 putative species, identified by morphology and sequence analysis of the ribosomal DNA internal transcribed spacer region. Taxa were grouped by genus into operational taxonomic units: Alternaria, Colletotrichum, and Diaporthe were the dominant genera isolated. Plant parts were analyzed separately using a multivariate community analysis of isolate counts per plant. The 14.3% fluxapyroxad and 28.6% pyraclostrobin fungicide spray significantly increased the proportion of Diaporthe isolates over no-spray controls, whereas the inverse occurred for foliar Alternaria isolates. In addition, seed harvested from fields with shorter-season varieties and sprayed with fungicide showed higher percentages of Diaporthe isolates than fields with no fungicide spray. In conclusion, soybean farmers may want to consider that the application of a QoI fungicide in the absence of disease pressure might adversely impact seed quality.