ITS Loci Research Articles

Characterization of Stemphylium spp. associated with tomato foliar diseases in Algeria

Leaf blight and spot caused by Stemphylium spp. and Alternaria spp. are the most common destructive tomato diseases in north-western Algeria. During 2018 growing seasons, more than 30% of samples collected from plants grown in greenhouses or open fields were infected with Stemphylium. Initial symptoms were small, multiple, irregular to oval, yellow leaf spots, which enlarged to brown lesions later. In these lesions, Stemphylium mostly co-occurred with Alternaria spp. Twenty nine Stemphylium isolates were characterized based on morphological features, and multi-locus phylogenies using ITS, gpd, and cmdA genomic loci. Five Stemphylium species (S. lycopersici, S. gracilariae, S. eturmiunum, S. vesicarium, S. lycii) were associated with tomato leaf spot, of which S. lycii is a new report for tomato. Pathogenicity tests on healthy 2-months-old tomato seedlings reproduced symptoms similar to those observed in tomato crops. The tested fungus isolates differed in pathogenicity. Two isolates of S. lycopersici were more aggressive than those of the other species, causing major lesions on tomato plants. The five identified Stemphylium species are reported for the first time as new pathogens for tomato in Algeria, and S. lycopersici, S. gracilariae, S. eturmiunum, and S. lycii as new species of Algerian mycoflora.

Population genetic structure of the elephant tick Amblyomma tholloni from different elephant populations in Kenya

Understanding factors that shape tick population genetic structure is important as they may be exploited in crafting strategies for vector control. Amblyomma tholloni, or “elephant tick” is a three-host tick whose adults preferentially parasitize African elephants. The aim of this study was to determine the influence of fragmentation of the host populations on the genetic structure of this tick species from different ecosystems in Kenya, using the second internal transcribed spacer (ITS-2) and mitochondrial cytochrome oxidase 1 (CO1) loci. Population genetic analysis of ticks collected from four elephant populations using ITS-2 and CO1 loci revealed high gene diversity. Gene diversity at the ITS-2 locus was 0.91 and the nucleotide diversity was, 0.01. ITS-2 gene diversity was highest in Laikipia-Samburu ecosystem (0.947) and lowest in Tsavo (0.80). The CO1 locus also had high gene diversity, 0.790, and low nucleotide diversity, 0.006, and like ITS-2, gene diversity was higher in Laikipia-Samburu ecosystem (1.00) and lower in Tsavo (0.286). There was a modest statistically significant genetic differentiation among the four tick populations based on ITS-2 (FST = 0.104, P < 0.001; ΦST = 0.105, P < 0.001), and a 10% of molecular variance attributed to genetic variation between populations. There was also statistically significant differentiation among tick populations using haplotype frequencies for CO1 locus (FST = 0.167, P < 0.001) accounting for 17% of genetic variance among populations, but not modelled genetic distances (ΦST = 0.029, P = 0.095) suggesting very recent genetic differentiation. In addition, populations of A. tholloni in Kenya had a significantly negative Tajima D and Fu & Li's F* and D* at the CO1 locus suggesting recent positive selection. The extensive acaricide use in livestock, which host the larval stage, could be driving purifying selection and genetic hitchhiking of the CO1 locus. However, tests sensitive to demography such as Fu's FS, Ramos-Onsins & Rozas's R2 and raggedness index r were statistically significant at the ITS-2 locus suggesting ancient demographic expansion. Elephant population fragmentation appears to shape the genetic structure of A. tholloni, while agro-ecological factors could influence the genetic diversity of ticks.

Sciaromiopsis sinensis (Amblystegiaceae), revised to a member of Brachythecium (Brachytheciaceae)

Sciaromiopsis comprises a single species, S. sinensis, endemic to China. The genus is currently placed in the Amblystegiaceae, solely on the basis of gametophytic characters since its sporophytes are unknown. As a semiaquatic plant, the character of a strong leaf costa and marginal borders can aid S. sinensis to adapt drought as while as aquatic conditions. In this study, the phylogenetic position of Sciaromiopsis was revisited based on morphological and molecular phylogentic evidences of three datasets, the plastid rps4 dataset with 346 mosses species, the ITS dataset with 104 moss species and 82 plastid protein-coding gene dataset with 32 moss species. According to the morphological characters of gametophyte, the stable pattern of pseudoparaphyllia, leaf cells smooth and single costa of Sciaromiopsis are consistent with key characteristics of the Brachytheciaceae, the ovate-lanceolate leaves, differentiated alar cells and rhomboidal median leaf cells are consistent with key characteristics of the Brachythecium. Maximum likelihood and Bayesian analyses of both datasets of the rps4 gene and the 82 plastid protein-coding genes clearly indicated that Sciaromiopsis is a member of Brachytheciaceae. Sciaromiopsis sinensis is either clustered in a well supported clade among species of Brachythecium, Koponeniella and Myuroclada in the rps4 tree, or strongly supported as the sister to Brachythecium rivulare in plastid 82 gene tree. Maximum likelihood analysis of the nuclear ITS locus suggested that S. sinensis is nested within Brachythecium with moderate support. Thus, S. sinensis should be considered as a member of the Brachythecium, the diagnostic character of its leaf margin bordered by 5–8 rows of linear cells making it an easy distinction from other species of Brachythecium.

Taxonomy and Phylogeny of Novel and Extant Taxa in Pleosporales Associated with Mangifera indica from Yunnan, China (Series I).

Pleosporales is the largest fungal order with a worldwide distribution in terrestrial and aquatic environments. During investigations of saprobic fungi associated with mango (Mangifera indica) in Baoshan and Honghe, Yunnan, China, fungal taxa belonging to pleosporales were collected. Morphological examinations and phylogenetic analyses of ITS, LSU, SSU, rpb2 and tef1-α loci were used to identify the fungal taxa. A new genus, Mangifericomes; four new species, namely Mangifericomes hongheensis, Neomassaria hongheensis, Paramonodictys hongheensis, and Paramonodictys yunnanensis; and six new host and country records, namely Byssosphaeria siamensis, Crassiparies quadrisporus, Paradictyoarthrinium aquatica, Phaeoseptum mali, Torula fici, and Vaginatispora amygdali, are introduced. Photoplates, full descriptions, and phylogenetic trees to show the placement of new and known taxa are provided.

Molecular survey and genetic diversity of Bartonella spp. in domestic cats from Paraguay.

Although Bartonella spp. is described in cats worldwide, little is known about the occurrence and genetic diversity of Bartonella spp. in cats from South America. To date, it has only been detected in cats from Brazil, Chile and Argentina. This study aimed to undertake a molecular survey and explore the genetic diversity of Bartonella spp. in domestic cats from Paraguay. A TaqMan real-time quantitative PCR (qPCR) targeting the nuoG gene (83 bp) for Bartonella spp. was used to screen 125 blood samples from cats in Asuncion, Paraguay. nuoG qPCR-positive samples were further submitted to conventional PCR assays based on the ITS (453- 717 bp), gltA (767 bp), ftsZ (515 bp), rpoB (333 bp), ribC (585-588 bp), and pap-31 (564 bp) loci. Positive samples were sequenced for species identification, phylogenetic, and haplotype analyses. Bartonella D.N.A. was present in 20.8% (26/125) cat blood samples, with low levels of Bartonella nuoG D.N.A. cPCR products targeting gltA, ftsZ, ITS, and rpoB loci from sixteen cats were successfully sequenced. However, all nouG qPCR-positive samples were negative for the ribC and pap-31 genes. Bartonella henselae [62.5% (10/16)] and Bartonella clarridgeiae [37.5% (6/16)] were identified among the sequenced samples. Upon phylogenetic analysis, B. henselae and B. clarridgeiae from Paraguay clustered with sequences detected in domestic and wild cats, dogs, and cat fleas worldwide. Two to four haplotypes of B. henselae and B. clarridgeiae in cats from Paraguay were observed, with some being exclusive and others shared with worldwide distributed haplotypes. Here, we report B. henselae and B. clarridgeiae for the first time in cats from Paraguay. Its circulation in cats suggests the need to consider Bartonellae when testing clinical samples from suspected infectious diseases in humans from Paraguay.

First report of Diplodia seriata, D. mutila, and Dothiorella omnivora associated with apple cankers and dieback in Rio Negro, Argentina.

Apple (Malus domestica Borkh.) is an important fruit crop in Río Negro, Argentina. In recent years, the frequency of canker and dieback symptoms have increased affecting different apple cultivars. In September 2014, a higher occurrence of cankers (29%) and dieback (9%) was observed in a commercial orchard of 10-year-old apple trees (n=210) cv 'Ital Red' in General Roca, Río Negro, Argentina (39°2'36.73"S - 67°32'44.55"W). Symptoms initially appeared as necrotic bark lesions on tree trunks and branches often associated with pruning wounds. Superficially, papyrus detachment of the bark and cracked bark were observed on the affected area. When the bark was removed, the diseased wood showed dark brown color. Cross sections of diseased branches revealed necrotic lesions that progress to branch death. Samples were collected from different symptomatic trees (n=30) and were superficially disinfected with 70% ethanol. Internal tissues (0.5 cm2) were excised from the advance margin of the necrotic lesions, plated on potato dextrose agar (PDA), and incubated at 22°C. Pycnidia were induced on sterilised pine needles overlaid on 2% water agar under near-UV light. Optimum temperature of culture growth on PDA was studied. According to their morpho-cultural characteristics, three different morphotypes were identified. The first, showed optimum growth at 30°C, had moderately dense white aerial mycelia and turned dark gray after 7 d. Conidia were ovoid, mostly aseptate, 20.8-25.6 × 8-11.4 µm (n=50) and hyaline to brown. The second, exhibited optimum growth from 25 to 30°C, was white to gray, with sparse to moderate aerial mycelium that turned dark olive green. Conidia were ovoid, 1-septate, 17.6-22.4 × 8.1-11.2 µm (n=50) and brown. Finally, the third showed optimum growth at 25°C, mycelium was grey to dark olive green. Conidia were oblong to ovoid with both ends rounded, aseptate and 1-septate at maturity, 20.8-24 × 11.2-14.4 µm (n=50), hyaline turned brown. Genomic DNA was extracted from one representative isolate of each morphotype and the ITS and EF1-α loci were amplified with the primer sets ITS1/ITS4 (White et al., 1990) and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. The nucleotide sequences indicated ≥99% identity to D. seriata (CBS 114796 and CBS 112555), D. mutila (CBS 302.36 and CBS 112553), and D. juglandis (CBS 188.87), reclassified as Dothiorella omnivora (Linaldeddu et al., 2016), for both DNA regions. The sequences were deposited in the GenBank database (MW596418, MW598375; MH665432, MK955889; MH665413, MK937229). To confirm pathogenicity, healthy 1-year-old twigs of adult apple trees were pruned and wounds of attached twigs immediately inoculated with 20 μL of conidial suspension (103 conidia.mL-1, n=9 per isolate) or sterile distilled water (control, n=9), and wrapped with Parafilm. The experimental design was randomized, and the experiment was repeated once. After 90 d, the area of lesion on all twigs inoculated was determined. D. mutila and Do. omnivora produced mean canker areas (65 and 73 mm2, respectively) significantly larger (p < 0.005) than D. seriata (48 mm2). No lesion occurred in the negative controls. Fulfilling Koch's postulates, fungi were reisolated from all inoculated twigs and no fungus was recovered from controls. To our knowledge, this is the first report of D. seriata, D. mutila, and Do. omnivora associated with apple canker and dieback in Argentina, which shows the need of study the role of these fungi in orchard health.

First report of Truncatella angustata causing leaf spot on oregano (Origanum vulgare) in Latvia

AbstractOregano is one of the most commonly cultivated aromatic and medicinal plants in Europe. There is a lack of information about fungi infecting oregano during the vegetation period. In autumn 2020, the diversity of fungi colonizing and damaging oregano leaves was surveyed in the ex‐situ collection held at the Latvia University of Life Sciences and Technologies. A fungus with appendage‐bearing conidia was isolated from oregano leaf tissue, causing brown lesions at the tip or edge of the leaf. The causal agent of the disease was identified as Truncatella angustata after the evaluation of morphology and culture characteristics. The identity of the species was confirmed based on multi‐locus phylogenetic analyses, using ITS, LSU, tef‐1α, tub2 and rpb2 loci, in combination with morphological data. The homologous regions from relevant T. angustata available in the GenBank were compared with T. angustata sequences obtained from oregano in this study. Pathogenicity tests performed on oregano leaves resulted in the same symptoms as observed in naturally‐infected plants. This is first report of T. angustata as a pathogen of oregano, as well as the first report on the occurrence of T. angustata on oregano in Latvia.

Biodiversity of South Indian tea clones with detection of plant-based adulterants in tea dust using DNA barcoding

Tea is by and large a highly penetrated product in south India. Hence the adulteration risk in tea dust gets hiked in the markets. We constructed a standard database using plant plastid markers (rbcL, matK, trnH-psbA, rpoC, rpoB, ycf 1) and nuclear (ITS2) locus from prominent south Indian tea clones representing Assam, China, and Cambod varieties. These barcodes were used as reference algorithm to investigate the authenticity of 10 sampled commercial tea dust by recovering its DNA barcodes using rbcL, matK, and ITS2 loci. PCR amplification success, sequencing efficiency, genetic polymorphisms, BLAST search, and phylogenetic analysis were performed to enhance genotypic information on south tea cultivars and in authenticating the commercial samples of Camellia sinensis. Findings suggest that the chloroplast and nuclear loci can identify tea plant at the genus and varietal level respectively and rbcL as the potential marker for detecting plant-based admixtures coupled with TA cloning after DNA barcoding.

Phlebotomus neglectus (Diptera: Psychodidae): New Insights on Its Presence in Iran Based on Three Independent Genetic Loci.

The idea of the existence of Phlebotomus (Larroussius) neglectus (Diptera: Psychodidae) Tonnoir, 1921 in Iran and the skepticism about the existence of Phlebotomus major s.str. Annandale, 1910 had been grown recently in the country. This study reports a combined analysis of mitochondrial and ribosomal DNA target regions of P. major s.l.Annandale, 1910, specimens collected from different parts of Iran. Two different morphotypes were found among the collected samples based on the shape of the aedeagus, ventrally located hairs of the coxite, and parameral sheets. One morphotype seemed similar to P. neglectus Tonnoir 1921 or P. major krimensis Perfiliv1966 (called here MI.N.K.); the other one was similar to P. neglectus and to some extent to P. notus Artemiev & Neronov 1984 (here called MII.N.NO). Cytochrome B, elongation factor 1-alpha, and internal transcribed spacer II loci were amplified, sequenced, and characterized. High sequence homology (98-100%) was observed between P. neglectus and these morphotypes, and phylogenetic analysis was also concordant. Phlebotomus neglectus sequences available in GenBank are located as the sister group of sequences here, particularly near to morphotype MII.N.NO. Moreover, ITS2 locus provides the maximum resolution for differentiation of two morphotypes. Based on achieving results, although a strong support for the presence of P. neglectus was provided, but it is too early to say that P. major s.str. does/does not exist in Iran. This question could be resolved by studying more samples and, most importantly, by comparing the topotypes of P. neglectus and P. major s. str. if possible in the future.

Comparative Comprehensive Analysis on Natural Infections of Hymenolepis Diminuta and Hymenolepis Nana in Commensal Rodents.

SummaryThis first comprehensive report from Punjab province of India relates to patho-physiological alterations alongwith morpho-molecular characterisation and risk assessment of natural infections of Hymenolepis diminuta and Hymenolepis nana in 291commensal rodents including house rat, Rattus rattus (n=201) and lesser bandicoot rat, Bandicota bangalensis (n=90). Small intestine of 53.61 and 64.95 % rats was found infected with H. diminuta and H. nana, respectively with a concurrent infection rate of 50.86 %. There was no association between male and female rats and H. diminuta and H. nana infections (ᵡ2 = 0.016 and 0.08, respectively, d.f.= 1, P>0.05), while the host age had significant effect on prevalence of H. diminuta and H. nana (ᵡ2 = 28.12 and 7.18, respectively, d.f.= 1, P≤0.05) infection. Examination of faecal samples and intestinal contents revealed globular shaped eggs of H. diminuta without polar filaments (76.50 ± 3.01μm x 67.62 ± 2.42 μm), while smaller sized oval eggs of H. nana were with 4 – 8 polar filaments (47.87 ± 1.95 μm x 36.12 ± 3.05 μm). Cestode infection caused enteritis, sloughing of intestinal mucosa, necrosis of villi and inflammatory reaction with infiltration of mononuclear cells in the mucosa and submucosa. Morphometric identification of the adult cestodes recovered from the intestinal lumen was confirmed by molecular characterisation based on nuclear ITS-2 loci which showed a single band of 269 bp and 242 bp for H. diminuta and H. nana, respectively. Pairwise alignment of the ITS-2 regions showed 99.46 % similarity with sequences of H. diminuta from USA and 100 % similarity with sequences of H. nana from Slovakia, Kosice.

Isolation of Metarhizium guizhouense and Metarhizium robertsii Strains from Soil-Exposed Amblyomma americanum (Acarina: Ixodidae) from Northwest Arkansas, USA

The lone star tick, Amblyomma americanum (L.) (Acarina: Ixodidae), is the most abundant tick species found in Arkansas and is involved in the transmission of pathogens of medical and veterinary importance. When not feeding, most non-nidicolous tick species shelter in the soil and leaf litter where they may be exposed to and potentially infected with entomopathogenic fungi that reside naturally in the soil. Entomopathogenic fungi in the genus Metarhizium Sorokīn (Hypocreales: Clavicipitaceae) have shown promise as biological control agents of ticks. Here, the first study to isolate and identify Arkansas-derived isolates of Metarhizium from A. americanum ticks is presented. We exposed 320 ticks artificially to native soil from Savoy. Of these soil exposed ticks, 2.5% of adults and 1.5% of nymphs displayed signs of infection with Metarhizium. Of the infected Savoy adults, 3.3% were females and 1.7% were males. Similarly exposed ticks from West Fork resulted in only 2.4% of nymphal ticks being infected with this fungus. Eight isolates of Metarhizium were cultured from infected ticks exposed to soil from these locations. Four of these Metarhizium isolates (3 from Savoy and 1 from West Fork) were identified to species by sequencing of the ITS locus and the EF1-α genes. Three Savoy strains (P10N1, P10AF1, and P2AM1) had identical sequences and were identified as Metarhizium robertsii (Bischoff, Rehner & Humber) (Hypocreales: Clavicipitaceae). The strain from West Fork (P9N2) was identified as Metarhizium guizhouense (Chen & Guo) (Hypocreales: Clavicipitaceae). The ITS and the EF1-α sequences of the Savoy strains showed 100% similarity to M. robertsii strains ARSEF 2575 and ART 500, respectively. The ITS and EF1-α sequences of the West Fork strain showed 99% similarity to M. guizhouense strains ARSEF 977 and CBS 258.90, respectively. This study demonstrates that entomopathogenic fungi M. guizhouense and M. robertsii are pathogenic to and can be isolated from A. americanum. Furthermore, the EF1-α genetic marker was shown to be a very effective tool for distinguishing different species of Metarhizium from ticks when used in conjunction with ITS sequence data. Standardizing the use of ticks in soil exposure methods for isolating entomopathogenic fungi could be useful for obtaining isolates that are highly virulent to A. americanum. The isolation and identification of Metarhizium spp. from A. americanum in Arkansas indicates that further exploration of entomopathogenic fungi as biological agents to control A. americanum is warranted.

The Morphology, Ultrastructure and Molecular Phylogeny of a New Freshwater Heterolobose Amoeba Parafumarolamoeba stagnalis n. sp. (Vahlkampfiidae; Heterolobosea)

Heterolobose amoebae are important members of marine, freshwater, and soil microbial communities, but their diversity remains under-explored. We studied the diversity of Vahlkampfiidae to improve our understanding of heterolobosean relationships and their representation in aquatic benthos. Using light and electron microscopy, and molecular phylogenies based on the SSU rRNA and ITS loci, we describe the fine morphology and evolutionary relationships of a new heterolobosean Parafumarolamoeba stagnalis n. sp. from a small pond in European Russia. Cells of P. stagnalis possess a clearly distinguishable anterior hyaline pseudopodium, eruptive movement, several thin and sometimes branched uroidal filaments, spherical cysts without pores and plugs, and mitochondria that have discoid cristae and are surrounded by cisternae of the endoplasmic reticulum. The genus Parafumarolamoeba has so far included a single species, Parafumarolamoeba alta from high-altitude soil in Tibet, which is morphologically distinct from P. stagnalis. Taxonomic description for a new Parafumarolamoeba species is therefore provided.

First reports of Phytophthora ramorum clonal lineages NA1 and EU1 causing Sudden Oak Death on tanoaks in Del Norte County, California.

A year of forest health surveys has led to the first detection of Phytophthora ramorum in Del Norte County followed by the first wildland detection of the EU1 clonal lineage (Grunwald et al. 2009) of this pathogen in California. In July 2019, leaves were sampled from two tanoaks (Notholithocarpus densiflorus) and 16 California bay laurels (Umbellularia californica) in Jedediah Smith State Park in Del Norte County, the northernmost coastal County of California. Leaves displayed lesions normally associated with Sudden Oak Death (SOD) caused by P. ramorum and were discovered during the citizen science-based survey known as SOD Blitz (Meentemeyer et al. 2015). Samples were surface sterilized using 75% Ethanol and plated on PARPH-V8 agar (Jeffers and Martin 1986). After plating, DNA was extracted and amplified using two P. ramorum-specific assays (Hayden et al. 2006, Kroon et al. 2004). Leaves from two tanoaks exhibiting twig die-back had typical SOD lesions along the midvein, gave positive PCR results and yielded cultures with colony morphology, sporangia and chlamydospores typical of the NA1 lineage of P. ramorum originally isolated in California from tanoaks and coast live oaks (Quercus agrifolia) (Rizzo et al. 2002). The ITS locus and a portion of the Cox-1 locus were sequenced from DNA extracts of each culture using primers DC6-ITS4 (Bonants et al. 2004) and COXF4N-COXR4N (Kroon et al. 2004), respectively. ITS sequences (GB MN540639-40) were typical of P. ramorum and Cox-1 sequences (GB MN540142-3) perfectly matched the Cox-1 sequence of the NA1 lineage (GB DQ832718) (Kroon et al. 2004). Microsatellite alleles were generated as described in Croucher et al. (2013) for the two Del Norte cultures and for eight P. ramorum cultures, representative of the four main multilocus genotypes (MLGs) present in California, namely c1 (Santa Cruz/Commercial Nurseries), c3 (San Francisco Bay Area), c2 (Monterey County), and c4 (Humboldt County) (Croucher et al. 2013). The two Del Norte MLGs were identical to one another and most similar to MLG c1, with a single repeat difference at a single locus. SSR results suggest the inoculum source may not be from Humboldt County, neighboring to the South, but from a yet unidentified outbreak, possibly associated with ornamental plants. Jedediah Smith State Park was surveyed for 12 months following the initial detection, however the pathogen has yet to be re-isolated in that location. In July 2020, SOD symptomatic leaves from two tanoak trees exhibiting twig cankers were collected 8 Km north of Jedediah Smith State Park, where three additional tanoak trees displayed rapidly browned dead canopies consistent with late stage SOD. Leaves were processed as above. Colonies from these samples produced chlamydospores and sporangia typical of P. ramorum on PARPH-V8 agar, but displayed a growth rate faster than that of NA1 genotypes and were characterized by aerial hyphae, overall resembling the morphology of EU1 lineage colonies (Brasier 2003). The EU1 lineage was confirmed by the perfect match of the sequence of a portion of the Cox-1 gene (GB MW349116-7) with the Cox-1 sequence of EU1 genotypes (GB EU124926). The EU1 clonal lineage has been previously isolated from tanoaks in Oregon forests, approximately 55 Km to the North (Grünwald et al. 2016), but this is the first report for California wildlands and will require containment and government regulations. It is unknown whether the EU1 strains in Del Norte County originated from Oregon forests or elsewhere.

Studies of canker and dieback of oak tree in China, with two Cytospora species described

AbstractOak (Quercus sp.) is an excellent tree species as a windbreak, for water conservation, and for fireproofing in forests in China. However, several trees of this genus were found to be suffering from various fungal diseases. In this study, we evaluated 15 fungal pathogens that can cause dieback and canker disease in oak in China, and discovered two Cytospora species. They were identified as Cytospora quercinum sp. nov. and C. vinacea, based on detailed morphological comparisons and phylogenetic analysis of ITS, LSU, act, rpb2, tef1‐α, and tub2 loci. This study is the first record of C. quercinum and C. vinacea as causal agents of dieback in oak based on pathogenicity tests conducted on 2‐year‐old plants in a greenhouse. In addition, this study also revealed the influence of different conditions on the growth rate of mycelia. Mycelial growth of C. quercinum and C. vinacea occurred at optimum temperatures of 20.1 and 20.8 °C, and optimum pH of 5.4 and 5.3, respectively. For these two species, utilization of glucose and fructose was highly efficient, and sucrose was the least efficient. The habitat of Quercus mongolica indicated that more attention and management are needed to prevent the occurrence of Cytospora disease in the summer in north‐eastern China, and in spring and autumn in eastern and northern China. This study contributes to the understanding of the species causing canker or dieback diseases in important economic forest trees, and provides useful information for effective disease management of oak trees in China.

Occurrence and infective potential of Colletotrichum gloeosporioides isolates associated to Citrus limon var Eureka

A collection of 37 fungi associated to Italian lemon plants with disease symptoms, was obtained. Ten genera including Aspergillus, Alternaria, Nigrospora, Lasiodiplodia, Dothideomycetes, Pleurostoma, Setosphaeria, Penicillium, Fusarium and Colletotrichum were identified by using ITS1–5.8S–ITS2, D1/D2 26S and COX1 loci. The last three genera were abundant on the damaged fruits, being Colletotrichum the more abundant (32.4 %). CaInt2 and CgInt primers support the identity of these isolates as C. gloeosporioides. Variability, inferred by rep-PCR and multilocus sequence analysis shows genetic differences among the C. gloeosporioides isolates. Infective profile evaluated in Colletotrichum isolates shows different leave infection percentages (26 to 60 %). SEM analysis showed mycelium, spores and appressoria on the leaves of selected Colletotrichum isolates. Specifically, the AL-05 and AL-13 isolates showed a high chitin deacetylase activity (CDA) peaking at 1.2 U/mg protein in AL-13. This is the first report on C. gloeosporioides infecting Italian lemon leaves in Mexico.

Inter- and Intra-Continental Genetic Variation in the Generalist Conifer Wood Saprobic Fungus Phlebiopsis gigantea

Phlebiopsis gigantea (Fr.) Jülich is a well-known generalist conifer wood saprobe and a biocontrol fungus used in several world countries to prevent stump infection by tree pathogenic Heterobasidion fungal species. Previous studies have reported the presence of regional and continental genetic differentiation in host-specific fungi, but the presence of such differentiation for generalist wood saprobes such as P. gigantea has not been often studied or demonstrated. Additionally, little information exists on the distribution of this fungus in western North America. The main purposes of this study were: (I) to assess the presence of P. gigantea in California, (II) to explore the genetic variability of P. gigantea at the intra and inter-continental levels and (III) to analyze the phylogeographic relationships between American and European populations. Seven loci (nrITS, ML5–ML6, ATP6, RPB1, RPB2, GPD and TEF1-α) from 26 isolates of P. gigantea from coniferous forests in diverse geographic distribution and from different hosts were analyzed in this study together with 45 GenBank sequences. One hundred seventy-four new sequences were generated using either universal or specific primers designed in this study. The mitochondrial ML5–ML6 DNA and ATP6 regions were highly conserved and did not show differences between any of the isolates. Conversely, DNA sequences from the ITS, RPB1, RPB2, GPD and TEF1-α loci were variable among samples. Maximum likelihood analysis of GPD and TEF1-α strongly supported the presences of two different subgroups within the species but without congruence or geographic partition, suggesting the presence of retained ancestral polymorphisms. RPB1 and RPB2 sequences separated European isolates from American ones, while the GPD locus separated western North American samples from eastern North American ones. This study reports the presence of P. gigantea in California for the first time using DNA-based confirmation and identifies two older genetically distinct subspecific groups, as well as three genetically differentiated lineages within the species: one from Europe, one from eastern North America and one from California, with the latter presumably including individuals from the rest of western North America. The genetic differentiation identified here among P. gigantea individuals from coniferous forests from different world regions indicates that European isolates of this fungus should not be used in North America (or vice versa), and, likewise, commercially available eastern North American P. gigantea isolates should not be used in western North America forests. The reported lack of host specificity of P. gigantea was documented by the field survey and further reinforces the need to only use local isolates of this biocontrol fungus, given that genetically distinct exotic genotypes of a broad generalist microbe may easily spread and permanently alter the microbial biodiversity of native forest ecosystems.

Diversity of Diaporthe species associated with melanose disease on citrus trees in Jiangxi Province, China

Citrus melanose is an important disease that seriously damages citrus production. It is caused by Diaporthe species, which are known as important pathogens, endophytes or saprobes with broad host ranges. Over 170 species of Diaporthe were reported around the world and the complexity of species structures makes it hard to be identified. Phylogenetic analysis based on translation elongation factor 1-ɑ (TEF1) gene, s-tubulin (TUB) gene, calmodulin (CaM) gene, histone (HIS) gene and ITS locus was a reliable method to identify Diaporthe species In this study, the species structure of Diaporthe species from different tissues within individual trees was investigated. Altogether, 494 single spore isolates were obtained, and 92 of them were selected to sequence the five loci (ITS, TEF1, HIS, TUB, CaM) for phylogenetic analysis. Based on the combined multi-locus dataset, phylogenetic trees constructed by using two methods (MP and ML) showed that D. citri was the dominant species no matter on fruits, leaves or twigs. Diaporthe species showed the greatest diversity on fruits and the least diversity on twigs. D. eres was only isolated from twigs. The results of this study may enhance our understanding for the species diversity of Diaporthe species on different tissues from same trees, such information would be useful for the control of Diaporthe diseases in the future.

Identification of Corynespora cassiicola causing leaf spot on Syringa species

ABSTRACTLilacs (Syringa spp.), a genus of trees and shrubs in the Oleaceae, are known for their beautiful, fragrant flowers and are widely cultivated as ornamentals. In September 2017, a new leaf spot disease was observed on S. oblata in the Sui and Tang Ruins Dynasties Botanical Garden in Luoyang, Henan Province, China. Leaf symptoms appeared as small brown lesions at earlier stages, which with age turned into suborbicular brown, faintly zonate and depressed. Fully mature lesions were mostly irregular to circular, concentric rings with taupe and dark brown bands. A total of 12 monoconidial isolates were obtained from S. oblata showing leaf symptoms. Colonies were grey to light brown and darker underneath on potato dextrose agar. Based upon morphological characteristics and multilocus phylogenetic analyses using ITS, TEF1 and RPB2 loci, isolates were identified as Corynespora cassiicola. Pathogenicity tests on leaves produced symptoms matching those described above and the fungus was re‐isolated, confirming Koch's postulates. This study represents the first report of C. cassiicola on lilacs in China.

DNA barcode-based phylogenetic assessment of selected mangroves from sundarbans delta and Kerala

India harbors the wealthiest biodiversity of mangroves, yet the utilization of sequence-based DNA barcodes for the characterization of mangrove diversity from the Indian coastal region is needed to be explored. In the present study, we assessed DNA barcode-based phylogenetics of mangroves from Sundarbans Delta and Kerala at the ITS locus of the nuclear genome. The phylogenetic analysis was performed using Bayesian inference, which proved to resolve the evolutionary affinities effectively. Our study presents mangroves’ systematic assessment with total 6 DNA sequences generated from 5 genera, and the first DNA barcode of Volkameria inermis and Avicennia marina from Payyanur Kerala, Avicennia alba and Suaeda maritima from the Sundarbans Delta was reported from ITS locus. The phylogenetic assessment confirmed that Avicennia belongs to the family Acanthaceae and placed the Volkameria inermis from Payyanur and Clerodendrum eriophyllum under the same monophyletic clade.

Sarcocystis neurona Transmission from Opossums to Marine Mammals in the Pacific Northwest

Increasing reports of marine mammal deaths have been attributed to the parasite Sarcocystis neurona. Infected opossums, the only known definitive hosts, shed S. neurona sporocysts in their feces. Sporocysts can contaminate the marine environment via overland runoff, and subsequent ingestion by marine mammals can lead to fatal encephalitis. Our aim was to determine the prevalence of S. neurona in opossums from coastal areas of Washington State (USA) and to compare genetic markers between S. neurona in opossums and marine mammals. Thirty-two road-kill opossums and tissue samples from 30 stranded marine mammals meeting inclusion criteria were included in analyses. Three opossums (9.4%) and twelve marine mammals (40%) were confirmed positive for S. neurona via DNA amplification at the ITS1 locus. Genetic identity at microsatellites (sn3, sn7, sn9) and the snSAG3 gene of S. neurona was demonstrated among one harbor porpoise and two opossums. Watershed mapping further demonstrated plausible sporocyst transport pathways from one of these opossums to the location where an infected harbor porpoise carcass was recovered. Our results provide the first reported link between S. neurona genotypes on land and sea in the Pacific Northwest, and further demonstrate how terrestrial pathogen pollution can impact the health of marine wildlife.