Internal Transcribed Spacer rDNA Research Articles

First Report of Verticillium Wilt Caused by Verticillium dahliae Infection on Chinese Cabbage in Korea.

Chinese cabbage (Brassica rapa L.) is one of the most important vegetables in Korea due to its role as the main ingredient for the making of Kimchi. In June 2014, disease symptoms of leaves wilt, dry, and drop off on Chinese cabbage were observed in a Chinese cabbage farm located at Taebeak (37°26'50.7"N 128°95'50.0"E), Gangwon province, Korea. This disease was observed on approximately 35% of the plants in the field, causing an almost 10% decrease in total production. At the early stage of infection, the color at the edge of the plant foliage changed from green to yellow. As the disease progressed, infected leaves wilted, dried off, and detached from the plant. Soft rot that occurred at the base of the leaf stem and root tissues caused the infected leaves to dry and fell off the plant. To identify the causal agent, a small piece of infected leaf tissues was sterilized with 1% sodium hypochlorite solution for 1 min and rinsed with sterile water before it was transferred onto potato dextrose agar (PDA) media. The plates were then incubated at 25°C for 10 days in the dark. Fungal colonies grown on PDA media were of white-creamy in color with an abundance of mycelia and later develop into black color due to the formation of microsclerotia embedded in the media. Microscopic examination showed conidiophores and phialides were both appeared in a verticillate arrangement, whereas conidia were hyaline, smooth-walled, and ellipsoidal to oval with average size 5.4×2.5 µm (n=100). Microsclerotia appeared in elongate to an irregularly spherical shape and greatly variable in size. The morphological attributes of the fungal isolate described above were comparable to the characteristics of Verticillium dahliae Kleb. (V. dahliae) described by Hawksworth and Talboys (1970), and V. dahliae isolated from Chinese cabbage in Japan reported in Kishi (1998). Pathogenicity test was performed by soaking twelve individual Chinese cabbage seedlings for 15 min into fungal pathogen conidial suspension (1x106 conidium/ml) before transferred into soil tray. The same number of non-inoculated seedlings on the soil tray was used as a control. Inoculated and control plants were then covered with a plastic bag for 24 hours to maintain high humidity before transferred into the greenhouse (25°C). Seven days post-inoculation (dpi), treated plant leaves turned yellow, and soft rot was observed. At 10-dpi, plant leaf tissues dried off and severe soft rot occurred. Pathogenicity test was repeated three times and consistent results were obtained. The re-isolated fungal pathogen from the inoculated plants showed identical morphological characteristics to the original isolates, thus fulfilling Koch's postulates. For further identification, PCR amplification targeting Internal Transcribed Spacer (ITS) and RNA polymerase II gene (RPB2) regions were performed (Liu et al., 1999; White et al., 1990). Each PCR product was sequenced and deposited in the GenBank under the accession LC549667 and LC061275, respectively. Sequence analysis using BLAST showed that the nucleotide sequences of ITS and RPB2 DNA fragments are 99-100% identical to the reference strain of V. dahliae available in the NCBI database (MG585719, HE972023, XM_009652520 and DQ522468, respectively). Therefore, based on the results of morphological and molecular analyses, the fungal pathogen isolated from Chinese cabbage in this study was identified as V. dahliae and deposited in the National Institute of Horticultural and Herbal Science germplasm collection (NIHHS 13-252). Recently, due to high demand and a more competitive price, more Chrysanthemum farmers in Korea switch their crops to Chinese cabbage. Interestingly, the occurrence of V. dahliae infection was also reported to occur in Chrysanthemum plants in Korea (Han et al. 2007), which indicates a serious problem ahead to these farmers. Therefore, in this current study, the identification of V. dahliae pathogenic to Chinese cabbage will provide vital knowledge for the development of disease management strategies to minimize the loss of crop production. To our knowledge, this is the first report that V. dahliae causes Verticillium wilt disease on Chinese cabbage in Korea.

First Report of Powdery Mildew Caused by Erysiphe viciae-unijugae on Vicia sativa subsp. nigra in Korea.

Vicia sativa subsp. nigra, also known as narrow-leaved vetch, belongs to the family Fabaceae and is distributed in different countries.Vetch is one of the most important annual forage legumes in the world; it is used as hay, grain, silage, green manure, and livestock fodder. In Korea, this plant is often used as cattle feed. In early May of 2019 and 2020, a powdery mildew was observed on leaves of V. sativa subsp. nigra plants in a garden of Chonnam National University, Gwangju, Korea. The first symptom appeared on the upper surface of the leaves as a small white spot. Within two weeks, the white spots of powdery mildew spread and covered the entire leaf. Voucher specimens were deposited at the Environmental Microbiology Laboratory Herbarium, Chonnam National University, Gwangju, Korea, under the accession numbers CNUFC PWV1 and CNUFC PWV2. The hyphal appressoria of the fungus were nipple-shaped, moderately lobed or multilobed, single or in opposite pairs. Measurements were made of 35 conidiophores and 55 conidia. Conidiophores arising from the upper part of mother cells, were cylindrical, 60.5 to 117.5 × 7.5 to 9.5 μm. The foot cells of the conidiophores were cylindrical, straight to slightly curved at the base, 23.5 to 38.7 × 7.3 to 9.2 μm, followed by 2-3 shorter cells. Conidia were formed singly, subcylindrical to oval, 32.5 to 48.7 × 13.5 to 19.7 μm. Conidia produced short to long germ tubes, terminating in lobed or unlobed appressoria. No chasmothecia were observed. The morphological characteristics of conidiophores and conidia were consistent with the description of Erysiphe viciae-unijugae in Shin (2000). However, it was not possible to identify the fungus only based on the asexual morph. In addition, the conidiophore and conidium shapes of E. baeumleri on the known host V. sativa were similar to those of E. viciae-unijugae (Braun and Cook 2012). Therefore, genomic DNA was extracted directly from the mycelia and conidia using a DNA prep kit (Solgent, Daejeon, Korea). The internal transcribed spacer (ITS) regions ofribosomal DNA (rDNA) and 28S rDNA from the two specimens were amplified using the primers ITS5 and PM6 (Takamatsu and Kano 2001), and LROR and LR3 (Vilgalys and Hester 1990), respectively. A BLASTn search with the ITS sequences indicated that CNUFC PWV1 (GenBank accession no. MT509395) and CNUFC PWV2 (MT509396) were 100% (503/503 bp) and 99.8% (502/503 bp) identical to those of E. viciae-unijugae (LC010086 and LC009962), respectively. The 28S sequences of CNUFC PWV1 (MT705629) and CNUFC PWV2 (MT705630) showed 99.8% (507/508 bp) and 99.4% (505/508 bp) identity values with E. viciae-unijugae (LC009976 and LC010029), respectively. Phylogenetic analyses based on the combined ITS and 28S rDNA sequences indicated that the two specimens, CNUFC PWV1 and CNUFC PWV2, formed a monophyletic clade together with sequences retrieved from E. viciae-unijugae. Therefore, the causal pathogen was identified as E. viciae-unijugae based on the results of morphological and molecular analyses. Pathogenicity tests were performed by gently pressing the infected leaves onto 10 young leaves of three healthy plants. A non-inoculated plant was used as a control. The plants were maintained in a greenhouse at 22 to 25°C, with relative humidity around 75%, and natural light. Powdery mildew symptoms were observed on the inoculated leaves after 10-14 days, whereas no symptoms were observed on the leaves of the control. The fungi on the inoculated leaves were morphologically identical to the fungi on the original infected leaves. Erysiphe viciae-unijugae has been reported to occur on Lathyrus spp. and Vicia spp. (Farr and Rossman 2020). To our knowledge, this is the first report of powdery mildew caused by E. viciae-unijugae on V. sativa subsp. nigra in Korea. The weather conditions, especially during May, are favorable for disease outbreaks. This disease may be a potential threat to feed production in Korea.

Symbiosis between river and dry lands: Phycobiont dynamics on river gravel bars

River gravel bars are dynamic and heterogeneous habitats straddling the transition between aquatic and terrestrial environments. Periodic flooding, low nutrient concentrations, frost, lack of stable sites, drought, and ground surface heat significantly influence the biota of these habitats. Mutualistic symbiosis may be a successful strategy for organisms to survive and proliferate under such harsh conditions. The lichen genus Stereocaulon was selected as a model symbiotic system from among the organisms living on river gravel bars. The goal of the current study was to determine the effect of this dynamic environment on phycobiont (i.e., green eukaryotic photobiont) community structure. We analyzed 147 Stereocaulon specimens collected in the Swiss Alps using Sanger sequencing (fungal internal transcribed spacer (ITS) rDNA, algal ITS rDNA, and algal actin type I gene) and analyzed 8 selected thalli and 12 soil samples using Illumina metabarcoding (ITS2 rDNA). Phytosociological sampling was performed for all 13 study plots. Our analyses of communities of phycobionts, lichens, bryophytes, and vascular plants indicated a gradual change in the phycobiont community along a successional gradient. The particularly large phycobiont diversity associated with Stereocaulon mycobionts included algae, here reported as phycobionts for the first time. Each of the two Stereocaulon mycobiont operational taxonomic units had a distinct pool of predominant phycobionts. The thalli selected for Illumina metabarcoding contained a wide range of additional algae, i.e., they showed algal plurality.

DNA barcoding of Pericopsis mooniana from two different populations in Indonesia based on rDNA ITS (Internal Transcribed Spacer)

A DNA barcoding is a taxonomy method that provide fast and acurate species identification, thus it can be applied to anticipate when a species can not be identified morphologically. This study aimed to assess molecular characteristics of Pericopsis mooniana from two wild populations in Indonesia using a barcoding marker rDNA ITS (Internal Transcribed Spacer) as additional information for establishing conservation strategy of this species. Leaf samples of P. mooniana were collected from natural forests at South East Sulawesi and South Kalimantan, and a germplasm orchard at Purwodadi Botanic Garden. Molecular characteristic was evaluated based on a pair of ITS primers that produce between 634-641 bp nucleotide. The sequence data were analyzed by Bioedit version 7.0.5.3 and Mega 7 software. Results showed that P. mooniana from South East Sulawesi and South Kalimantan were clustered together, while P. mooniana from Purwodadi Botanic Garden were separated with only one base difference from those two population. In conclusion, ITS barcoding in this study shows a diffences between P. mooniana collected from South East Sulawesi and South Kalimantan with P. mooniana that originally from Papua. A further study using more barcoding marker and more population could be approached to obtain more information on molecular characters of P. mooniana populations that will useful for establishing conservation and tree breeding program of this species.

Bioremediation of Crude Oil by Rhizosphere Fungal Isolates in the Presence of Silver Nanoparticles.

Background: This research work focuses on the utilization of indigenous fungi for in situ bioremediation of crude oil in the presence of silver nanoparticles. Methods: Two fungi belonging to two different genera showed promising crude oil-degrading abilities. Fungal isolates were identified based on internal transcribed spacer rDNA sequence analysis. Gas chromatography-mass spectrometry analysis of the crude oil remaining in the culture medium after seven days was performed. The response surface method (RSM) designed by Box-Behnken was used to establish a mathematical model. Inter-simple sequence repeat (ISSR) primers were used to examine the genetic variation of fungal isolates. Results: Gas chromatography-mass spectrometry (GC-MS) analysis after seven days showed that the optimum biodegradation of crude oil was 57.8%. The crude oil degradation rate was significantly affected by a temperature of 30 °C, pH value of 7, crude oil concentration of 4 g/L, a 1:1 ratio between A. flavus AF15 and T. harzianum TH07, and an silver nanoparticle (AgNP) concentration of 0.05 g. Molecular characterization in fungal isolates is extremely valuable when using ISSR markers. Conclusions: Two fungal isolates showed promising crude oil-degrading abilities with positive effect of low concentrations of AgNPs on biodegradation. RSM is an efficient mathematical method to optimize the microbial biodegradation of crude oil.

Etiology of Halo Blight in Michigan Hopyards.

Michigan's hop acreage ranks fourth nationally, but the state's growers contend with unique disease challenges resulting from frequent rainfall and high humidity. In August 2018, a Michigan hop grower reported necrosis and blighting of foliage and shattering of cones resulting in yield loss. Irregular-shaped lesions developed on leaves, surrounded by a halo of chlorotic tissue, and cone bracts became brown. Pycnidia were observed in symptomatic tissue. The goal of this study was to identify and characterize the causal agent of symptoms in leaf and cone tissue. In symptomatic leaves, 15 of 19 isolates recovered had 96.4% internal transcribed spacer rDNA (ITSrDNA) homology with Diaporthe nomurai. Bayesian and maximum likelihood analyses were performed on a subset of isolates using ITSrDNA, histone H3, beta-tubulin, and elongation factor 1 alpha. Bootstrap and posterior probabilities supported a unique cluster of Diaporthe sp. 1-MI isolates most closely related to the Diaporthe arecae species complex, Diaporthe hongkongensis, and Diaporthe multigutullata. Diaporthe sp. 1-MI was pathogenic in detached leaf and whole plant assays. Single-spore isolates from pycnidia originating from cones and leaves shared 100% ITSrDNA homology with Diaporthe sp. 1-MI obtained from the lesion margins of leaves collected in 2018. The distribution of Diaporthe sp. 1-MI was widespread among 347 cones collected from 15 Michigan hop yards and accounted for >38% of fungi recovered from cones in three hop yards. Diaporthe sp. 1-MI causing halo and cone blight presents a new disease management challenge for Michigan hop growers.

First Report of Dieback Caused by Lasiodiplodia pseudotheobromae on Ormosia pinnata in China.

Ormosia pinnata (Lour.) Merr. is an important tree used for landscape and plant recovery of barren slopes in China. During an investigation of plant disease on landscape trees in 2018, a dieback was observed on O. pinnata trees in Guangzhou, Guangdong Province, China. Symptoms were characterized by initial dryness of the twigs and eventual death of the whole branch of the tree. Isolations from symptomatic branches yielded 13 isolates including two main morphotypes. Pathogenicity tests showed that isolate GDOP1 from Type I caused dieback of O. pinnata. Based on morphological characteristics and molecular analysis of the internal transcribed spacer rDNA (ITS1-5.8S-ITS2) and partial sequence of the translation elongation factor 1α (EF1-α), the fungus causing dieback on O. pinnata was identified as Lasiodiplodia pseudotheobromae. This is the first report of L. pseudotheobromae infecting O. pinnata in the world.

First Report of Erysiphe adunca Causing Foliar Powdery Mildew on Poplar in China

Hybrid poplars are important economic plants and sources of biofuel all over the world. Poplars are subjected to a wide range of diseases (Cellerino 1999). In September 2019, powdery mildew was observed on the hybrid poplar Populus davidiana × P. alba var. pyramidalis, a fast-growing elite poplar cultivar widely planted in the northeast of China, in one poplar nursery room in the Northeast Forestry University in China (45.7266°N, 126.6380°E). Disease incidence during September and October was only 5 to 15% but gradually increased to 100% during November and December. The symptom of white circular powdery spots with diameters of up to 12 mm appeared on the upper surface of the mature leaves of poplar plants over 40 days old. Under nursery conditions (50 to 65% humidity, 20 to 25°C, and 16-h/8-h day/night cycles), the diameter of a mildew spot could develop from merely visible to 10 mm in 3 days. The mildew site was observed with a stereomicroscope (Leica DVM6S, Germany). A single fungal species demonstrating characteristics of the obligate pathogens Erysiphe spp. was observed. The conidiophores grew straight upward on the leaf surface until reaching 102 to 165 μm in length (n = 30). Then, even-looking, ellipse-shaped conidia were developed, forming a singular, curved string of up to tens of conidia, on top of each conidiophore. Conidia (n = 30) measured 29.4 to 35.1 × 18.1 to 21.0 μm. Appressoria were lobed or nipple-shaped and appeared in pairs or singular. The germ tubes were slightly curly unbranched tubes pointing at one direction near the long axis of the conidium. We carefully collected the mildew by scraping it off the upper surfaces of fresh diseased leaves with a scalpel and extracted the genomic DNA. The internal transcribed spacer rDNA region was amplified according to the method of Siahmard et al. (2017). The resulting nucleotide sequence (deposited in GenBank as MN998572) displayed 99.84% identity with that of Erysiphe adunca (KY653179.1). Hence, we identified the causal pathogen of this poplar powdery mildew as an E. adunca strain. Furthermore, an Mcm7 sequence (deposited as MN998573) and a Chs sequence (deposited as MN998574) were cloned according to the method of Ellingham et al. (2019). A voucher specimen and the sequence information have been deposited in the Northeast Forestry University Culture Collection (NECC) and named as NECCFP001. Pathogenicity was tested in the nursery, by merely placing healthy 40-day-old plants next to diseased plants (n = 10, with a 15 cm distance between plants), and by pressing the mildew sites of diseased leaves onto each mature leaf of the healthy plants (n = 10, also with 15 cm distance). The same symptoms appeared on heathy plants after 10 to 15 days, and 6 to 8 days, respectively. The induced mildew was confirmed morphologically to be NECCFP001 by microscopic examination. E. adunca occurs on aspen, willow, and Populus spp. throughout temperate regions of the Northern Hemisphere and forms fruiting bodies (chasmothecia) to overwinter (Horst 2001; Sinclair and Lyon 2005). However, chasmothecia were not observed in our case, suggesting no sexual morph under the nursery conditions. Incidents of powdery mildew on hybrid poplars caused by Phyllactinia populi had been reported in Jiangsu and Shandong provinces of China (Zhao et al. 2013). This is the first report of E. adunca causing poplar powdery mildew in Heilongjiang, China.

A Molecular Phylogenetic Study of Clarohilum henningsii (Mycosphaerellaceae, Fungi) on Cassava from Indonesia based on the ITS rDNA Sequence

Leaf spot is a common disease of cassava in Indonesia and other tropical countries. The present study aims to determine two isolates isolated from leaf spot of cassava in Indonesia using a molecular phylogenetic analysis based on internal transcribed spacer (ITS) rDNA sequence and morphological examination. The results showed that the two isolates belong to Clarohilum henningsii. This study revealed the phylogenetic position of C. henningsii from Indonesia, where in the previous studies, it has been reported as Cercospora cassavae, C. manihotis, C. henningsii, or Passalora henningsii based on morphological examinations.

First find of Cadophora antarctica Rodr.-Andrade, Stchigel, Mac Cormack & Cano in the Arctic

Cadophora antarctica Rodr.-Andrade, Stchigel, Mac Cormack & Cano was isolated from spoil tip of coal mine in the Arctic, on the territory of the Svalbard archipelago, and is represented by strain IVA-206. Macro- and micromorphology of the isolate were examined along with partial sequences of Internal transcribed spacer rDNA region (ITS1-5.8S-ITS2) and D1/D2 region of 28S rDNA (LSU). The isolate C. antarctica IVA-206 had a number of features that distinguished it from the strain C. antarctica CBS 143035 from Antarctica. Colonies of Arctic strain had darker pigmentation, ramoconidia and conidia were larger, and the optimal growth temperature was higher. As a result of our study, we first discovered the microfungi C. antarctica Rodr.-Andrade, Stchigel, Mac Cormack & Cano in the Arctic. Our study shows that C. antarctica Rodr.-Andrade, Stchigel, Mac Cormack & Cano is a bipolar species found in both the Arctic and Antarctic region.

Problems, Limitations, and Challenges in Species Identification of Ascomycota Members on the Basis of ITS Regions

The internal transcribed spacer (ITS) region is regarded as a formal fungal primary barcode with a high probability of the correct identification for a broad group of fungi. ITS sequences have been widely used to determine many fungal species and analysis of rDNA ITS is still one of the most popular tools used in mycology. However, this region is not equally variable in all groups of fungi; therefore, identification may be problematic and result in ambiguous data, especially in some species-rich genera of Ascomycota. For these reasons, identification based on rDNA ITS is usually complemented by morphological observations and analysis of additional genes. Reliable species identification of Ascomycota members is essential in diagnosing plant diseases, verifying air quality and the effectiveness of agronomic practices, or analyzing relationships between microorganisms. Therefore, the present study aimed to verify, using specific examples, the extent to which ITS sequence analysis is useful in species identification of pathogens and saprobionts from Ascomycota and demonstrate problems related to such identification in practice. We analyzed 105 ITS sequences of isolates originating from air and plant material. Basic local alignment search tool (BLASTn) significantly contributed to the reliable species identification of nearly 80% of isolates such as <em>Arthrinium arundinis</em>, <em>Beauveria bassiana</em>, <em>Boeremia exigua</em>, <em>Cladosporium cladosporioides</em>, <em>Epicoccum nigrum</em>, <em>Nigrospora oryzae</em>, <em>Sclerotinia sclerotiorum</em>, or <em>Sordaria fimicola </em>and members of the genera <em>Alternaria </em>and <em>Trichoderma</em>. However, for most isolates, additional morphological observations, information regarding the isolate origin and, where possible, a PCR with species-specific primers were helpful and complementary. Using our practical approach, we determined that ITS-based species identification and comparative analysis with GenBank sequences significantly helps identifying Ascomycota members. However, in many cases, this should be regarded as suggestive of a taxon because the data usually require the use of additional tools to verify the results of such analysis.

The true nature of Ganoderma in Iran: Taxonomy based on ITS and mtSSU rDNA

AbstractThe genus Ganoderma Karst. has broad‐spectrum usage in biotechnology, medicine and the food industry. The complexity of the morphology within species has led to uncertain identification in the past, but recent advancements in molecular identification methods have provided scientists with the opportunity to better understand the taxonomy of the species. The present study attempts for the first time to elucidate the distinctiveness of the Ganoderma species growing in Iran concerning those elsewhere in the world based on mitochondrial small subunit ribosomal DNA (mtSSU rDNA) and internal transcribed spacer (ITS) sequence data. The results disclosed that the G. lucidum Karst. samples collected in Iran are more similar to the European Ganoderma species than to the Asian (Chinese) one used in this study.

Phylogenetic placement of Tritirachium strains from the URM culture collection originally founded by Augusto Chaves Batista (1916-1967) in Brazil, and the description of T. batistae sp. nov.

Twenty-seven Tritirachium strains were present in the URM culture collection originally founded in Brazil by Augusto Chaves Batista. Fifteen freshly-prepared cultures were obtained from these original strains preserved under mineral oil. DNA was extracted for analysing phylogenetic relationships using the sequence information available from Tritirachium type materials and reference strains. Phylogenetic analysis using the internal transcribed spacer (ITS) ribosomal DNA sequences revealed that eight of these strains belong to the same clade as of T. oryzae and that the four other strains belong to the same clade as of T. candoliense. The strain URM 38, which was previously identified as “T. brumptii”, appeared to exist as a single lineage, related to T. roseum and T. candoliense. Based on morphological features and multi-locus phylogenetic analysis, including the analyses of ITS and LSU rDNA, and rpb2 sequences, we propose that URM 38 belongs to the new species T. batistae. This novel species exhibited velutinous to cottony colonies of varying colour, septate hyphae without clamp connections, conidiophores reduced to conidiogenous cells, conidiogenous cells with a distinct sympodial rachis, and single-cell conidia that was globose to subglobose, obovoid, smooth, and hyaline. The morphological features of species accepted in Tritirachium are included in this study.

Role of Root and Stem Base Fungi in Fraxinus angustifolia (Vahl) Dieback in Croatian Floodplain Forests

Large-scale ash (Fraxinus spp.) dieback caused by the fungus Hymenoscyphus fraxineus has been a major concern throughout Europe for more than two decades. Most of the related research has been focused on Fraxinus excelsior L., and there is still little information on fungal involvement in the dieback of Fraxinus angustifolia Vahl, especially in roots and stem bases, which play an important role in decline progress and tree stability. The objectives of this study were to identify fungi present in visually healthy and symptomatic wood tissues in basal parts of narrow-leaved ash trees in different decline phases, in order to determine the possible role of these fungi and their importance in the dieback process. The stem bases and roots of 90 trees in three different health categories, determined based on crown defoliation, were sampled in natural stands affected by ash dieback. Isolated fungal cultures were identified based on the rDNA ITS (Internal transcribed spacer) region and their association with tree health status was analyzed. In total, 68 different fungal taxa were confirmed, including Hymenoscyphus fraxineus and Armillaria spp., which were mainly present in roots, although overall in lower frequencies than on common ash in other studies. Most frequently isolated fungal taxa, which encompassed 51% of all obtained isolates, were Trichoderma spp., Ilyonectria robusta, Fusarium solani, Cladosporium cladosporioides, and Diaporthe cotoneastri. Their associations with tree health categories and presence in both symptomatic and visually healthy wood indicate that they act as opportunistic pathogens and early colonizers of weakened ash tissues. Research also revealed that, although the extent of crown defoliation and presence of root and stem necroses were associated, basal symptoms occurred on a number of trees with healthy looking crowns, meaning they can develop independently.

Characterization of Leaf Spot Pathogens from Several Spinach Production Areas in the United States

Leaf spot diseases have become a major concern in spinach production in the United States. Determining the causal agents of leaf spots on spinach, their prevalence and pathogenicity, and fungicide efficacy against these pathogens is vital for effective disease management. Spinach leaves with leaf spots were collected from Texas, California, Arizona, and South Carolina from 2016 to 2018, incubated in a moist chamber, and plated on potato dextrose and tryptic soy agar media. Fungal and bacterial colonies recovered were identified based on morphology and sequence analysis of the internal transcribed spacer rDNA and 16S rRNA, respectively. Two predominant genera were isolated: (i) Colletotrichum spp., which were identified to species based on sequences of both introns of the glutamate synthetase (GS-I) and glyceraldehyde-3-phosphate dehydrogenase (gapdh-I) genes; and (ii) Stemphylium spp., identified to species based on sequences of the gapdh and calmodulin (cmdA) genes. Anthracnose (Colletotrichum spinaciae) and Stemphylium leaf spot (Stemphylium vesicarium and S. beticola) were the predominant diseases. Additional fungi recovered at very limited frequencies that were also pathogenic to spinach included Colletotrichum coccodes, C. truncatum, Cercospora beticola, and Myrothecium verrucaria. All of the bacterial isolates were not pathogenic on spinach. Pathogenicity tests showed that C. spinaciae, S. vesicarium, and S. beticola caused significant leaf damage. The fungicides Bravo WeatherStik (chlorothalonil), Dithane F-45 (mancozeb), Cabrio (pyraclostrobin), and Merivon (fluxapyroxad and pyraclostrobin) were highly effective at reducing leaf spot severity caused by an isolate of each of C. spinaciae and S. vesicarium, when inoculated individually and in combination.

Peronospora monardae, Hyaloperonospora daughtreyae and H. iberidis: new species associated with downy mildew diseases affecting ornamental plants in the United States

Species in the genera Hyaloperonospora and Peronospora (Oomycota, Peronosporaceae) are obligate biotrophic pathogens responsible for destructive downy mildew diseases of ornamental horticulture plants worldwide. Throughout the past decade, newly emergent downy mildew diseases have become increasingly prevalent, but often the identity of the pathogen is unknown or poorly defined. In this study we set out to identify the downy mildew pathogens infecting three widely grown ornamental plants: Agastache spp. (hyssops), Tarenaya hassleriana (syn. = Cleome hassleriana; spider-flower) and Monarda didyma (bee balm or bergamot). Phylogenetic analysis of the internal transcribed spacer rDNA and cox2 revealed two new species, which are described here as Peronospora monardae infecting both Monarda didyma and A. mexicana, and Hyaloperonospora daughtreyae infecting T. hassleriana. One sample of Peronospora collected from Agastache sp. ‘Bolero’ did not cluster with P. monardae, but its relationship to other species in the clade was not sufficiently resolved for taxonomic determination. Phylogenetic analysis also supported the combination of Peronospora iberidis, a pathogen of Iberis spp. (candytuft), into Hyaloperonospora. To assess intrasample ITS sequence variation from P. monardae, data were generated using high-throughput amplicon sequencing (HTAS). Multiple haplotypes were identified from the P. monardae samples, with less than 1% intrasample variation observed. From a core set of haplotypes shared across the HTAS the variant frequencies could not be completely explained by heterozygosity alone.

Low internal transcribed spacer rDNA variation in New Zealand Bonamia ostreae: evidence for a recent arrival.

Bonamia ostreae is a haplosporidian parasite of oysters that was first reported to occur in the Southern Hemisphere in 2015 in the New Zealand flat oyster Ostrea chilensis. Until that report, B. ostreae had been restricted to populations of O. edulis within the Northern Hemisphere. This large range extension raised questions regarding B. ostreae dispersal, including whether B. ostreae is a recent introduction and from where it originated. The whole 18S rRNA gene of New Zealand B. ostreae revealed 99.9-100% sequence homology to other published B. ostreae 18S rDNA sequences. Internal transcribed spacer (ITS) rDNA sequences (n = 29) were generated from New Zealand B. ostreae and compared to published B. ostreae sequences from 3 Northern Hemisphere sites: California, USA (n = 18), Maine, USA (n = 7), and the Netherlands (n = 6) to investigate intraspecific variation. Low ITS rDNA variation was observed from New Zealand B. ostreae isolates, and high levels of variation were observed from Northern Hemisphere B. ostreae sequences. We hypothesise that the low ITS rDNA diversity found in New Zealand B. ostreae is the result of a founder effect resulting from a single introduction from a limited number of propagules. The high level of ITS rDNA variation from the Northern Hemisphere prevented inferences of dispersal origins. New Zealand B. ostreae were genetically differentiated from all sites, and additional genetic data are required to better determine the origin of B. ostreae in New Zealand.

Isolation, Characterization and Targeted Metabolic Evaluation of Endophytic Fungi Harbored in 14 Seed-Derived Hypericum Species.

Medicinal plants of the genus Hypericum are rich sources of bioactive naphthodianthrones, which are unique in the plant kingdom, but quite common in fungal endophytes. Cultivable endophytic fungi were isolated from 14 different Hypericum spp. originating from seeds grown under in vitro conditions and further acclimated to outdoor conditions. Among 37 fungal isolates yielded from the aerial and underground plant organs, 25 were identified at the species level by the fungal barcode marker internal transcribed spacer rDNA and protein-coding gene region of tef1α. Ten of them were isolated from Hypericum spp. for the first time. The axenic cultures of the isolated endophytes were screened for the production of extracellular enzymes, as well as bioactive naphthodianthrones and their putative precursors by Bornträger's test and HPLC-HRMS. Traces of naphthodianthrones and their intermediates, emodin, emodin anthrone, skyrin, or pseudohypericin, were detected in the fungal mycelia of Acremonium sclerotigenum and Plectosphaerella cucumerina isolated from Hypericum perforatum and Hypericum maculatum, respectively. Traces of emodin, hypericin, and pseudohypericin were released in the broth by Scedosporium apiospermum, P. cucumerina, and Fusarium oxysporum during submerged fermentation. These endophytes were isolated from several hypericin-producing Hypericum spp. Taken together, our results reveal the biosynthetic potential of cultivable endophytic fungi harbored in Hypericum plants as well as evidence of the existence of remarkable plant-endophyte relationships in selected non-native ecological niches. A possible role of the extracellular enzymes in plant secondary metabolism is discussed.

Molecular identification of rust disease on Acacia mangium collected from West Java, Indonesia

Acacia mangium is one of wood producing species widely planted in industrial and small scale forest. The only rust disease that has been reported on A. mangium is the phyllode rust caused by Endoraecium digitatum (syn. Atelocauda digitata). Some revision had been made for E. digitatum and some new species had been proposed on acacias. However, the information for Indonesian isolates is still limited. We found rust disease on A. mangium plantation in Tasikmalaya, West Java that had not been reported previously. Morphological identification is quite difficult to determine the species of those rust pathogen. Therefore, a molecular approach is needed. This study was aimed to identify the rust disease of A. mangium based on sequence data of internal transcribed spacer rDNA. The DNA samples of rust pathogen were isolated using CTAB methods followed with ITS amplification using specific rust primer and sequenced. BLAST analysis based on ITS fragments showed that rust pathogen has 98% identity to the E. auriuliforme. Meanwhile phylogenetic analysis showed that the rust pathogen on A. mangium was more closely related to the E. auriculiforme.

Mollisiaceae: An overlooked lineage of diverse endophytes.

Mollisia is a taxonomically neglected discomycete genus (Helotiales, Leotiomycetes) of commonly encountered saprotrophs on decaying plant tissues throughout temperate regions. The combination of indistinct morphological characters, more than 700 names in the literature, and lack of reference DNA sequences presents a major challenge when working with Mollisia. Unidentified endophytes, including strains that produced antifungal or antiinsectan secondary metabolites, were isolated from conifer needles in New Brunswick and placed with uncertainty in Phialocephala and Mollisia, necessitating a more comprehensive treatment of these genera. In this study, morphology and multigene phylogenetic analyses were used to explore the taxonomy of Mollisiaceae, including Mollisia, Phialocephala, and related genera, using new field collections, herbarium specimens, and accessioned cultures and sequences. The phylogeny of Mollisiaceae was reconstructed and compared using the nuc internal transcribed spacer rDNA (ITS) barcode and partial sequences of the 28S nuc rDNA (LSU) gene, largest subunit of RNA polymerase II (RPB1), DNA topoisomerase I (TOP1), and the hypothetical protein Lipin/Ned1/Smp2 (LNS2). The results show that endophytism is common throughout the Mollisiaceae lineage in a diverse range of hosts but is infrequently attributed to Mollisia because of a paucity of reference sequences. Generic boundaries within Mollisiaceae are poorly resolved and based on phylogenetic evidence the family included species placed in Acephala, Acidomelania, Barrenia, Bispora, Cheirospora, Cystodendron, Fuscosclera, Hysteronaevia, Loramyces, Mollisia, Neopyrenopeziza, Obtectodiscus, Ombrophila, Patellariopsis, Phialocephala, Pulvinata, Tapesia (=Mollisia), and Trimmatostroma. Taxonomic novelties included the description of five novel Mollisia species and five novel Phialocephala species and the synonymy of Fuscosclera with Phialocephala, Acidomelania with Mollisia, and Loramycetaceae with Mollisiaceae.