Internal Transcribed Spacer Region Research Articles

First report of Colletotrichum scovillei causing anthracnose fruit rot of pepper in Ohio.

In August 2019, the Ohio State University Vegetable Pathology laboratory received multiple bell and banana pepper fruits (Capsicum annuum, cvs. unknown) from Columbiana County, Ohio. The grower reported a disease incidence of 100% and severity of 70% in fruits across their pepper fields. Fruit lesions were brown, sunken, and covered with orange-colored sporulation. On banana peppers, the lesions mainly affected the blossom end of the fruits, while the lesions were distributed randomly on bell pepper fruits. Pieces of diseased tissue were cut from the fruit and surface sterilized in 0.5-0.6 % sodium hypochlorite, rinsed in sterile water, blotted dry, and placed on potato dextrose agar. All of the fungal cultures recovered were cottony, pale gray-green with shades of orange on the underside of the mycelial mat. Two representative isolates, SM209-19 (bell pepper) and SM210-19 (banana pepper), were grown on oatmeal agar to induce sporulation. Pink-orange concentric rings containing acervuli and conidia were present on the oatmeal agar plates after one week of growth at 22◦C (12-h dark/light). Conidia (n=29) were hyaline, aseptate, cylindrical in shape, and had an average length of 10.5 µm (std. dev. = 1.3 µm) and width of 4.1 µm (std. dev. = 0.6 µm) (Fig.1). DNA was extracted from both isolates using a DNeasy Plant Kit (Qiagen Inc, Germantown, MD), and partial sequences of the internal transcribed spacer (ITS) region, -tubulin 2 gene (TUB2), and glyceraldehyde-3-phosphate dehydrogenase gene (GDPH) were amplified by PCR with the following primers: ITS4/ITS5 (White et al. 1990), Bt1a/Bt1b (Glass et al. 1995), and GDF1/GDR1 (Guerber et al. 2003), and squenced. The ITS region of both isolates SM209-19 and SM210-19 (PP280815 and PP280816, respectively) showed 100% identity with C. scovillei (Cs) isolate LJTJ35 (KP748226). The partial sequences of GDPH, (PP320348, PP320349, respectively) showed 99% sequence identity with the Cs CBS 126528 (JQ948597) and 100% identity with Cs HP1 (MT826948) The partial sequences of TUB2 (PP472464 and PP472465, respectively) had 100% sequence similarity with the Cs HP1 and Cs CBS 126528 (MT826951, JQ949918 respectively). Pathogenicity was tested in a greenhouse experiment on blossoming bell pepper plants (cv. Carmen) by spraying 10 ml of 1 X 105 conidia/ml suspension onto flower blooms (nine plants per isolate). Control pepper plants were mock inoculated by spraying 10 mL of sterile deionized water. The plants were re-inoculated one week later. Brown, sunken lesions with orange sporulation developed on the fruits of inoculated plants 21 days after the initial inoculation (Fig. 2), while the mock-inoculated plants did not produce any symptoms. Culturing from symptomatic fruits on PDA, following the same method described above, produced fungal colonies with the same morphological traits previously described. C. scovillei causing anthracnose on pepper has been described in the US (Toporek et al. 2021), Brazil (Caires et al. 2014), China (Zhao et al. 2016), and different South Asia Countries (Khalimi et al. 2019). Open-field peppers are produced in Ohio on more than 5,400 acres, with a value of more than $53 million, with anthracnose being one of the most severe fungal diseases reducing yield. This newly reported Colletotichum species could represent a further threat for this crop. Further studies evaluating fungicide sensitivity and efficacy against this pathogen will be of crucial importance for disease management.

Myxotrichum persicum sp. nov., and two new combinations in the genus Myxotrichum

We describe Myxotrichum persicum sp. nov. as a seed endophyte of Aegilops triuncialis in Lorestan province of Iran, using morphological traits and sequences of the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS) and partial nuclear 28S ribosomal DNA (LSU) regions. Myxotrichum persicum is differentiated from its closest relative, M. deflexum, by possessing smaller ascomata bearing much shorter, usually unbranched appendages, and smaller, ellipsoidal to fusiform, smooth-walled ascospores. Based on morphology and the multilocus phylogeny of ITS, LSU, MCM7 and RPB1, we herein propose to combine two species of Malbranchea, namely M. circinata and M. flavorosea, into the genus Myxotrichum as Myxotrichum circinatum and M. flavoroseum, respectively.

Fungal Trunk Diseases Causing Decline of Apricot and Plum Trees in the Czech Republic.

Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer region, partial beta-tubulin, and translation elongation factor 1-α genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31%), followed by Biscogniauxia (13%), Thelonectria (10%), Eutypa (9%), Dothiorella (7%), Diplodia (6%), and Diaporthe (6%). The most frequent endophytic genus was Aposphaeria (17%). The pathogenicity of six fungal species (Cadophora daguensis, Collophorina africana, Cytospora sorbicola, Dothiorella sarmentorum, Eutypa lata, and E. petrakii var. petrakii) to four Prunus spp. was evaluated, and Koch's postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata, which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii and D. sarmentorum. Japanese plum (Prunus salicina) and almond (P. amygdalus) were the most susceptible hosts, while apricot (P. armeniaca) was the least susceptible host in the pathogenicity trial.

Survey of potential fungal antagonists of Coffee Leaf Rust (Hemileia vastatrix) on Coffea arabica in Hawai'i, USA.

Hemileia vastatrix, causal agent of coffee leaf rust (CLR), is an aggressive pathogen of coffee plants worldwide. Conventional fungicides play a major role in the suppression of this disease, but a recent shift toward eco-friendly farming practices has occurred and additional novel, effective, and sustainable strategies for CLR control are needed. Naturally occurring fungal antagonists could be well-positioned to meet this demand, but these fungi need to be isolated and tested for efficacy to identify organisms with potential. In this study, a survey of fungi associated with CLR lesions in four districts of Hawai'i Island, HI, USA (Kona, Ka'ū, Hāmākua, and Hilo) was conducted. Coffee leaves infected with CLR were collected from 22 locations and over 600 lesions were plated on ½ APDA and CTC 4T media. DNA was extracted from purified isolates and the internal transcribed spacer region (ITS) was sequenced and analyzed by BLASTn. In total, 194 isolates comprising 50 taxa were recovered. Several of the genera are known antagonists of CLR or other plant pathogens, including Simplicillium, Akanthomyces, Cladosporium, Fusarium, and Clonostachys. The wide diversity of fungi associated with CLR lesions provide a wealth of possibilities for identifying potential CLR antagonists that could serve as a valuable tool for coffee farmers as part of an integrated pest management plan.

Loop‐mediated isothermal amplification (LAMP) assays for Aedes and Culex species and evaluation of a simple DNA preparation method for field application

AbstractThe spatial pattern of many pathogens that are transmitted by mosquitoes (Diptera: Culicidae) is changing due to globalization and climate change. Thus, surveillance of mosquito vectors is becoming increasingly widespread as basis for risk assessment and control. Species identification by loop‐mediated isothermal amplification (LAMP) assays is simple, fast and reliable. Specific primers for several mosquito species are available (Aedes aegypti, Aedes albopictus, Aedes geniculatus, Aedes japonicus, Aedes koreicus, species of the Anopheles funestus group and An. gambiae complex). In the present work, LAMP assays targeting the rDNA internal transcribed spacer region were developed for Ae. cretinus, a minor sister taxon of Ae. albopictus, and for Culex pipiens/Culex quinquefasciatus and Culex torrentium. The specificities of the primers designed in silico were confirmed by in vitro tests with DNA form non‐target species. Further, the release of DNA from mosquito stages (eggs, larvae, pupae, adults) was investigated, revealing that an incubation for 5 min at 80°C in water is suitable for LAMP. With this method, one specimen (egg, larva, pupa, adult) of a target species could be detected among 49 non‐targets. Thus, the assays are suitable for fast and reliable identification of mosquito species of all life stages by colour change visible to the naked eye, and they are operable under field conditions.

Endophytic species of Nigrospora from grasses and shrubs of Shadegan International Wetland, with new species and records from Iran.

The"Shadegan International Wetland" (SIW) is one of the wetlands internationally recognized in the Ramsar convention. The vegetation of this wetland ecosystem consists of mostly grasses and shrubs that host a large number of fungi including endophytes. In this study, Nigrospora isolates were obtained from healthy plants of this wetland and its surrounding salt marshes and identified based on morphological features and multilocus phylogenetic analyses based on three DNA loci, namely the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS), β-tubulin (tub2), and elongation factor 1-α (tef1-α). Accordingly, the following Nigrospora species were identified: N. lacticolonia, N. oryzae, N. osmanthi, N. pernambucoensis and a novel taxon N. shadeganensis sp. nov., which is described and illustrated. To the best of our knowledge, 10 new hosts for Nigrospora species are here reported, namely Aeluropus lagopoides, Allenrolfea occidentalis, Anthoxanthum monticola, Arthrocnemum macrostachyum, Cressa cretica, Halocnemum strobilaceum, Seidlitzia rosmarinus, Suaeda vermiculata, Tamarix passerinoides, and Typha latifolia. Moreover, the species N. lacticolonia and N. pernambucoensis are new records for the mycobiota of Iran.

Exophiala amphawaensis sp. nov., a novel black yeast isolated from the gut of amphipods in Thailand.

The genus Exophiala is polymorphic, able to transition between yeast, hyphal and pseudohyphal forms. Species of the genus Exophiala are ubiquitous fungi that are distributed in various environments around the world. During a survey of fungal diversity in the gut of amphipods (Floresorchestia amphawaensis and undescribed Dogielinotid amphipods) from the Amphawa estuary, Samut Songkhram province, Thailand, five black yeast strains (DMKU-MG01, DMKU-MG07, DMKU-MG08, DMKU-HG10 and DMKU-FG04) were identified as representing a novel taxon on the basis of a combination of morphological and molecular phylogenetic features. The five strains did not produce filamentous hyphae or pseudohyphae. Only budding yeast cells were observed. On the basis of the phenotypic characteristics and the results of molecular analyses of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the five strains were identified as representing a novel species via applied nucleotide pairwise analysis. They differed from the most closely related species Exophiala alcalophiala by 3.54 % nucleotide substitutions (20 nucleotide substitutions in 572 bp) in the D1/D2 domains of the LSU rRNA gene. Moreover, the sequences of the ITS region of the five strains differed from those of the most closely related species E. alcalophiala, by 7.44-9.62 % nucleotide substitutions, and Exophiala halophiala, by 7.2-7.53 % nucleotide substitutions. The results of phylogenetic analyses based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene confirmed that the five black yeast strains represented a single novel species of the genus Exophiala. In this study, Exophiala amphawaensis sp. nov. is proposed to accommodate these strains. The holotype is TBRC 15626T and the isotype is PYCC9020. The MycoBank accession number of the novel species is MB 851477.

Occurrence of Stem Rot on Acacia mangium Caused by Amauroderma subresinosum.

Acacia mangium has the characteristics of developed root system, nitrogen fixation and soil improvement, fast growth and high yield, and improvement of soil fertility. It is often used as a windbreak tree species in rubber plantations, a highway shade tree, for coastal and mountain restoration in Hainan . In October 2021, a stem rot disease with an incidence of 3% was found in Baisha city(19°22'18″N,109°16'58″E), Hainan Province, China. In the early stage of the disease, the crown showed chlorotic leaves, followed by defoliation. In later stages, whole tree dieback was observed. The basal tissue of the stem of the diseased tree had white rot, and black-brown basidiocarps were observed about 1 m away from the ground. The basidiocarps surface of fresh was disinfected with 75 % ethanol, the epidermal tissue was removed, and the inner tissue blocks were transferred to PDA medium. After culturing in dark at 28°C for 3 days, a colony with white aerial mycelium was isolated and designated: HNBSMZXS20211011001. The basidiocarp was dark brown, sessile, mostly one-year-old, and the cap is nearly semi-circular, wooden, slightly shiny, with a size of 14.7 to 18.1cm × 9.5 to 10.1 cm. The base is thick (5 to 6.5 cm) and the edge is thin (0.3 to 0.7 cm). The basidiospores are oval, 10.7 to 13.65μm × 6.7 to 9.06μm in size, with a double-layer wall. The outer wall is transparent and the inner wall is light yellow. The basidiospores contain 1~2 oil droplets. The morphological features are consistent with those of Amauroderma subresinosum (Murrill) Corner (Zhang et al., 2000). The basidiocarps and type strain cultures were stored as accessions in the Laboratory of Plant Pathogen Fungus Biology, Hainan University. For pathogenicity tests, sawdust culture medium was used (soft sawdust 82%, wheat bran 15%, glucose 2%, gypsum 1%, mixed with water in proportion, sterilized at 121°C for 40min). The mycelium plug from a fresh culture (d=5mm) was taken from the edge of the colony of type strain, and transferred to the sterilized sawdust medium. When mycelium has colonized the media, it was used to inoculate plants. Media without mycelium was used as a control. Naturally growing seedlings (three year old) of A.mangium were selected from the teaching nurseries of Hainan University (20°6'25''N,110°32'24''E). First, 75 % alcohol was sprayed on the stem of the base of A.mangium for surface disinfection. After the surface was dried, a slight wound (about 4×2cm) was made on the surface with a sterilized scalpel. A inoculated and control sawdust media rods were tightly attached to the wound, moistened with cotton balls soaked in sterile water, and then fixed with plastic wrap, and the outer layer was wrapped with newsprint. Inoculation and controls were replicated three times. Two months after inoculated, the stems of the plants inoculated with the isolated fungus grew white hyphae and showed white rot symptoms, and the leaves became chlorotic and defoliated with complete tree decline in six months, which was consistent with the original symptoms observed. By comparison, white callus had grown on the edge of the stem wounds of the control plants. The same fungus was re-isolated from the inoculated plants and confirmed as A.subresinosum based on the internal transcribed spacer (ITS), the ribosomal large subunit(LSU), and the translation elongation factor 1-α(EF1-α) gene sequence, the fungus was not isolated from control plants thus fulfilling Koch's postulates. The ITS region of r-DNA, the ribosomal large subunit(LSU), the translation elongation factor 1-α gene(EF1-α) were amplified using ITS1/ITS4(White et al. 1990), LR0R/LR5(Hu et al. 2021), EF1-983F/EF1-1567R(Buckley et al. 2005) primers, respectively. The sequences of ITS (OQ674500), LSU (OQ674502) and EF1-α gene (OQ883944) were submitted to GenBank. Through with BLAST, the identities of the ITS, LSU and EF1-α sequences to A.subresinosum (GenBank Accession no. ITS: LC176755; LSU: MK119903 and EF1-α: MK121572) was 99.82%; 99.15% and 99.82%, respectively, the identities were more than 99 %. It was reported that A.subresinosum could infect Casuarina equisetifolia and Areca catechu(Chen et al., 2016; Cheng. 2017; Wu et al., 2019). However, this is the first report of Amauroderma subresinosum causing stem rot of Acacia mangiumin Hainan, China. This report will facilitate field diagnosis and provide scientific reference for further research on the disease.

Wickerhamiella lachancei f.a. sp. nov., a novel ascomycetous yeast species isolated from flowers of Lantana camara in India.

Two isolates representing a novel species of the genus Wickerhamiella were obtained in India from nectar of flowers of Lantana camara, an ornamental exotic species native to Central and South America. Phylogenetic analyses of the D1/D2 domain of the 26S large subunit (LSU) rRNA gene, internal transcribed spacer (ITS) region, and physiological characteristics, supported the recognition of the novel species, that we designate Wickerhamiella lachancei sp. nov (MycoBank no. MB851709), with MCC 9929T as the holotype and PYCC 10003T as the isotype. Considering pairwise sequence similarity, the type strain of the novel species differs from the type strain of the most closely related species, Wickerhamiella drosophilae CBS 8459T, by 16 nucleotide substitutions and two gaps (3.9 % sequence variation) in the D1/D2 region (560 bp compared) and 28 nucleotide substitutions and five gaps (7.22 % sequence variation) in the ITS region (444 bp compared).

Lambertella (Rutstroemiaceae, Helotiales) from Northern Thailand

ABSTRACT During our surveys for discomycetes from 2019 to 2021 in forests and plantations in northern Thailand, several lambertella-like specimens were found. Morphological observation and BLAST search confirmed that seven newly collected specimens belong to Lambertella. Further phylogenetic analyses using maximum likelihood and Bayesian inference analysis based on combined the internal transcribed spacer region of rDNA (ITS), large subunit region of 28S rRNA (LSU), small subunit region of 18S rRNA (SSU) and RNA polymerase II gene (RPB2) sequence data and morphological examination coupled with chemical reactions confirmed seven Lambertella species. Lambertella aurantiaca and L. himalayensis were established as new geographical and host records, and the other five specimens, L. chiangraiensis, L. fusoidea, L. phanensis, L. sessilis and L. takensis, were introduced as novel species. The highlight of the current study is the contribution of the morphological description for Lambertella aurantiaca since its introduction in 1964, emending the morphological criteria for Lambertella sensu stricto, and providing sequence data for all Lambertella species described in the current study.

Prevalence and molecular characterization of Hysterothylacium species infecting Pandora (Pagellus erythrinus) in the Mediterranean Sea of Egypt

Species of the genus Hysterothylacium are aquatic roundworms (nematodes) belonging to the family Raphidascarididae. Some species in this family are known to be associated with zoonotic diseases in humans after they consume their parasitic larvae in raw or undercooked fish. The aim of this research was to report the prevalence, morphology, and molecular characteristics of Hysterothylacium species in Pagellus erythrinus. A total of Two hundred fish were purchased from the fish market in Damanhour, Beheira Province, between December 2021 and November 2022 and subjected to examination. For molecular characterization, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA and the mitochondrial cytochrome oxidase subunit 2 (COX-2) gene were used. Hysterothylacium species were morphologically described and identified from the intestine of Pagellus erythrinus in Beheira Province, Egypt. The PCR amplified 1087 bp and 629 bp of the target sequences of the ITS region and COX-2 gene, respectively. Sequence analysis revealed the Hysterothylacium thalassini species. The identified species provided novel biological data for the Hysterothylacium nematode in Pagellus erythrinus. The prevalence of Hysterothylacium species recovered from the intestine was 55%. The highest prevalence of 72% has been reported in summer compared to the lowest prevalence of 38% in the winter. Females had a higher prevalence of 61.8% than males, with 44.2%. The first detection, prevalence, and molecular characterization of H. thalassini in Pagellus erythrinus from Beheira Province, Egypt, was presented in this study.

First Report on Apothecium Deformity of Morchella importuna Caused by Alternaria alternata in China.

Morel mushrooms (Morchella spp.) are highly regarded globally for their distinctive texture and savory flavor. In 2022, the cultivation area for morel mushrooms in China reached nearly 20,000 hectares, with predominant cultivars including M. sextelata, M. importuna and M. exima (Bian et al., 2024). In March 2022, however, deformities of friting bodies were observed in M. importna at morel mushroom farms in Huaihua city (28.43°N, 110.47°), China, with an incidence rate ranging from 5% to 10%. The disease symptoms begin with the invasion of the hymenium of morel mushroom by white cotton-like mycelia, ultimately resulting in halted fruiting body growth and the manifestation of anomalous fruiting body morphology. Infected samples were collected from the morel growers. Following sterilization with 75% ethanol of the surrounding tissue of infected samples, the white hyphae from the morel lesions were picked out using a dissecting needle, and incubated onto potato saccharose agar medium supplemented with 60 mg/L streptomycin at 25°C. Studies showed that seven out of nine fungal isolates exhibiting identical morphological features rapidly grew on the same culture medium described above, reaching a length of 75 mm in 4 to 5 days at 25°C. The white and thick hyphal colonies of these isolates gradually filled with brown spore powder. Generally, the conidia of the hyphal colonies were polyblastic with protrusions at the tips, measuring 75 to 165 × 36 to 50 μm (n = 30) in width and length, displaying colors varying from light reddish brown to grayish brown, and possessing one or five septa. To confirm the identity of the pathogen, the region of the internal transcribed spacer region (ITS), 28S nuclear ribosomal large subunit (LSU), and RNA polymerase II second largest subunit (rpb2) genes of the representative isolate H2 were amplified by PCR (Taguiam, et al. 2021). The generated ITS (OR338304), rpb2 (OR452112) and LSU (OR338334) from the isolate H2 had 98-100% similarity to the Alternaria alternata strains ATCC 6663 and CBS 880.95 in BLASTn analysis. ITS, rpb2 and LSU sequences were assembled using Sequence Matrix, and their homogeneity was assessed with PAUP (Vaidya et al., 2011). Bayesian (MrBayes-3.2.7a) and maximum-likelihood (RAxML1.3.1) methods, utilizing the best fit GTR+G+I model obtained from MrModeltest 2.3, were employed for phylogenetic analysis (Aveskamp et al. 2010). Based on morphological characteristics and phylogenetic analysis, the isolate H2 was identified as A. alternata. In the second year post-disease, disease-free morels, with a height of 3 cm, were cultivated in field greenhouses and used for test. A 15 ml suspension (1 × 106 conidia/ml) was applied to 15 young fruiting bodies and their corresponding substrate soil. The results showed that the reappearance of white cotton-like mycelia and deformed M. importuna fruiting bodies within 7 days post-inoculation with the spore suspension, as opposed to the controls. The isolates (H2-1, H2-2 and H2-3) were reisolated from the infected tissues and identified as A. alternata based on its morphological features and phylogenetic analyses. In this study, a similar investigation was previously conducted on cultivated quinoa (Chenopodium quinoa) in Eastern Denmark (Colque-Little et al., 2023). This study marks the first documentation of A. alternata causing deformities in M. importuna fruiting bodies. These deformities occur under conditions of high-temperature (>22°C) and high humidity (>88%). Our findings provide crucial insights for managing A. alternata in M. importuna cultivation in China.

Phenotypic Characteristics, Pathogenicity, and Molecular Identification of Hypomyces perniciosus Causing Wet Bubble Disease of Edible Mushrooms

Abstract The research aimed to determine the phenotypic characteristic, pathogenicity and molecular characterization of Hypomyces perniciosus isolates responsible for wet bubble disease of the white button mushroom Agaricus bisporus. Phenotypic characteristics such as colony appearance, mycelium texture, and pigmentation were studied on four different agar media, i.e., potato dextrose agar (PDA), malt extract agar (MEA), Czapek-Dox yeast agar (CYA), and Sabouraud dextrose agar (SDA), after eight days of incubation. Additionally, the growth rate of the tested isolates was studied depending on the pH of the medium. Fungal isolates showed the highest mycelial growth on MEA and SDA at pH 6.0. However, on CYA at pH 7.0, the mycelium exhibited the worst growth. Isolate identification and genetic relationship analysis were carried out using internal transcribed spacer region sequencing and the random amplified polymorphic DNA method. The research confirmed that all Polish isolates belong to the species H. perniciosus, and the genetic diversity is relatively low. Phylogenetic analyses revealed three subgroups of H. perniciosus isolates. The first group included three genetically distinct isolates with a similarity coefficient in the range of 0.76–0.85 to isolates CBS 815.73 and CBS 322.52. The second group was divided into two subgroups and included 16 isolates with a genetic similarity range of 0.91 to 1.0 to CBS 815.73 and CBS 322.52 isolates. Furthermore, the eight genetically similar isolates exhibited the greatest pathogenicity towards A. bisporus.

Functional ecology and evolution of terrestrial and epiphytic species of Rhododendron section Schistanthe (Ericaceae)

Epiphytes are often characterized by small size, specifically adapted to a relatively dry and nutrient-poor habitat. However, this epiphyte syndrome is derived from studies on species-rich and habitat-dominating groups such as orchids and bromeliads. To better understand general trait syndromes of epiphytes, it is important to analyze plant groups that are more transitional in the evolution of epiphytism e.g., the Australasian vireyas (Rhododendron sect. Schistanthe; Ericaceae). Here, we studied the morphology, ecophysiology, and anatomy of 44 species of Rhododendron section Schistanthe from living collections and obtained data for 288 species related to their "life form" (holoepiphytes [= obligatory epiphytes], facultative epiphytes and terrestrial congeners) from the literature. For phylogeny and time-calibrated tree, we used the nuclear ITS (Internal Transcribed Spacers) region. Using different statistics and phylogenetic comparative methods, our results support the hypothesis that holoepiphytic vireyas are smaller in size than the terrestrial ones. Terrestrial vireyas are found at higher minimum and maximum elevation than either facultative or holoepiphytic congeners. We demonstrate that the diversification of tropical Rhododendron is not related to the evolution of the epiphytic life form. Regarding the evolution of traits that are important for water economy, holoepiphytic vireyas do not differ much from their terrestrial relatives indicating that evolutionary steps for specialization towards an holoepiphytic habit are in their initial stages in Rhododendron. Given the large species diversity of the group conclusions based on the subset used here must be considered preliminary. However, our results provide the basis for more detailed future studies.

First Reports of Cladosporium tenuissimum Causing Leaf spots on Hydrangea macrophylla in Anhui province in China.

Forever Summer Hydrangea (Hydrangea macrophylla) is a common flowering plant in the Yangtze River Valley area of China, and it is widely cultivated globally (Chen et al. 2015). In July 2023, H. macrophylla leaves exhibiting visible diseased lesions were reported in a nursery in Wuhu, Anhui Province, China. The incidence reached 40% in a 0.2 ha area. The primary disease symptom was multiple irregular necrotic spots (0.5 to 1 mm in diameter) appearing on the leaves. These spots on the leaves were faded yellow around the perimeter and grayish brown in the center.). 15 leaf samples were sterilized with 75% alcohol and rinsed three times in sterile distilled water, then transferred to antibiotic-added potato dextrose agar (PDA) for incubation at 27°C. The colonies were fluffy, flocculent, or hairy, dark green, gray-green to gray-brown in color, and spreading or protruding punctate with a colorless halo on PDA. The conidiophores were brown to dark brown, smooth or rough surface, mostly unbranched, clearly differentiated, erect or curved. The conidia displayed a light brown to brown hue, lemon shape, fusiform, elongated ellipsoid or others with obvious spore markings and spore umbilicus. Genomic DNA was extracted from fungal colonies on infected leaves of three collections separately (Braun et al. 2003) and the internal transcribed spacer regions (ITS), actin (ACT) genes and partial translation elongation factor-l-alpha (EF) were amplified and sequenced using the primers ITS1/4 (Yin et al. 2012), ACT-512F/ACT-783R and EF 1-728F/986R (Carbone and Kohn 1999), respectively. DNA sequences of isolates were identical and deposited in GenBank (accession no. OR362754 for ITS, OR611929 for ACT and PP209106 for EF). The consensus sequences from ITS, EF and ACT showed 100%, 98.98% and 100% identical to Cladosporium strains (accession no. OQ186140.1, MT154169.1 and OL322092.1), respectively. To confirm the pathogenicity of the isolates, hydrangeas were planted in 15-cm pots containing commercial potting mix (one plant/pot). Three healthy plants were inoculated at the five to eight leaf stage by spraying 50 μL of the isolate conidial suspension (4 × 106 spores/mL) on healthy leaves. Three plants treated with sterile distilled water were used as controls. After inoculation, all plants were placed in a humidity chamber (>95% relative humidity, 26°C) for 48 h and then transferred to a greenhouse at 22/27°C. All inoculated leaves exhibited symptoms similar to those observed in the nursery 10 days after inoculation, while no symptoms were observed for control leaves. The fungus was re-isolated and confirmed to be C. tenuissimum. Based on the above morphological characterization and molecular identification, the causal agent for this leaf spot disease was identified as C. tenuissimum. Although C. tenuissimum has been reported to cause disease on H. paniculata in northern China (Li et al.2021), this is the first time that C. tenuissimum has been found on H. macrophylla in southern China. This new disease of H. macrophylla caused by C. tenuissimum is a threat to urban greening and is worth further investigation.

Conserved DNA sequence analysis reveals the phylogeography and evolutionary events of Akebia trifoliata in the region across the eastern edge of the Tibetan Plateau and subtropical China

BackgroundThe eastern edge of the Qinghai‒Tibet Plateau (QTP) and subtropical China have various regions where plant species originate and thrive, but these regions have been the focus of very few integrative studies. Here, we elucidated the phylogeographic structure of a continuous and widespread Akebia trifoliata population across these two regions.ResultsSixty-one populations consisting of 391 genotypes were examined to assess population diversity and structure via network distribution analysis, maximum likelihood phylogenetic tree reconstruction, divergence time estimation, demographic history inference, and ancestral area reconstruction of both conserved internal transcribed spacer (ITS) and chloroplast (rps16) DNA sequences. The results showed that the ITS region was more variable than the rps16 region and could be suitable for studying intraspecific phylogeography. The A. trifoliata population displayed high genetic diversity, genetic differentiation and obvious phylogeographical structure, possibly originating on the eastern QTP, expanding during the last glacial-interglacial cycle, diverging in the early Pleistocene and middle Pleistocene, and extensively migrating thereafter. The migration route from west to east along rivers could be largely responsible for the long-distance dispersal of this species, while three main refuges (Qinba Mountains, Nanling Mountains and Yunnan-Guizhou Plateau) with multiple ice shelters facilitated its wide distribution.ConclusionsOur results suggested that the from west to east long migration accompanying with the minor short reciprocal migration in the south-north direction, and the three main refuges (the Qinba Mountains, Nanling Mountains and Yunnan-Guizhou Plateau) contributed to the extant geographical distribution of A. trifoliata. In addition, this finding also strongly reduced the discrepancy between glacial contraction and postglacial expansion and the in situ survival hypothesis by simultaneously considering the existence of many similar climate-related ecological niches and migration influences.

First report of the barley root-knot nematode, Meloidogyne naasi Franklin, 1965 from Pennsylvania, with new host report.

Meloidogyne naasi Franklin, 1965, the barley root-knot nematode, was originally found in field crops such as cereals, grasses, and sugar beet (Beta vulgaris L.) in England and Wales, (Franklin,1965). This nematode is one of the most significant root-knot nematodes impacting grains in European countries (Santos et al. 2020). Among root-knot nematode species, M. naasi, exhibits a distinct preference for grasses, with documented impacts on turfgrasses leading to reduced growth and vigor (Skantar et al., 2023; Cook and Yeates, 1993). In September 2022, root-knot nematode females and second-stage juveniles (J2) were recovered from roots of fowl manna grass, Glyceria striata (Lam.) Hitchc., during a nematode survey on natural vegetation at the Allegheny National Forest (41°30'13.8"N 79°09'46.2"W). Second-stage juvenile specimens were recovered from soil using sugar centrifugal flotation (Jenkins, 1964). Small galls with egg masses were dissected from fowl manna grass roots originally collected from the surveyed areas. In parallel, five plants of non-infected fowl manna grass were placed in a pot in the greenhouse using naturally nematode-infested soil collected from the same forested area. Small galls and female specimens recovered from these plants were dissected and processed for further analyses. Female and J2 were fixed in 3% formaldehyde solution and processed to glycerin (Golden, 1990; Hooper, 1970). The specimens were examined by light microscopy, morphometric measurements, and molecular markers, which included the D2-D3 region of the large ribosomal subunit 28S, and the rDNA internal transcribed spacer region (ITS). The perennial pattern of five females analyzed morphologically were consistent to the patterns observed for M. naasi. The perennial patterns had coarse ridges on the cuticle in dorsal region forming broken irregular lines around anal and phasmid area. We also noted a prominent fold that covered some of the anus and showed a curved line between vulval slit and phasmids, typical of M. naasi. The area around the vulval area had a few or no striae except for a few lines radiating from the vulval slit as in the original description. Measurements of ten J2 had a body length ranged between 380 and 410 µm, stylet 11-13 µm, tail 50-70 µm long with a hyaline tail terminus between 12-22 µm in length, 4 lines in the lateral field, a and c ratio between 29.23-35.91 and 5.79-7.9 fitting the original description by Franklin, 1965 and others populations found in the USA (Skantar et al., 2023). The matrix codes for the female specimes are A32, B324, C3, D3 and for J2's A2, B21, C123, D1, E3, F12 (Subbotin et al., 2021). The amplified DNA fragments were sequenced, resulting in an 726 bp fragment flanked by the D2-D3 primers (PP097762), while for the ITS primers an 634 bp fragment was obtained (PP092043). Both generated sequences for the specimens collected in Pennsylvania revealed >99% similarity to M. naasi sequences deposited at GenBank, and therefore, validating the morphological analyses. Based on both morphological and molecular analyses the specimens collected in the state of Pennsylvania were identified as M.naasi. To our knowledge, this is the first report of this species from this state and being associated with naturally infected fowl manna grass.

Unique photosynthetic strategies employed by closely related Breviolum minutum strains under rapid short-term cumulative heat stress.

The thermal tolerance of symbiodiniacean photo-endosymbionts largely underpins the thermal bleaching resilience of their cnidarian hosts such as corals and the coral model, Exaiptasia diaphana. While variation in thermal tolerance between species is well documented, variation between conspecific strains is understudied. We compared the thermal tolerance of three closely related strains of Breviolum minutum represented by two internal transcribed spacer region 2 profiles (one strain B1-B1o-B1g-B1p and the other two strains B1-B1a-B1b-1g) and differences in photochemical and non-photochemical quenching, de-epoxidation state of photopigments, and accumulation of reactive oxygen species under rapid short-term cumulative temperature stress (26-40°C). We found that B. minutum strains employ distinct photoprotective strategies, resulting in different upper thermal tolerances. We provide evidence for previously unknown interdependencies between thermal tolerance traits and photoprotective mechanisms which include a delicate balancing of excitation energy and its dissipation through fast relaxing and state transition components of non-photochemical quenching. The more thermally tolerant B. minutum strain (B1-B1o-B1g-B1p) exhibited an enhanced de-epoxidation that is strongly linked to the thylakoid membrane melting point and possibly membrane rigidification minimising oxidative damage. This study provides an in-depth understanding of photoprotective mechanisms underpinning thermal tolerance in closely related strains of B. minutum.

Enterocytozoon bieneusi in Wild Rats and Shrews from Zhejiang Province, China: Occurrence, Genetic Characterization, and Potential for Zoonotic Transmission.

Globally, Enterocytozoon bieneusi has been detected in humans and various animal hosts. Wild rats and shrews have the potential to act as carriers of E. bieneusi, facilitating the parasite's transmission to humans and domestic animals. We aimed to investigate the prevalence of E. bieneusi in 652 wild rats and shrews from Zhejiang Province, China, by amplifying the internal transcribed spacer (ITS) region of rDNA through polymerase chain reaction (PCR). To determine animal species, we amplified the Cytochrome b (Cyt-b) gene in their fecal DNA using PCR. Furthermore, we determined the genotype of E. bieneusi by amplifying the ITS region of rDNA through PCR. Genetic traits and zoonotic potential were evaluated using similarity and phylogenetic analyses. Suncus murinus (n = 282) and five rat species, Rattus losea (n = 18), Apodemus agrarius (n = 36), Rattus tanezumi (n = 86), Rattus norvegicus (n = 155), and Niviventer niviventer (n = 75), were identified. The average infection rate of E. bieneusi was 14.1% (92/652) with 18.1% (51/282) in S. murinus and 11.1% (41/370) in rats (27.8% in R. losea, 22.2% in A. agrarius, 10.5% in R. tanezumi, 8.4% in R. norvegicus, and 8.0% in N. niviventer). Thirty-three genotypes were identified, including 16 known genotypes. The most commonly known genotypes were HNR-VI (n = 47) and Peru11 (n = 6). Type IV, KIN-1, SHW7, and HNPL-II were each found in two samples, while Macaque4, CH5, K, Henan-III, Henan-V, HNP-II, HNPL-I, HNPL-III, HNHZ-II, and HNHZ-III were each found in one sample. Additionally, 17 novel genotypes were discovered: WZR-VIII (n = 5), WZR-I to WZR-VII, WZR-IX to WZR-XII, and WZSH-I to WZSH-V (n = 1 each). Those 33 genotypes were divided into three groups: Group 1 (n = 25), Group 2 (n = 3), and Group 13 (n = 5). The initial report underscores the extensive occurrence and notable genetic diversity of E. bieneusi in wild rats and shrews from Zhejiang province, China. These results suggest that these animals play a pivotal role in the transmission of E. bieneusi. Furthermore, animals carrying the zoonotic genotypes of E. bieneusi pose a serious threat to residents.

Genetic diversity and population structure of Fusarium udum in India and its correlation with pigeonpea wilt incidence.

In a study conducted in India, 50 Fusarium isolates were collected from pigeonpea growing regions and extensively examined for their cultural and morphological characteristics. These isolates exhibited significant variations in traits including growth rate, mycelial growth patterns, color, zonation, pigmentation, spore size, and septation. Subsequently, 30 isolates were chosen for pathogenicity testing on eight pigeonpea genotypes. Results showed distinct reactions, with four genotypes displaying differential responses (ICP8858, ICP8859, ICP8862, and BDN-2), while ICP9174 and ICP8863 consistently exhibited resistance and ICP2376 and BAHAR remained susceptible to wilt disease. To study the interaction between Fusarium isolates and pigeonpea host differentials (HDs), an additive main effects and multiplicative interactionanalysis was conducted. The majority of disease incidence variation (75.54%) was attributed to HD effects, while Fusarium isolate effects accounted for only 1.99%. The interaction between Isolates and HDs (I × HD) contributed 21.95% to the total variation, being smaller than HD but larger than I. Based on HD reactions, isolates were classified into nine variants, showing varying distributions across pigeonpea growing states, with variants 2 and 3 being prevalent in several regions. This diversity underscores the need for location-specific wilt-resistant pigeonpea cultivars. Furthermore, genetic analysis of 23 representative isolates, through internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-αgene sequencing, revealed three major clusters: Fusarium udum, Fusarium solani, and Fusarium equiseti. These findings hold potential for developing location-specific wilt-resistant pigeonpea cultivars and enhancing disease management strategies.