Comparative genomics reveals ample evidence to Ganoderma sinense cultivars for molecular identification and new FIP exploration.

The first dikaryotic genome of Ganoderma cultivar Zizhi S2 (56.76 Mb, 16,681 genes) has been sequenced recently. 98.15% of complete BUSCOs were recovered in this genome assembly and high-confidence annotation rate improved to 91.41%. Collinearity analysis displayed the nuclear genome were 80.2% and 93.84% similar to reference genome of G. sinense at nucleotide and amino acid levels, which presented 8,521 core genes and 880 unique orthologous gene groups. Among that, at least six functional genes (tef1-α, ß-tubulin, rpb2, CaM, Mn-SOD and VeA) and a newly discovered fip gene were highly similar 99.27% ∼100% to those in reference genome. And the mt-LSU, mt-SSU and 13 PCGs in their mitogenome were also highly conserved with 99.27%-99.87% and 99.08%-100% identity, respectively. So that, this cultivar Zizhi S2 is confirmed conspecific with Ganoderma sinense (NCBI: txid1077348). The new fip gene (MN635280.1_336bp) existing a novel mutation which can be reflected on the phylogenetic tree and 3-dimensional model topology structure.

Changes in fungal taxonomy: mycological rationale and clinical implications.

Numerous fungal species of medical importance have been recently subjected to and will likely continue to undergo nomenclatural changes as a result of the application of molecular approaches to fungal classification together with abandonment of dual nomenclature. Here, we summarize those changes affecting key groups of fungi of medical importance, explaining the mycological (taxonomic) rationale that underpinned the changes and the clinical relevance/importance (where such exists) of the key nomenclatural revisions. Potential mechanisms to mitigate unnecessary taxonomic instability are suggested, together with approaches to raise awareness of important changes to minimize potential clinical confusion.

Mitochondrial genome provides species-specific targets for the rapid detection of early invasive populations of Hylurgus ligniperda in China.

BACKGROUND: Hylurgus ligniperda, a major international forestry quarantine pest, was recently found to have invaded and posed a serious threat to the Pinus forests of the Jiaodong Peninsula in China. Continuous monitoring and vigilance of the early population is imperative, and rapid molecular detection technology is urgently needed. We focused on developing a single-gene-based species-specific PCR (SS-PCR) method. RESULTS: We sequenced and assembled the mitochondrial genome of H. ligniperda to identify suitable target genes. We identified three closely related species for detecting the specificity of SS-PCR through phylogenetic analysis based on 13 protein-coding genes (PCGs). Subsequently, we analyzed the evolution of 13 PCGs and selected four mitochondrial genes to represent slow-evolving gene (COI) and faster-evolving genes (e.g. ND2, ND4, and ND5), respectively. We developed four species-specific primers targeting COI, ND2, ND4, and ND5 to rapidly identify H. ligniperda. The results showed that the four species-specific primers exhibited excellent specificity and sensitivity in the PCR assays, with consistent performance across a broader range of species. This method demonstrates the ability to identify beetles promptly, even during their larval stage. The entire detection process can be completed within 2-3 h. CONCLUSIONS: This method is suitable for large-scale species detection in laboratory settings. Moreover, the selection of target genes in the SS-PCR method is not affected by the evolutionary rate. SS-PCR can be widely implemented at port and forestry workstations, significantly enhancing early management strategies and quarantine measures against H. ligniperda. This approach will help prevent the spread of the pest and effectively preserve the resources of Chinese pine forests.

Rare Case of Echinostoma cinetorchis Infection, South Korea.

A woman in South Korea who underwent a colonoscopy for occasional gastrointestinal discomfort had 4 adult flukes of Echinostoma cinetorchis showing 37 collar spines around the oral sucker recovered from the terminal ileum through the ascending colon. Partial gene sequencing showed high identity with E. cinetorchis.

What are we imaging? Software tools and experimental strategies for annotation and identification of small molecules in mass spectrometry imaging.

Mass spectrometry imaging (MSI) has become a widespread analytical technique to perform nonlabeled spatial molecular identification. The Achilles' heel of MSI is the annotation and identification of molecular species due to intrinsic limitations of the technique (lack of chromatographic separation and the difficulty to apply tandem MS). Successful strategies to perform annotation and identification combine extra analytical steps, like using orthogonal analytical techniques to identify compounds; with algorithms that integrate the spectral and spatial information. In this review, we discuss different experimental strategies and bioinformatics tools to annotate and identify compounds in MSI experiments. We target strategies and tools for small molecule applications, such as lipidomics and metabolomics. First, we explain how sample preparation and the acquisition process influences annotation and identification, from sample preservation to the use of orthogonal techniques. Then, we review twelve software tools for annotation and identification in MSI. Finally, we offer perspectives on two current needs of the MSI community: the adaptation of guidelines for communicating confidence levels in identifications; and the creation of a standard format to store and exchange annotations and identifications in MSI.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at the National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM), and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n = 8) and A. levis (n = 1), from brain abscess (n = 4), respiratory tract (n = 3), and corneal scraping (n = 2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the internal transcribed spacer ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed the lowest minimum inhibitory concentrations (MICs) for triazoles and the highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin, and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.

We describe clinical, phenotypic, and genotypic characteristics of Acrophialophora species. This species causes mild infection to fatal infection in immunosuppressed individuals. Triazoles are effective in treating such infections.

Discovery of Encyclometra bungara (Digenea: Encyclometridae) in a new host (Enhydris enhydris) from Thailand and Cambodia through morphological and molecular identification.

The genus Encyclometra is one of the two genera in family Encyclometridae, known for parasitising the oesophagus, stomach and intestine of snakes. Among Encyclometra, the species present are: Encyclometra colubrimurorum, Encyclometra japonica, Encyclometra asymmetrica and Encyclometra bungara. Species discrimination within Encyclometra has predominantly relied on morphological differences, such as the length of the caeca and the position of the testes. Morphological overlaps exist among these species making species discrimination challenging. Additionally, the use of molecular information has been limited for Encyclometra. To determine the Encyclometra species infecting Enhydris enhydris from Thailand and Cambodia, morphological and molecular identification was conducted. Morphological characters and measurements were obtained from 30 Encyclometra adults, and they were compared with previous studies of other Encyclometra species. Novel sequences of E. bungara were generated using the nuclear 18S and 28S ribosomal RNA genes, and the mitochondrial cytochrome c oxidase subunit 1 gene. Our results revealed that the specimens could be morphologically identified as E. bungara, with support from molecular information obtained from the phylogenies of the 3 genetic markers employed. Molecular analysis showed that the Encyclometra specimens were distinct from E. colubrimurorum and E. japonica. Through morphological and molecular identification of the Encyclometra specimens found in E. enhydris from Thailand and Cambodia, we describe and provide a record of E. bungara in a new host and new locality. Additionally, novel molecular sequences were generated, revealing the phylogenetic position of E. bungara within the superfamily Gorgoderoidea.

Characterization of initial CLABSI culture results to support antimicrobial de-escalation in pediatric GI inpatients.

OBJECTIVES: Central Line-associated Bloodstream Infections (CLABSIs) pose a serious mortality and morbidity risk. An institutional protocol was developed for the evaluation and empirical antibiotic treatment of possible CLABSIs. The potential impact of de-escalating antimicrobial therapy based on initial Gram stain and molecular identification was assessed. METHODS: All positive blood cultures from patients admitted to the gastroenterology service at a large pediatric medical center were collected from 1/1/14 to 12/31/20. Cultures that were negative, repeated, or causative organisms that were unable to be identified with susceptibility data were excluded. Timepoints and organism(s) from each culture were recorded. Polymicrobial cultures were classified as containing only gram-positive organisms (polymicrobial GP), only gram-negative organisms (polymicrobial GN), or mixed spectrum. RESULTS: During the 6-year period, 361 positive blood cultures were included in the study. Single isolates were identified in 79.5% (287/361) of cultures. Polymicrobial cultures from confirmed central line source accounted for 15.0% (54/361), with 6.4% (23/361) Polymicrobial GP, 4.4% (16/361) Polymicrobial GN, and 4.2% (15/361) being mixed-spectrum cultures. Both organism types were detected on initial gram-stain in 40% (6/15) of the mixed-spectrum cultures, another 26.7% (4/15) had the opposite-spectrum organism identified within an average of <3 h and the remaining 33.3% (5/15) had the opposite-spectrum organism identified by culture growth. CONCLUSIONS: Polymicrobial mixed-spectrum cultures accounted for <5% of positive blood cultures and most isolates were identified within 3 h of first positivity. This may allow for further investigation of early de-escalation of therapy for this population and limit antimicrobial exposure.

Phylogenetic evidence of a possible Trichuris globulosa species complex in Arabian camels from Kuwait.

During a 1-year study, Trichuris adults were obtained after necropsy of Arabian camels (Camelus dromedarius) from a slaughterhouse in Kuwait. Morphological and molecular identification was performed to confirm the identity of the Trichuris specimens obtained from C. dromedarius. Fifteen male Trichuris specimens were selected, and molecular identification was performed using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA, 16S ribosomal RNA genes and the nuclear internal transcribed spacer 2 (ITS2) region. Through phylogenetic analysis, 2 distinct groups were obtained using the mitochondrial genes, where group 1 showed a close relationship to Trichuris globulosa while group 2 showed a close relationship to Trichuris ovis, providing molecular evidence of a possible T. globulosa species complex. Additionally, the nuclear ITS2 region did not provide enough resolution to distinguish between the 2 groups of Trichuris specimens. Observation of morphological characters revealed variations in the shape of the male spicule sheath, where specimens present either a globular posteriorly truncated swelling or the absence of posteriorly truncated swelling. Moreover, the variations in male spicule sheath does not corroborate with the results of molecular data, suggesting the limited use of this character for identification of T. globulosa. In conclusion, molecular analysis suggests a possible species complex in T. globulosa, with the mitochondrial genetic markers successfully differentiating between the 2 groups. The limited use of the male spicule sheath as a diagnostic character for identification of T. globulosa is suggested.

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

Pectate lyase genes from Radopholus similis and their application in pathotype identification.

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes. KEY POINTS: • Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed. • The expression of Rs-pels is different in two pathotypes of Radopholus similis. • A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Two proteocephalid cestodes in the fish Malapterurus electricus and Heterobranchus bidorsalis from Lake Nasser, Egypt: a morphological, molecular, and histopathological study.

Despite the importance of the electric catfish (Malapterurus electricus) and the African giant catfish (Heterobranchus bidorsalis) in the foodweb of Lake Nasser, Egypt, little is known about their diseases and parasitic fauna. This work describes, for the first time, cestodiasis in M. electricus and H. bidorsalis. Corallobothrium solidum and Proteocephalus sp. were identified morphologically and molecularly from M. electricus and H. bidorsalis, respectively. Using PCR, sequencing, and phylogenetic analysis, the two cestodes shared rRNA gene sequence similarities yet were unique and the two new sequences for the proteocephalid genera were submitted to the GenBank database. The prevalence of infection was 75% and 40% for the two fish species, respectively. Infections significantly increased in the summer and spring and were higher in female fish than in male fish. The intestine was the preferred site of the two adult cestodes. However, in the case of C. solidum some larval cestodes were found outside the intestine in between the skin and abdominal musculature, attached to the mesentery, and within intestinal tunica muscularis. Desquamation of the intestinal epithelium and inflammation at the site of infection in addition to congestion of the intestinal wall of the tapeworm infected fish were evident, indicating that C. solidum and Proteocephalus sp. impacted the infected fish. The larval stages of C. solidum attempted to penetrate the intestine and sometimes they were encircled within fibrous layers infiltrated with inflammatory cells. The infected fish's musculature was free of cestode infections. Preventive measures should be implemented to prevent the spread of infections.

Dasyrhynchus giganteus plerocercoids encysting in the musculature of Indian halibut (Psettodes erumei): seasonal prevalence, morpho-molecular characterization, and histopathological alterations.

This study investigated the prevalence, morphology, molecular identification, and histopathological effects of larval tapeworms (plerocercoids) infecting the skeletal muscles of the Indian halibut (Psettodes erumei) collected from the coastal waters of the Arabian Gulf. Numerous oval or round blastocysts, measuring 13-26 mm, were found embedded within the muscular tissues of the Indian halibut, rendering the fish unsuitable for human consumption. Morphological and molecular analyses identified the plerocercoids as Dasyrhynchus giganteus (family Dasyrhynchidae), with an overall prevalence of 15.4%. The seasonal prevalence was the highest in summer (14.6%), followed by spring (10.6%), winter (4.4%), and autumn (3.5%). Infection rates increased with fish size. Histopathological examination revealed fibrous connective tissue capsules surrounding the larvae, causing muscular atrophy and degenerative changes, with few inflammatory eosinophilic cells. Molecular and phylogenetic analysis of the 28S rDNA gene sequences confirmed the specimens as D. giganteus, clustered closely with other sequences of D. giganteus with 100% bootstrap values. This study provided valuable insights into the parasitic infection dynamics, seasonal variation, molecular identification, and histopathological effects, highlighting the importance of monitoring fish for food safety and public health implications.

Molecular Identification of Acrossocheilus jishouensis (Teleostei: Cyprinidae) and Its Complete Mitochondrial Genome.

Molecular identification, such as DNA barcoding, is a useful tool that is widely applied in distinguishing species. To identify the cyprinid Acrossocheilus jishouensis, which was previously known to be restricted to only its type locality, we conducted molecular identification of this species based on 23 samples in five localities. Molecular identification based on the mitochondrial COI gene sequence showed that the morphologically similar samples from the five populations were all A. jishouensis, as the mean genetic distances between populations were very small (0.1-1.6%); thus, the distribution of this species was substantially expanded. The whole mitochondrial genome of one sample was also assembled, which was 16,594 bp in length and consisted of 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and one control region. All PCGs began with ATG except the COI gene, which started with GTG; seven PCGs used the complete stop codon TAA, while four terminated in T(AA) and two ended with TAG. The overall base composition reflected a higher proportion of A+T than G+C and a positive AT-skew and negative GC-skew pattern except for the opposite in ND6. Phylogenetic relationships inferred using BI and ML methods revealed that both Acrossocheilus and Onychostoma were nonmonophyletic, which indicated that the traditional diagnoses between these two genera need to be assessed further. The results of this study not only expanded the known distribution ranges of A. jishouensis, but also provided a valuable data resource for future molecular and evolutionary studies of Acrossocheilus and other cyprinids in Barbinae.

The first report of digenean infecting short mackerel (Rastrelliger brachysoma) from Chon Buri Province, the Gulf of Thailand.

Rastrelliger brachysoma (Bleeker, 1851), the short mackerel, is a dietary staple and of significant economic demand in Southeast Asia and Thailand. However, the demand for short mackerel has precipitated an overfishing crisis, leading to a depletion of fish stocks. Overfishing, coupled with parasitism, may result in a decline in the population of R. brachysoma. Digenetic trematode infection is prevalent in marine fish and has a considerable impact on the overall health of the fish. Here, to identify digenetic trematodes infecting R. brachysoma, we aim to determine the identity, prevalence, and intensity of digenean infections in R. brachysoma from the Gulf of Thailand. A total of 194 short mackerel were obtained from Chon Buri Province, where digeneans were isolated and identified. The molecular identity of the digeneans was confirmed using the nuclear 28S rRNA gene. Of the 194 short mackerel, 100% were found to be infected with digeneans, comprising of Lecithocladium, Prodistomum, Opechona, and Aphanurus. Lecithocladium was the most prevalent (98%) and had the highest intensity of infection (37 mean intensity), followed by Prodistomum (75% prevalence and 17 mean intensity). Our study thus presents the first evidence of digeneans infecting the economically important short mackerel from the Gulf of Thailand. The high infection rate of digenetic trematodes may have implications on the health of R. brachysoma, further driving their population decline. These data underscore the importance of safeguarding fisheries resources in the Gulf of Thailand, and downstream conservation efforts are crucial for evidence-based management decisions to safeguard the long-term sustainability of fish resources.

Molecular identification and genetic diversity of zoonotic hookworm infections in domestic dogs from northeastern, Thailand.

Hookworm infections remain a significant public health concern in tropical and subtropical regions, including Thailand. This study investigated the species and genetic diversity of hookworm infections in domestic dogs from northeastern Thailand. The molecular analysis focused on amplifying and sequencing specific regions of ribosomal RNA genes (ITS1-5.8S-ITS2 region) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene in hookworm larvae recovered from 21 domestic dog stool samples. Among 21 larvae (one larva per infected dog) analyzed, 14 had sequences identical to Ancylostoma caninum, and 7 showed sequences almost identical to Ancylostoma ceylanicum. Phylogenetic analysis of cox1 sequences placed A. caninum and A. ceylanicum in separate clades. The median-joining network of A. caninum cox1 sequences from Thailand showed high haplotype diversity and belonged to the same cluster as sequences from Australia while forming separate clusters from those of A. caninum samples from the USA. The available published A. ceylanicum cox1 sequences (n = 33), in combination with seven sequences in the present study, represented 15 haplotypes distributed among three clusters. Interestingly, A. ceylanicum sequences from dogs and humans shared the same haplotypes. These findings are crucial for recognizing the potential for zoonotic transmission, highlighting the necessity for targeted control measures, and increasing awareness among pet owners and healthcare professionals to mitigate the risk of hookworm transmission to humans.

Phylogeny and ultrastructure of Elthusa raynaudii (Isopoda, Cymothoidae) firstly recorded from the invasive silver cheeked toadfish (Lagocephalus sceleratus) (Gmelin 1789) in eastern Mediterranean Sea coast.

With the opening of the Suez Canal in 1869, many changes have occurred in the Mediterranean Sea ecosystem so became a home to many invasive Lessepsian marine species that have migrated from the Red Sea. About 500 marine species including pufferfish have immigrated and rapidly established a population in the Mediterranean Sea causing significant impact on its ecosystem and fisheries sector. The parasitic fauna of these pufferfish has scarcely been studied in the Mediterranean Sea and also in their native habitat. During this surveillance study on the invasive pufferfish species from the Egyptian Mediterranean coast, the female cymothoid isopod Elthusa raynaudii was detected from the branchial cavity and also in the buccal cavity of 23.9% of the examined Lagocephalus sceleratus. The isolated isopod species was firstly identified and described through electron microscopy and molecular phylogeny based on the sequences of mitochondrial 16S rRNA gene. Additionally, the description of eggs, embryonic stage, and manca of E. raynaudii was firstly provided. The pathological impact on the infested fish tissues was investigated and revealed curling and loss of secondary gill lamellae in addition to mucous exudates in between the gill filaments and granuloma formation in the gill arch. The study provided the first report of L. sceleratus as a new host for the isopod E. raynaudii collected from the Egyptian Mediterranean coast as a new locality record. The role of the Lessepsian invasive pufferfish in transmitting parasites to the native fish species was discussed.

Molecular characterizations of Cryptosporidium spp. in brown rat (Rattus norvegicus) from an animal feedlot in Xinjiang, China.

Cryptosporidium infection is a common occurrence in rodents worldwide. In this study, 435 wild brown rats were captured from an animal feedlot in Xinjiang, China, with a fecal sample obtained directly from the rectal contents of each rat. The DNA extracted from these fecal samples was analyzed for Cryptosporidium spp. using PCR targeting the SSU rRNA gene. The prevalence of Cryptosporidium infection in brown rats was found to be 5.5% (24 out of 435). Interestingly, the infection rates varied among different animal enclosures, with rates of 0% in the chicken coop (0/51), cowshed (0/3), and varying rates in other areas including the sheepfold (6.1%, 6/98), the pigsty (7.6%, 10/132), the dovecote (7.0%, 5/71), and outdoor environments (3.8%, 3/80). The study identified three species and one genotype of Cryptosporidium, namely C. occultus (n = 10), C. parvum (n = 4), C. ditrichi (n = 1), and Cryptosporidium rat genotype IV (n = 9). Additionally, two of the C. parvum isolates were successfully subtyped as IIdA19G1 (n = 2) at the gp60 gene. These results offer valuable insights into the prevalence and genetic diversity of Cryptosporidium in brown rats within the region.

Description, identification, and growth of Tuber borchii Vittad. mycorrhized Pinus sylvestris L. seedlings on different lime contents.

Tuber borchii forms ectomycorrhiza with oaks, hazel, and pines, including Pinus sylvestris. However, its ectomycorrhiza morphotype with P. sylvestris was not comprehensively described so far, and molecular analyses are missing despite a high danger of misidentification of T. borchii ectomycorrhiza with other closely related and less valuable truffle species. We described for the first time the morphology and anatomy of T. borchii-P. sylvestris ectomycorrhiza using differential interference contrast technique and semi-thin sections in combination with molecular confirmation of identity. Color of ectomycorrhiza is reddish to dark brown, and morphotypes are unevenly but densely covered by warts-bearing pin-like cystidia. All layers of the hyphal mantle are pseudoparenchymatous with outer mantle layer formed of epidermoid cells. T. borchii ectomycorrhiza was identified by a molecular comparison with fruitbodies used for inoculation and its respective ectomycorrhizae. T. borchii has a wide ecological amplitude. To get a better insight in mycorrhization requirements, we investigated growth of P. sylvestris and its ectomycorrhiza infection rate with T. borchii in substrate with different lime content. The mycorrhization of P. sylvestris with T. borchii in the mycorrhization substrate and cultivation in greenhouse conditions was successful, with colonization of P. sylvestris varying between 36.5 and 48.1%. There was no significant correlation of mycorrhization to applied lime contents, and consequently to pH in substrate, while the increased levels of lime improved growth of the P. sylvestris seedlings.

Heliconia subulata, a New Host of Colletotrichum tropicale Causing Anthracnose in China.

Heliconia subulata is a common ornamental plant, it has been widely planted in southern China for greening parks, roads, and residential areas. H. subulata plants with spots on their leaves were observed in East Coast Wetland Park (18°16'53.37″N, 109°30'19.36″E), Sanya City, Hainan Province, China on Aug. 31, 2023. The symptoms of the leaves are irregular gray-white, spots, that develop into brown and black, with yellow halos at the disease-health junction. Following an on-the-spot investigation, it was found that the incidence of the disease was 40 to 50%. The leaves were disinfected with 70% ethanol for 1 min, rinsed with sterile water 3 times, disinfected for 1 min with 0.1% HgCl2, rinsed with sterile water 3 times, dried, put on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. The red conidia pile was selected from the culture, dispersed in sterile water and diluted to 20 µL containing 1 to 2 conidia. After absorbing 20 µL spore suspension for many times and inoculating it on the new PDA plate, five pure cultures of single spore, J-1-1 to J-1-5, were obtained. After 7 days of growth, the colonies were grayish aerial mycelium on the front and light orange conidia on the reverse. The white aerial mycelia, conidia, acervulus, and appressorium were observed (Supplementary Fig. S1). The morphological characteristics showed that the isolate had the same characteristics as the previously described Colletotrichum spp. (Wang et al. 2021). The genomic DNA of isolates J-1-1 and J-1-5 were extracted by Fungal DNA Kit (OMEGA bio-tek, Guangzhou, China). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and ß-tubulin 2 genes (TUB2) were amplified by primers ITS1/ITS4, GDF/GDR, and Bt2a/Bt2b, respectively (Weir et al. 2012). Based on sequencing and gene sequence alignment analysis, it was found that the consistency between the ITS sequences of isolates J-1-1 and J-1-5 was 99.82%. The consistency between GADPH and TUB2 sequences was 100%. The gene sequences of isolates J-1-1 and J-1-5 were submitted to GenBank with accession numbers PP455510/PP455511 (ITS), PP510210/PP510211 (GADPH) and PP510212/PP510213 (TUB2) respectively. Based on the BLAST analysis, the three sequences were more than 99% identical to those of the C. tropicale strain FC1 (ITS: MT192648, GAPDH: MT155819, TUB2: MT199874; Duan et al. 2022). A phylogenetic tree was constructed by MEGA 11 based on the ITS, GADPH, and TUB2 gene sequence by the maximum-likelihood method. The results showed that the isolates J-1-1 and J-1-5 were clustered with C. tropicale CBS:124949 (Supplementary Fig. S2). Based on morphological and molecular biological analysis, two isolates were identified as C. tropicale. To further test the pathogenicity of isolates J-1-1 and J-1-5, spore suspensions (1×106 conidia/mL) were prepared and 20 µL spore suspensions were inoculated on the leaves of healthy H. subulata potted plants stabbed with sterile toothpicks. Three leaves were inoculated in each treatment, and sterile water was inoculated as a control. The treated plants were placed in an incubator with a temperature of 28℃, relative humidity of 90%, and light/dark (12h/12h). After 15 days, the spore suspension treatment showed the same symptoms as the naturally diseased H. subulata plants in the field, but the leaves treated with sterile water were not infected (Supplementary Fig. S1). The morphology of the isolates obtained from diseased leaves was the same as that of isolates J-1-1 and J-1-5 on the PDA plate. To our knowledge, this is the first report of H. subulata, a new host of C. tropicale causing anthracnose in China.