ITS Region Research Articles

Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs.

Comparative Assessment of colorimetric assays in evaluating intracellular drug susceptibility of Leishmania tropica against conventional antileishmanial drugs.

DNA barcoding of Culicoides biting midges (Diptera: Ceratopogonidae) and detection of Leishmania and other trypanosomatids in southern Thailand

BackgroundBiting midges of the genus Culicoides play an important role in the transmission of pathogenic arboviruses and parasites. Thailand has documented more than 100 species of Culicoides; however, several cryptic species complexes remain to be clarified. Recent studies in areas with leishmaniasis indicate that several species of Culicoides might be potential vectors of Leishmania in the subgenus Mundinia, but evidence supporting the hypothesis is still lacking. Therefore, the diversity of Culicoides biting midges and their potential role as vectors of leishmaniasis in southern Thailand remains uncertain.MethodsFemale Culicoides biting midges were collected using Centers for Disease Control and Prevention (CDC) ultraviolet (UV) light traps from four locations within leishmaniasis-affected areas in three provinces of southern Thailand, including Nakhon Si Thammarat, Krabi, and Surat Thani. Culicoides species were identified based on the morphology of wing spot patterns and subsequently confirmed by cytochrome c oxidase subunit I (COI) Sanger sequencing. A potential cryptic species was classified using an integrative taxonomic approach associated with DNA barcoding identification by Barcode of Life Database (BOLD) and Basic Local Alignment Search Tool (BLAST) searches. Furthermore, three different methods of species delimitation, namely ASAP [Assemble Species by Automatic Partitioning], TCS [Templeton, Crandall, and Sing], and PTP [Poisson Tree Processes], were employed to verify the sequences into the molecular operational taxonomic unit (MOTU). Detection of Leishmania and other trypanosomatid parasites was performed by polymerase chain reaction (PCR) based on the ITS1 region and small subunit SSU ribosomal RNA (rRNA) gene, followed by Sanger sequencing and haplotype diversity analysis. The identification of host blood sources was carried out using host-specific multiplex PCR.ResultsA total of 716 unfed midges and 159 blood-fed specimens were morphologically identified into 25 species belonging to five subgenera (Avaritia, Hoffmania, Meijerehelea, Remmia, and Trithecoides) and four species groups (Clavipalpis, Ornatus, Shermani, and Shortti). Two unidentified specimens were classified into two subgenera (Trithecoides and Avaritia). The DNA barcoding identification exhibited an 82.20% success rate. Species delimitation analyses demonstrated the presence of cryptic species complexes, categorized into six species: Culicoides actoni, C. orientalis, C. huffi, C. palpifer, C. clavipalpis, and C. jacobsoni. Furthermore, 6.42% of the Culicoides biting midges tested positive for Leishmania DNA in three sampling sites in Nakhon Si Thammarat and Surat Thani provinces (with no positive results in Krabi province). Furthermore, the sympatric infection of Leishmania martiniquensis and Leishmania orientalis was identified in several Culicoides species in Ron Phibun and Phunphin districts in Nakhon Si Thammarat and Surat Thani, respectively. In contrast, L. orientalis was detected in Sichon district, Nakhon Si Thammarat province. A genetic diversity analysis revealed high haplotype diversity and relatively low nucleotide diversity in both parasite populations. Additionally, Crithidia sp. and Crithidia brevicula were detected in Culicoides peregrinus and Culicoides subgenus Trithecoides. The analysis of the host blood meal from Ron Phibun also demonstrated that Culicoides had fed on cows, dogs, and chickens, and mixed blood preferences for humans and cows or chickens and cows were detected.ConclusionsThe findings of the present study demonstrate the presence of mixed blood hosts and co-circulation of L. martiniquensis and L. orientalis in Culicoides in areas of leishmaniasis, as well as cryptic species of Culicoides biting midges, through an integrative taxonomic approach. These findings support the hypothesis that Culicoides biting midges may serve as potential vectors in southern Thailand, and vector diversity is a contributing factor to the risk of zoonotic transmission.Graphical

Exploring the antifungal activities of secondary metabolites of Trichoderma harzianum against the leaf spot pathogen of turmeric Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.

The present study aimed to evaluate the antifungal activity of Trichoderma harzianum against Colletotrichum gloeosporioides, the causal agent of turmeric leaf spot. The pathogen was isolated from symptomatic turmeric leaves and identified using morphological traits and amplification of the ITS region, followed by sequencing. Pathogenicity was confirmed via Koch's postulates. Various biocontrol agents were tested in vitro, and T. harzianum exhibited the highest inhibition (75.92%) in dual culture assays. Crude culture filtrates of T. harzianum also inhibited the pathogen in a seeded agar assay, showing 22.22% inhibition. GC-MS profiling was carried out to identify the bioactive compounds responsible using the culture filtrates of T. harzianum, the pathogen, and their interaction. A total of 129 metabolites were identified, including phenolic acids, fatty acids, esters, and phthalates with reported antimicrobial activity. The interaction revealed compounds common to both organisms, suggesting biochemical cross-talk. This study demonstrates the potential of T. harzianum as an effective biocontrol agent against C. gloeosporioides and highlights key antifungal metabolites involved. Further characterisation of these compounds may support the development of bioformulations for eco-friendly disease management.

First Report of Powdery Mildew of Cocculus orbiculatus (L.) DC. var. mollis Caused by Erysiphe pseudolonicerae in China

The genus Cocculus comprises diverse medicinal plants in China, including two species C. laurifolius DC. and C. orbiculatus (L.) DC., and one variety C. orbiculatus (L.) DC. var. mollis (Kunming Institute of Botany, Chinese Academy of Sciences. 1983). In January 2023, severe powdery mildew symptoms were found on C. orbiculatus var. mollis at the Huaxi Campus (26°23'04'' N, 106°37'57'' E) of Guizhou Normal University, Guiyang, China. The incidence observed was ~95% among 80 C. orbiculatus var. mollis plants. Infected leaves presented chlorotic to necrotic areas. Mycelium grew mainly on the adaxial surface of leaves. Hyphae were hyaline and 4–10 μm wide. Hyphal appressoria were lobed to multilobed, and solitary or in opposite pairs. Conidiophores were erect, straight to somewhat flexuous and 55–130 µm long (n = 40). Foot cells were subcylindrical to curved-sinuous at the base, followed by 2–4 cells. Conidia formed singly and were ellipsoid to ovoid in shape with dimensions of 28.8–54 × 13.3–17 µm (n = 50). Based on these morphological characteristics, the powdery mildew fungus (named as GZCO-1) was provisionally identified as an Erysiphe species (Bradshaw et al. 2023). To identify the species, the ITS and LSU regions were amplified using the primer pairs PM10/PM28R (Bradshaw and Tobin 2020); the CAM, GAPDH, GS, and RPB2 region were amplified using the primer pairs PMCAM1/PMCAM4R, PMGAPDH1/PMGAPDH3R, GSPM2/GSPM3R, and PMRpb2_4/ PMRpb2_6R, respectively (Bradshaw et al. 2022); the TUB region was amplified using the primers BTF5b/BTR7a (Ellingham et al. 2019). The obtained 1364-bp ITS-LSU sequence (GenBank accession no. PQ818141), 282-bp CAM sequence (PQ822234), 316-bp GAPDH sequence (PQ822231), 468-bp GS sequence (PQ822235), 763-bp RPB2 sequence (PQ822232), and 733-bp TUB sequence (PQ822233) were deposited in GenBank. Using a phylogenetic tree based on the concatenated ITS-LSU, CAM, GAPDH, GS, RPB2, and TUB sequences (Bradshaw et al. 2023), the isolate GZCO-1 was grouped in the same clade as E. pseudolonicerae. To perform pathogenicity tests, leaves of three healthy potted C. orbiculatus var. mollis plants were inoculated by gently pressing with diseased leaves. Non-inoculated plants served as controls. All plants were incubated in a greenhouse at 25 ± 2°C with 80% relative humidity. Similar powdery mildew symptoms appeared on inoculated plants 12 days after inoculation, whereas no symptoms were observed on control plants. The fungus from inoculated C. orbiculatus var. mollis plants was morphologically identical to that on originally diseased plants. In addition, ITS-LSU sequences of the fungus from the artificially inoculated plants shared 100% identity with PQ818141. Based on the morphological and molecular characterization, the powdery mildew fungus was identified as E. pseudolonicerae. This powdery mildew on this host has previously been reported from China (Bradshaw et al. 2024), however this is the first report supported by sequence analyses.

Intestinal microbiome profile of the brown rock sea cucumber (Holothuria glaberrima) using ITS and 16S rDNA amplicons from direct mechanical, enzymatic, and chemical metagenomic extraction.

Using direct mechanical, enzymatic, and chemical extraction methods, the intestinal microbiome of the marine invertebrate Holothuria glaberrima was obtained. ITS and 16S rDNA regions were sequenced to enrich and investigate the prokaryotic and fungal diversity profiles from different anatomical regions within the sea cucumber's intestinal biology.

NEW SPECIES OF AXINE ABILDGAARD, 1794 (MONOGENOIDEA: AXINIDAE) INFECTING GILL LAMELLAE OF ATLANTIC FLYINGFISH, CHEILOPOGON MELANURUS (VALENCIENNES) (EXOCOETIDAE) IN THE WESTERN ATLANTIC OCEAN WITH PHYLOGENETIC ANALYSIS AND COMMENTS ON THE FEMALE GENITALIA OF AXINE SPP.

We collected specimens of Axine trickyvagina Brule and Bullard n. sp. (Monogenoidea: Axinidae) from the gill lamellae of Atlantic flyingfish, Cheilopogon melanurus (Valenciennes) (Exocoetidae) in the north-central Gulf of America. Specimens of the new species were heat-killed and formalin-fixed for morphology, and others were preserved in 95% EtOH for DNA extraction and sequencing of the 28S gene and ITS1 region. The new species differs from all congeners by the combination of having a long haptor (∼40-50% of the total body length), a male copulatory organ with 10-15 spines, and a genital atrium having bilateral spinous patches each with 18-25 spines. Our study of specimens of the new species, type and voucher specimens representing 8 congeners, and all published accounts of all congeners revealed that the terminal female genitalia of Axine spp. comprises 2 ducts (a multi-chambered vagina that lacks a sclerite plus an accessory duct with a sclerotized nozzle comprising its opening) that open into a common female genital atrium. Descriptions of Axine spp. published from 1794 through 2023 failed to recognize the vaginal duct and accessory duct as distinct components and unanimously misinterpreted the accessory duct's sclerotized nozzle as a "vaginal spine." The phylogenetic analysis inferred from 28S rDNA sequences placed our sequence in a clade of other Mazocraeidea spp. and sister to a nonugen sequence ascribed to Axine japonicaPrice, 1946 (GenBank LC799038). We recovered Axinidae as sister to Heteromicrocotylidae, Heteraxinidae, and Microcotylidae. The present study is the first published description of an axinid from a flyingfish in the western Atlantic Ocean.

Circumscription of African Dipcadi (Asparagaceae, Scilloideae) species based on ITS sequence data and morphology

Species circumscriptions in the genus Dipcadi were evaluated based on both morphological and molecular data, with an emphasis on African taxa. DNA was extracted and the ITS region amplified for 33 Dipcadi accessions, and an additional 18 ITS sequences were obtained from GenBank. Phylogenetic analyses based on Maximum Likelihood and Bayesian Inference were congruent. The species D. marlothii and D. glaucum are recovered as monophyletic, and their status is also supported by morphological data. Other species are either paraphyletic (D. viride), polyphyletic (D. longifolium, D. serotinum, and D. erythraeum), or in unresolved clades with other taxa (D. gracillimum and D. platyphyllum). Future studies should include additional loci and more material to improve the resolution of species relationships and to properly delimit non-monophyletic taxa.

Case study of non-lethal sampling for plant-pollinator networks via barcoding and metabarcoding on bumble bees in Germany

1. Insect decline is threatening ecosystem stability, making information about foraging preferences of pollinators a vital piece of information to acquire. A powerful emerging tool to study pollinator foraging behaviour is pollen-metabarcoding. This usually involves lethal sampling of insects. 2. Here, we propose a new, non-lethal way of sampling DNA for the analysis of pollen loads of bumble bees as well as the pollinator. The new methodology does not significantly harm the insect and is easy to implement in a wide range of study designs. The tool is cheap and easy to acquire, can easily be used in the field and has the potential to become a powerful tool in studying plant-pollinator interactions. 3. To test its feasibility, plant-pollinator networks were analysed using metabarcoding of the ITS2 region. Plants flowering at the time of collection were also recorded as a reference comparison. 4. Bumble bees with ambiguous morphology were additionally identified, based on COI barcoding. 5. With the workflow developed here, it is possible to gain knowledge about plants and their pollinators in a non-lethal way without reducing population sizes. This makes this method particularly suitable for endangered and protected species.

Additional Records of Potensaphelenchus stammeri (Nematoda: Aphelenchoididae) in Association With Spondylis buprestoides (Coleoptera: Cerambycidae) Across Europe

ABSTRACTSince the detection in Portugal of the pinewood nematode (PWN—Bursaphelenchus xylophilus), in 1999, annual field surveys have been conducted to monitor its spread. These surveys include sampling symptomatic and dead pine trees, mainly Pinus pinaster Aiton, to assess and control the pine wilt disease. A complementary approach involves the use of chemical lures to capture insect vectors of the genus Monochamus (Coleoptera, Cerambycidae), the primary PWN carriers. The lure used (Galloprotect 2D Plus) includes specific pheromones for Monochamus, but also pine volatiles and bark beetles' pheromones. As part of a collaborative study, insects caught in traps placed in Portugal (969 insects, during 2023) and Poland (272 insects, during 2024) were surveyed for the presence of nematodes belonging to the Aphelenchida. In both countries, Spondylis buprestoides (Coleoptera, Cerambycidae) were caught (195 and 150 insects, respectively) and were found to be the only carrying vector of Potensaphelenchus stammeri (Nematoda: Aphelenchoididae). The general morphology of both females and males agreed with the original description for P. stammeri, namely the male spicules and the conspicuous morphology of the female tail. Molecular analysis, performed by sequencing the partial 18S rDNA gene, a fragment spanning the interspacer ITS1 and ITS2 regions and the partial 28S rDNA gene, further confirmed the results of the morphological analysis. This finding highlights the unique relationship between P. stammeri and the insect S. buprestoides and their large distribution in Europe.

Comparative phylogenetic analysis of key chloroplast and nuclear DNA regions of some Brassica species

Abstract The genus Brassica includes the economically valuable vegetables and oilseed crops. Understanding the phylogenetic relationships among these species is vital for plant breeding and evolutionary biology research. In this study, the phylogenetic relationships among some Brassica species were comprehensively analyzed using four chloroplast DNA regions (psbA-trnH, matK, rbcL, and rpl32-trnL) and three nuclear ribosomal DNA regions (ITS1, ITS2, and 5.8S). The resolution of each gene region at the species differentiation level was evaluated. The effectiveness of different gene regions in resolving phylogenetic relationships was also compared. The findings revealed that the matK, rpl32-trnL, and ITS regions provided strong genetic relationships, particularly among species such as B. rapa and B. juncea, as well as B. nigra and B. carinata. Among the chloroplast DNA regions, matK and psbA-trnH effectively resolved variety-level relationships. In contrast, nuclear ribosomal DNA regions (ITS1, ITS2, and 5.8S) provided a higher resolution at the species level. Phylogenetic trees constructed using the Maximum Likelihood method and appropriate analytical models provided well-supported clades and evolutionary relationships among Brassica species. Additionally, genetic relationships were visualized using principal component analysis (PCA), and the results of the phylogenetic analyses were confirmed. PCAs were broadly consistent with the phylogenetic trees and revealed genetic differences and similarities among the species. Multiple gen regions played a complementary role in resolving phylogenetic relationships. This study highlighted the importance of focusing on multiple gene regions instead of a single gene region.

First report of Diaporthe humulicola causing stem blight in hop plants in Massachusetts.

Humulus lupulus L., the common hop plant, is a commercially cultivated crop used as a beer flavoring agent that has been grown in Massachusetts since the 1600s (Machado et al., 2019 ; Rumney, 1998). In 2018, Diaporthe leaf spot, caused by the fungal pathogen Diaporthe humulicola, was found at hopyard research plots in Connecticut, followed by reports in multiple New York counties in 2023 (Allan-Perkins et al., 2020; Sharma et al., 2023). Additionally, Diaporthe halo blight, caused by the same fungal pathogen, was reported in Prince Edward Island (PE), Canada (Foster et al., 2024). In August of 2021, severe stem blight symptoms with visible fungal fruiting bodies were observed on >50% of plants at a hopyard in Franklin County, MA. Brown spots were observed on stems. Severe infection presented as widespread dark browning on stems, leaves, and cones. Samples were collected from the hop cultivars Mt Rainier (N=5), Magnum (N=1), and Teamaker (N=6). Under brightfield microscopy (400x) conidia congruent with Diaporthe spp. were observed on stem samples. Diseased stems from all cultivars were peeled and cut into 2 to 4 mm2 pieces, then surface sterilized in 70% ethanol for 60s, and 1% NaOCl for 60s. Samples were rinsed in sterile water, dried for 3 min and placed on potato dextrose agar (PDA). Plates were incubated at 25℃ for 5 days. 11 isolates were obtained from stem tissues of 11 plants. Isolates were cultured on PDA. 10 isolates had morphological traits consistent with the pycnidium of holotype UAMH 12076 (Allans-Perkins et al, 2020). Isolate sequences were identical, using one reinfection tests were performed on detached stem segments obtained from field grown mature plants (≥ 1 year) of cvs. Cascade and Magnum.3 wounding methods and an untreated control were used for pathogenicity testing to simulate field conditions. Each treatment had 6 replicates per cultivar and all treatments had an agar plug of inoculum applied to them, then were incubated at room temperature. Wounding with a knife involved peeling off the outer tissue layer, insect damage was simulated by using a needle to puncture the stem 3 times, and friction damage from hop support ropes was mimicked by rubbing rope against the stem. Plugs were removed 5 days post-application. Pathogenicity was observed as fungal growth and necrosis of plant tissue on all non-control methods in all samples 5 days post plug removal, fulfilling Koch's postulates. The pathogenic organism was reisolated from all inoculated tissues, except the control and identified using morphological characteristics and ITS sequence analysis. DNA amplification of the ITS region was performed according to the methods of White et al. (1990). A maximum likelihood phylogenetic tree based on the region was constructed using the MEGA10 program (Saitou & Nei, 1987). Sequence data was collected from NCBI BlastN, then aligned and trimmed. The ITS sequences (NCBI Acc. Nos. PQ555192, PQ555193, PQ555194) from the 3 isolates (UMASS001, UMASS002, UMASS003) matched 100% (543/543bp) with those of the type specimen of D. humulicola, which causes Diaporthe leaf spot and halo blight, collected in CT (NCBI Acc. No. MN152929) and 2 ex-type sequences from CT and Canada respectively (NCBI Acc. Nos. MN152927, NR172855).This is the first report of D. humulicola associated with stem blight disease. Our findings indicate that the same pathogen that infects stems can also infect cones and leaves. Therefore, Diaporthe disease management strategies should target all plant parts.

Morphological, Physiological, Biochemical, and Molecular Characterization of Fungal Species Associated with Papaya Rot in Cameroon.

Post-harvest decay of Carica papaya L. is the primary cause of deterioration in papaya quality and the low economic impact of this sector in Cameroon. Field surveys conducted by teams from the Ministry of Agriculture and Rural Development (MINADER) in Cameroon have primarily associated these decays with fungal attacks. However, to date, no methodological analysis has been conducted on the identification of these fungal agents. To reduce post-harvest losses, rapid detection of diseases is crucial for the application of effective management strategies. This study sought to identify the fungal agents associated with post-harvest decay of papaya cv Sunrise solo in Cameroon and to determine their physiological and biochemical growth characteristics. Isolation and pathogenicity tests were performed according to Koch's postulate. Molecular identification of isolates was achieved by amplification and sequencing of the ITS1 and ITS4 regions. Phylogenetic analysis was based on the substitution models corresponding to each fungal genus determined by jModeltest, according to the Akaike information criterion (AIC). Fungal explants of each identified species were subjected to variations in temperature, pH, water activity, and NaCl concentration. The ability to secrete hydrolytic enzymes was determined on specific media such as skimmed milk agar for protease, peptone agar for lipase, and carboxymethylcellulose for cellulase. These experiments allowed the identification of three fungi responsible for papaya fruit decay, namely Colletotrichum gloeosporioides, Fusarium equiseti, and Lasiodiplodia theobromae. All three pathogens had maximum mycelial growth at a temperature of 25 ± 2 °C, pH 6.5, NaCl concentration of 100 µM, and water activity (aw) equal to 0.98. The three fungal agents demonstrated a strong potential for secreting cellulases, lipases, and proteases, which they use as lytic enzymes to degrade papaya tissues. The relative enzymatic activity varied depending on the fungal pathogen as well as the type of enzyme secreted. This study is the first report of F. equiseti as a causal agent of papaya fruit decay in Cameroon.

Prevalence of Neofusicoccum parvum Associated with Fruit Rot of Mango in South Italy and Its Biological Control Under Postharvest Conditions

Botryosphaeriaceae species were recently found to be responsible for heavy mango crop losses worldwide. In 2020, mango fruit samples showing fruit decay symptoms were collected from Glenn, Kent, Irwin, Palmer, Brokaw 2, and Gomera 3 accessions in 4 orchards located in Sicily (Italy). A molecular analysis of the ITS and tub2 regions performed on 41 representative isolates allowed for the identification of mainly Neofusicoccum parvum and occasionally Botryosphaeria dothidea (1/41) as the causal agents of fruit decay. Pathogenicity proofs were satisfied for both fungal pathogens. Ripe and unripe Gomera 3 mango fruits were used to compare the virulence among the N. parvum isolates. Postharvest experiments performed on Gomera 3 fruits and by using different biocontrol agents (BCAs) showed that the performance of treatments in reducing fruit decay depends on N. parvum virulence. The data show that unregistered Wickerhamomyces anomalus WA-2 and Pichia kluyveri PK-3, followed by the trade bioformulate Serenade™ (Bacillus amyloliquefaciens QST713), were the most effective in managing mango fruit rot. This paper shows, for the first time, the potential of different BCAs, including Trichoderma spp., for the controlling of postharvest decay caused by N. parvum on mango fruits.

First Report of Rhizoctonia solani AG 2-1 Causing Root and Bulb Rot on Hymenocallis glauca in Mexico.

In Mexico, there are 29 native species of the genus Hymenocallis, including H. glauca, which is characterized by a bulb that stores carbohydrates, giving energy for the emergence of foliage and floral scapes; it is the most cultivated species and holds economic value as a potted plant and cut flower (Leszczyñska-Borys and Borys, 2001). In September 2024, a survey was conducted at the Center for Research in Horticulture and Native Plants (18°55'55.6"N 98°24'01.4"W) at UPAEP University, where there was an average temperature of 25 °C and 75% relative humidity (RH) for 10 consecutive days. Approximately 30-day old H. glauca seedlings exhibited symptoms of root and bulb rot in a 0.4 ha area, with a 45% disease incidence. Symptoms included root and bulb rot with constriction at the base of the bulb and the presence of brown mycelia. Symptomatic tissues from 50 seedlings were collected, cut into 5 mm pieces, sterilized with 3% NaClO for a minute, rinsed with sterile distilled water, and placed in Petri dishes with potato dextrose agar (PDA) medium. Samples were incubated in the dark for six days at 28 °C. An isolate was obtained from each diseased seedling using the hyphal tip method. After six days, the colonies consisted of white mycelium that turned brown with age. Right-angle branching hyphae were observed, with slight constriction at the base of the branches. The hyphae were multinucleate, containing four to nine nuclei per cell. After 15 days, some isolates produced dark brown sclerotia. Based on these morphological characteristics, isolates were tentatively identified as Rhizoctonia solani Kühn (Parmeter, 1970). To confirm the anastomosis group (AG), two isolates (RsHg4 and RsHg8) were selected for molecular identification. Genomic DNA was extracted using the CTAB protocol. The ITS region was amplified and sequenced (White et al. 1990) in both isolates, and the sequences were identical. Thus, only the sequence of isolate RsHg8 was deposited in GenBank (PQ524600). BLAST analysis of the partial ITS sequence (639 bp) showed 99.84% similarity with R. solani AG 2-1 isolate (GenBank: JF792354) (Mercado et al. 2012). Phylogenetic analysis of AGs sequences allowed assignment of the isolate RsHg8 to the AG 2-1 clade. Pathogenicity was confirmed by inoculating 50 30-day old H. glauca seedlings, grown in pots with sterile substrate. A 5 mm diameter PDA plug colonized with mycelium from the RsHg8 isolate was placed on each bulb, 10 mm below the soil surface. For control treatment, a PDA plug without fungal growth was placed on the bulb of 25 seedlings. The inoculated seedlings were incubated in a greenhouse at 28 °C and 90% RH. After six days, inoculated seedlings showed root and bulb rot with constriction at the base of the bulb. No symptoms were observed in controls. Fungus was re-isolated from the inoculated seedlings and characterized both morphologically and molecularly, yielding identical results as described above and identified as R. solani AG 2-1, thereby fulfilling Koch's postulates. Pathogenicity tests were repeated thrice. R. solani AG 2-1 has been reported infecting Allium tuberosum in Hokkaido, Japan (Misawa and Kuninaga, 2013), and Allium cepa in Morrow, Oregon (Patzek et al. 2013). To the best of our knowledge, this is the first report of R. solani AG 2-1 causing root and bulb rot in H. glauca in Mexico. Data on diseases affecting this plant is scarce, highlighting the importance of this research in developing integrated management strategies and preventing pathogen spread.

Current Methods in Synovial Fluid Microbiota Characterization: A Systematic Review.

Evidence suggests that a cross-talk between the gut microbiota and joint health exists in a paradigm known as the gut-joint axis. Recent studies have also reported the presence of microorganisms potentially involved in the pathogenesis and progression of arthritis in synovial joints, previously believed to be sterile. This systematic review describes in detail the methodologies employed to characterize the microbiota in human synovial fluid (SF). A literature search was conducted in PubMed, Embase, and Web of Science up to 5 February 2025. Nine studies aimed to characterize the SF microbiome using next-generation sequencing or polymerase chain reaction. Eight studies detected bacterial DNA in SF. However, significant heterogeneity and incomplete reporting in methodologies, including sample collection and preparation, contamination management, DNA extraction and amplification, sequencing technology, targeted 16S rRNA or ITS regions, and bioinformatics processing, limit the comparability and significance of findings. Given the potential implications for understanding arthritis mechanisms and developing targeted treatments, a standardized methodological and reporting approach in SF microbiota characterization is needed to enhance the reproducibility and the relevance of results.

Study on the changes in the microbiome before and after seed embryo after-ripening of Fritillaria cirrhosa.

Microorganisms play an important role in the embryonic development of plant seeds; however, there are no existing reports on the microbial communities associated with Fritillaria cirrhosa before and after embryo after-ripening. In this study, the microbial communities of Fritillaria cirrhosa seeds before and after after-ripening were analyzed using the Illumina MiSeq platform, targeting the V4-V5 region of the bacterial 16S rRNA gene and the ITS1 and ITS2 regions of fungal ribosomal RNA. The results showed that bacterial communities were more susceptible to environmental stress and exhibited greater fluctuations compared to fungal communities, as reflected in higher diversity and significant changes in the relative abundance of dominant genera and species. After embryo after-ripening, the dominant fungal genera were Botrytis (SBAR, 29.35%), Tetracladium (SBAR, 15.86%), Ilyonectria (SBAR, 15.35%), and Mrakia (SBAR, 13.14%), while the dominant bacterial genera were Pseudomonas (SBAR, 26.69%) and Stenotrophomonas (SBAR, 16.30%).Prediction results suggested that the bacterial communities with sharply increased relative abundance after embryo after-ripening may interact with seeds through various pathways, including carbohydrate metabolism, absorption and utilization of nitrogen (N), sulfur (S), phosphorus (P), and iron (Fe), as well as secretion of antibiotics, vitamins, cytokinins, and amino acids. Functional validation revealed that most culturable fungi with sharply increased relative abundance had cellulase-degrading abilities, while most of the bacterial isolates were capable of absorbing and utilizing C, N, S, P, and Fe elements. Microbial co-occurrence network analysis indicated that the microbiome after embryo after-ripening formed an unstable, expansive, and rapidly changing network. In summary, this study revealed the overall dynamics of the microbiome in Fritillaria cirrhosa seeds after embryo after-ripening and identified key microbial taxa exhibiting sharp shifts in relative abundance. This work provides a foundational understanding of the microbial succession associated with seed embryo after-ripening in Fritillaria cirrhosa, which may support seed after-ripening and germination, and enhance seed stress resistance.

Pseudosperma umbonatum sp. nov. from Himalayan forests of Pakistan

A new species, Pseudosperma umbonatum was collected from different localities of Himalayan region of Khyber Pakhtunkhwa and Punjab in Pakistan. The species is characterized by its fibrillose pileus, ovoid to ellipsoid basidiospores (7.2–7.9 × 4.7–5.4 µm), and clavate to fusiform or ventricose septate cheilocystidia. Molecular phylogenetic analysis of DNA sequences from ITS and LSU regions supported it as a new species.

Phylogeny and new sectional classification for the Cape Clade of the genus Indigofera (Fabaceae: Indigofereae)

AbstractThe genus Indigofera in the Greater Cape Floristic Region (GCFR) comprises a diverse assortment of species. Almost 90% of the region's Indigofera species belong to the Cape Clade, while the remaining species are scattered among the other three globally distributed Indigofera clades. As a prelude to a species‐level revision, we aimed to revise the sectional classification of the Cape Clade, making use of molecular and morphological data. We present near‐complete sampled phylogenies representing ca. 95% of species within the GCFR, using the nuclear ITS region and three plastid regions. Ancestral state reconstructions identified several morphological characters that, as unique suites of traits, can help to distinguish different sections/subsections, rather than single diagnostic traits. A total‐evidence phylogeny based on both morphology and molecular data strongly supports the recognition of eight sections within the Cape Clade. Indigofera sect. Brachypodae, sect. Digitatae, sect. Juncifoliae, sect. Oligophyllae and sect. Productae are maintained and refined from previous sectional classifications. Indigofera cytisoides, I. merxmuelleri and I. nudicaulis are circumscribed into three new monospecific sections (I. sect. Cytisoidae, sect. Merxmuelleranae, sect. Nudicaules), based on suites of unique morphological characters. Indigofera sect. Digitatae and sect. Brachypodae are the most diverse sections, and are each further divided into four subsections.

High Nitrogen Fertilization Suppresses Dollar Spot in Amenity Turfgrass Through Promoting Oxalic Acid-Degrading Bacteria.

High nitrogen (N) fertilization can suppress dollar spot (Clarireedia spp.) disease of amenity turfgrass. However, golf course superintendents avoid high N fertilization due to the reduced playability of putting greens and concerns over increased cost and environmental contamination issues. Understanding how N fertilization suppresses dollar spot could result in novel control strategies that do not rely on high N fertilization or repeated pesticide applications. In 2017, we sampled a previous 3-year study conducted in Madison, WI, on turfgrass treated with either 0, 4.9, and 29.3 kg N/ha/yr. Dollar spot was assessed every two weeks during the study period, and microbiome samples were collected from each plot at 6 different time points during 2017. The bacterial 16s rRNA gene and fungal ITS region were sequenced using high-throughput sequencing, and the frc gene copy number was quantified using droplet digital PCR. The frc gene was previously established as a biomarker for oxalic acid-degrading bacteria. The 29.3 kg N/ha treatment reduced disease severity and had higher frc gene expression than lower N rates. Prokaryotic alpha diversity significantly declined under 29.3 kg N/ha, while fungal diversity remained relatively stable. N treatment significantly affected prokaryotic beta diversity but not fungal. The results suggest that N may have a role in selecting microbes that degrade oxalic acid (OA), an important virulence factor of C. jacksonii. Identifying the specific organisms involved in OA degradation may result in new biocontrol strategies and improve the sustainable management of dollar spot.

Phylogenetic analyses and bioactivities of endophytic Pestalotiopsis from Amazonian Species

The genus Pestalotiopsis shows its potential for bioactivity applications; this potential may originate from the close relationship with its host species. In this study, we verified the efficiency of the ITS regions for phylogenetic analysis of Pestalotiopsis species isolated as endophytes associated with basidiomycetes. Endophytic fungal strains were isolated from the genera Gustavia, Rollinia, Euterpe, Myrcia, Arrabidaea and a species of Pinus. A part of this investigation was to evaluate the degree of proximity between the isolated strains and their hosts, suggesting that proximity to the host plant could improve the chance to produce bioactive compounds. Secondary metabolites were tested for larvicidal, cytotoxic and, antifungal activity. This work demonstrates the capacity of the genus Pestalotiopsis to produce substances with larvicidal activity against larvae (L3) of Aedes aegypti. In addition, there are other biological activities, showing that the genus is a producer of biologically active substances with potential pharmacological interest.