Pathogenicity Tests Research Articles

First Report of Choanephora cucurbitarum Causing Blossom Blight on Passion Fruit (Passiflora edulis Sims) in China.

Passion fruit (Passiflora edulis Sims), which widely grows in tropical and subtropical regions (Wang et al., 2023; Xu et al., 2023), is an economically important fruit with large-scale cultivation in Guangxi, China. In September 2022, symptoms of blossom blight were observed on passion fruit (cultivar Tainong No. 1) in the commercial planting areas in Guangxi (23°15'9''N, 108°3'30''E). The incidence of this disease was approximately 30% in a field with a 2.5 ha planting area. Initially, there were water-soaked spots on the infected flowers, which progressed to a wet rot under high humidity, and the diseased flowers were covered with white mycelia and dark masses of sporangiophores bearing brown to black sporangia and sporangiola. Three fungal isolates (C9-23-1, C9-23-2, and C9-23-3) were isolated from three symptomatic blossoms, respectively. Colonies on potato dextrose agar (PDA) medium were white and cottony, and later became pale yellow, covering the 90 mm plate in three days at 28°C. Sporangiophores were aseptate, hyaline, and unbranched. Sporangia were globular to ellipsoid, brown to dark brown, 96.1 to 102.1 µm in length, and 88.6 to 95.8 µm in width (n =15). Sporangiospores from sporangia were brown to dark brown, ellipsoid to ovoid, distinctly longitudinally striate, about 7.4 to 17.8 µm wide and 11.0 to 25.8µm long (n = 30). Sporangiola were brown to dark brown, oval or spindle-shaped, 7.4 to 17.8 µm wide, and 15.5 to 29.8 µm long (n=30). These characteristics were consistent with the descriptions of Choanephora cucurbitarum (Park et al., 2016). To confirm the identity, the internal transcribed spacer (ITS) region and the large subunit (LSU) of ribosomal DNA (rDNA) were amplified and sequenced with the universal primer ITS1/ITS4 and LROR /LR5 (White et al.1990; Rehner & Samuels 1994), respectively. The BLAST results showed that the ITS and LSU sequences shared 99.83% to 100% identity with the corresponding sequences of C. cucurbitarum strain CBS 178.76. Moreover, a phylogenetic tree based on the two genes revealed that all three isolates were closest to C. cucurbitarum. Based on these results, the isolates were identified as C. cucurbitarum. Then pathogenicity tests were conducted by spraying six flowers with a sporangiospore suspension (1 × 105 spore/mL), which were then kept in the greenhouse (28°C, 85% to 95% RH). Control flowers were sprayed with sterile water. After three days, sporulation of C. cucurbitarum was present on all symptomatic flowers similar to that was observed in the field, while control flowers were asymptomatic. The isolates from artificially infected plants were confirmed as C. cucurbitarum through morphological identification, thus fulfilling Koch's postulates. C. cucurbitarum has a wide range of hosts, causing fruit and blossom rot in cucurbits and other plants, including cabbage, ice plant, beans, potato, cauliflower, okra and lettuce, Pinellia ternata, Crotalaria spectabilis, Crotalaria breviflora, etc ( Alfenas et al., 2018; Min et al., 2017; Oliveira et al., 2021; Ryu et al., 2022; Wang et al., 2021). However, to our knowledge, this is the first report of C. cucurbitarum causing blossom blight on passion fruit in China and worldwide. Guangxi is the biggest producer of passion fruit in China. Many growers in the region are reporting similar symptoms in their passion fruit plantations. This study will provide valuable information for growers and plant pathologists to improve the management of blossom blight.

Pseudomonas boreofloridensis sp. nov., and Pseudomonas citrulli sp. nov., isolated from watermelon in Florida.

Three fluorescent bacterial strains, K1, K13 and K18, were obtained from watermelon (Citrullus lanatus) foliage symptomatic of bacterial leaf spot of cucurbits in Florida. The strains underwent phenotypic characterization, including LOPAT (levan production, oxidase activity, pectolytic activity on potato, arginine dihydrolase production and hypersensitive response (HR) on both tobacco and tomato) and pathogenicity testing on watermelon and squash seedlings. Whole-genome sequencing of the isolates was performed, and multi-locus sequence analysis (MLSA) utilizing housekeeping genes gltA, rpoD, gapA and gyrB placed the isolates into two distinct clades within the Pseudomonas genus. Average nucleotide identity based on blast (ANIb) was used to compare the isolates to Pseudomonas reference genomes. Using ANIb, the closest relatives to the novel strains were identified as Pseudomonas wayambapalatensis (K1 : 82.58%; K13 : 83.77%) and Pseudomonas kilonensis (K18 : 87.16%), although ANIb values were below the 95% threshold for species delineation. DNA-DNA hybridization (genome-genome distance calculation method), comparison to the online Type Genome Server, Biolog biochemical profiling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were also unable to identify the isolates as any known species of Pseudomonas. Based on the combination of genetic and phenotypic data, we conclude that these isolates represent two novel Pseudomonas species, for which we propose the names Pseudomonas boreofloridensis sp. nov. (K1, K13T, NCPPB 4759=LMG 33364) and Pseudomonas citrulli sp. nov. (K18T, NCPPB 4761=LMG 33365). The specific epithet boreofloridensis was chosen for the geographic location of isolation (northern Florida), while citrulli designates the host of origin (C. lanatus).

Morphological and molecular identification of Schizophyllum commune causing storage bulb rot disease of Lanzhou edible lily in China and its biological characteristics

Lily bulb rot disease has harmed edible lily in recent years, resulting in yield losses in China. As a results, both morphological and molecular techniques must be used to confirm the etiology of storage bulb rot disease on lily bulbs. Lily bulbs with indications of rot symptoms during storage were gathered in Lanzhou, Gansu Province, China. A strain was isolated and its morphologically characterized as a Schizophyllum commune specie. Pathogenicity tests further confirmed that the strain caused apparent S. commune symptoms on lily bulbs which were consistent with those seen in the field. The pathogenicity of S. commune to lily bulb was 100%, and morphological identification showed that the mycelia of the pathogen were white and villous, with septate, branched, and acicular bodies and obvious lock-shaped joints. The mycelia had uneven thickness, ranging from 1.03 to 3.06 μm, and turned gray-white at the later growth stage. Moreover, the pathogen formed a mycelial column on potato dextrose agar (PDA) medium, which formed a coral-like fruiting body primordium, which split, forming pileus for spore production. The spores were colorless and cylindrical, with an oblique tip and a size of 4.6–7.2 μm × 2.2–2.7 μm. The isolate was deduced as based on phylogenetic analysis with 2 genes (ribosomal DNA-ITS and LSU) as well as morphological characteristics and cultural features, the isolate was identified S. commune. Soluble starch, yeast extract, temperature of 30°C, pH 7, relative humidity of 100%, and complete dark were shown to be the optimum culture conditions for surface mycelium growth. In conclusion, this is the first report of S. commune causing bulb rot of edible lilies in China. The study provides a basis for more effective control strategies for lily bulb rot disease.

Case report: Satralizumab as an adjunctive therapy for AQP-4 antibody and MOG antibody dual-negative optic neuritis in a third-trimester pregnancy case

The treatment of demyelinating optic neuritis (DON) in pregnant patients is challenging, especially when there is poor or no response to intravenous methylprednisolone pulse (IVMP) therapy or adjunctive treatments such as intravenous immunoglobulin (IVIG) therapy. We herein report a case of a 28-year-old pregnant woman who experienced sequential severe vision loss in both eyes. She presented to a local hospital with the main complaint of sudden, painless vision loss in the left eye and was diagnosed with DON in the left eye. However, she did not receive orbital MRI or IVMP therapy due to safety concerns. Upon admission to our hospital, her visual acuity was 20/30 in the right eye and there was no light perception in the left eye. Her right eye vision deteriorated rapidly, declining to 20/1,000 one day after the admission. The ophthalmic examination revealed a normal anterior segment and a swollen optic disk in the right eye and a dilated pupil with a relative afferent pupillary defect and a swollen optic disk in the left eye. The serological tests for common pathogens, including the aquaporin-4 antibody (AQP-4 Ab), myelin oligodendrocyte glycoprotein antibody (MOG-Ab), and other common autoantibodies, were all negative. The patient was clinically diagnosed with DON in both eyes and received 7 days of IVMP therapy and 4 days of IVIG therapy, but showed no visual improvement. A three-dose regimen of satralizumab 120 mg was then administered subcutaneously during the acute stage of DON, in combination with a slowly tapered oral methylprednisolone regimen. Moreover, 2 months after the first injection of satralizumab, the patient naturally gave birth to a healthy female infant weighing 2,305 g at 36 weeks and 1 day of gestation. Her visual acuity improved to 20/500 in both eyes and slightly increased to 20/320 in both eyes 2 months later. Her visual acuity remained stable during subsequent follow-up visits. The infant was fed formula milk powder and developed normally. No systemic or ocular side effects related to satralizumab therapy were observed in the patient or her fetus during the 9-month follow-up. Our findings in this case suggest that satralizumab may be a safe and efficient adjunctive therapy for pregnant patients with DON who poorly respond to IVMP and IVIG therapy, even in cases of dual-negative AQP-4 Ab and MOG-Ab.

Rio Mamore Hantavirus Endemicity, Peruvian Amazon, 2020.

To explore hantavirus infection patterns in Latin America, we conducted molecular and serologic hantavirus investigations among 3,400 febrile patients from Peru during 2020-2021. Reverse transcription PCR indicated that a patient from Loreto, in the Peruvian Amazon, was positive for Rio Mamore hantavirus (serum, 3.8 × 103 copies/mL). High genomic sequence identity of 87.0%-94.8% and phylogenetic common ancestry with a rodent-associated Rio Mamore hantavirus from Loreto in 1996 indicated endemicity. In 832 samples from Loreto, hantavirus incidence based on IgM ELISA of pooled Sin Nombre (SNV) and Andes virus (ANDV) nucleoproteins and immunofluorescence assay-based end-point titration using SNV/ANDV/Hantaan/Puumala/Saarema/Dobrava/Seoul hantaviruses was 0.5%. Across 3 ecologically distinct departments in Peru, SNV/ANDV IgG ELISA/IFA-based reactivity was 1.7%, suggesting circulation of antigenically distinct New World hantaviruses. Testing for arboviruses, nonendemic pathogens, and antigen-free ELISA corroborated nonspecific reactivity in 2 IgG and several IgM ELISA-positive serum samples. Hantavirus diagnostics and surveillance should be strengthened in Peru ad across Latin America.

Distribution and Antimicrobial Susceptibility Profiles of Pathogens in Patients With Esophageal Cancer From 2013 to 2022: A Retrospective Study.

Pathogenic microbial infections are closely related to the development and prognosis of esophageal cancer. The distribution and resistance of pathogens in different diseases are regional and gradually change over time. This study aimed to determine the distribution and drug resistance of pathogens isolated from patients with esophageal cancer and provide a reference for the rational use of antibiotics. The results of strain identification and antimicrobial susceptibility testing of pathogens in patients with esophageal cancer from January 2013 to December 2022 at our hospital were retrospectively analyzed. SPSS Statistics 26.0 (IBM) and R software 4.3.1 were used for data analysis. In total, 2322 non-repetitive pathogens were isolated from 14,037 samples. Of all strains, 1713 (73.77%) were Gram-negative bacteria, 483 (20.80%) were Gram-positive bacteria, and 126 (5.43%) were fungi. The top 10 pathogens were Pseudomonas aeruginosa (19.81%), Stenotrophomonas maltophilia (12.88%), A. baumannii (9.91%), Klebsiella pneumoniae (9.82%), Staphylococcus aureus (7.54%), Candida albicans (3.92%), Staphylococcus epidermidis (3.19%), Escherichia coli (3.14%), Enterococcus faecalis (2.97%), and Serratia marcescens (2.15%). The isolation rate of S. maltophilia showed an upward trend (p < 0.05). The resistance rates of P. aeruginosa, S. maltophilia, A. baumannii, and Enterobacteriaceae bacteria to some common antibiotics showed a tendency to change (p < 0.05), and 2019 became a turning point to some extent. All common Gram-positive pathogens were sensitive to vancomycin, except for three Enterococcus spp. isolates that showed intrinsic resistance. The prevalence of MRSA was 65.14% (114/175) in this study. In addition, the resistance rates of MRSA and MSSA to moxifloxacin, ciprofloxacin, levofloxacin, erythromycin, clindamycin, and penicillin were significantly different (p < 0.001). Pathogens are diverse in patients with esophageal cancer, with the most common being P. aeruginosa, followed by S. maltophilia. The pathogens exhibited different patterns of resistance. Antibiotics should be used rationally according to pathogen resistance patterns.

Characterization of Bacterial Blight Pathogen on Rice Plants in Sidoarjo

Rice (Oryza sativa L.) is a staple crop for all Indonesian people. Rice production in Indonesia in 2019 experienced a decline in production. Plant disease attacks are a factor that influences the decline in rice production. Bacterial leaf blight is an important disease in rice plants that can cause yield losses of up to 50%. Characterization of disease-causing bacteria needs to be carried out to develop appropriate, effective and efficient control in suppressing the development of bacterial leaf blight. The aim of this research was to characterize the morphological and physiological characteristics of the bacteria that cause bacterial leaf blight. The method used in this research included the isolation of bacteria that cause bacterial leaf blight disease carried out on rice plantations in Pulungan Village, Sedati, Sidoarjo. Identification is carried out through macroscopic and microscopic observations, analysis of physiological properties through the gram staining test, fermentative oxidative test, yellow colony test on YDC media, urease test, hypersensitivity test, and pathogenicity test. The results of the research show that the bacteria that cause bacterial leaf blight have the morphological characteristics of being yellow, round and shiny. The physiological characteristics of the bacterial isolate are gram-negative bacteria, are oxidative, yellow in YDC media, and react negatively in the urease test.

EFFECTIVENESS OF TRICHODERMA SPECIES IN THE MANAGEMENT OF SUGARCANE WILT (FUSARIUM SACCHARI)

Sugarcane is a major cash crop in Pakistan. It has a great contribution in world’s economy. Many diseases cause many losses in yield of sugarcane. Fungi are considered as abundant traitorous pathogens that obstruct the viable growth of sugarcane crop. Wilt disease caused by fungal pathogen Fusarium sacchari caused many losses in sugarcane production in Pakistan. Fusarium sacchari is a causative fungus of wilt in sugarcane. Biocontrol agent Trichoderma spp. used in the management of sugarcane wilt. These bio‐agents help in diseases management and play important role in increasing the crop yield. For this reason, these bio‐agents are used for biological maintaenace of crop diseases and research globally. The use of PDA medium in vitro revealed antagonistic activity of various Trichoderma isolates against the sugarcane wilt pathogen Pathogenicity test also executed to check the most aggressiveness strain of Fusarium spp. to cause disease in sugarcane plant. In vivo, Trichoderma multiply on different substrates and corn Cobbs is good byproduct Trichoderma multiply on it and mix in soil of pots. Pot experiment performed with different treatments to control wilt disease by using of antagonist’s to check disease incidence and disease reduction.

Leaf Spot Caused by Alternaria spp. Is a New Disease of Grapevine

In this study, we carried out large-scale leaf spot symptom observation on vineyards in the Krasnodar Krai of Russia and determined their distribution. The incidence and severity of leaf spot were higher on the Euro-American grapevine hybrids (Bianka, Levokumskij, Avgustin, Moldova, Pervenets Magaracha, Dunavski lazur). A total of 433 isolates that belonged to the genus Alternaria were isolated from samples with leaf spot. Pathogenicity testing confirmed the ability of the representative isolates to cause necrosis on the grapevine. The isolates of Alternaria sp. were typed by the loci of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), Alternaria allergen a1 (Alt a1), β-tubulin (tub), and translation elongation factor (tef1). Isolates from grapevine causing leaf spot were shown to cluster with isolates of Alternaria. alternata (Fr.) Keissl. and Alternaria. arborescens E.G. Simmons species complex. Of the fungicides tested to inhibit Alternaria growth, the most effective were mixtures, such as pyrimethanil and fluopyram, cyprodinil and fludioxonil, and those that included difenoconazole. The results of the study expand our knowledge of the biodiversity of Alternaria sp. fungi and can be used to limit the spread of Alternaria leaf spot of the grapevine.

Bacteriophage-Activated DNAzyme Hydrogels Combined with Machine Learning Enable Point-of-Use Colorimetric Detection of Escherichia coli.

Developing cost-effective, consumer-accessible platforms for point-of-use environmental and clinical pathogen testing is a priority, to reduce reliance on laborious, time-consuming culturing approaches. Unfortunately, a system offering ultrasensitive detection capabilities in a form that requires little auxiliary equipment or training has remained elusive. Here, a colorimetric DNAzyme-crosslinked hydrogel sensor is presented. In the presence of a target pathogen, DNAzyme cleavage results in hydrogel dissolution, yielding the release of entrapped gold nanoparticles in a manner visible to the naked eye. Recognizing that Escherichia coli holds high relevance within both environmental and clinical environments, an E. coli-responsive DNAzyme is incorporated into this platform. Through the optimization of the hydrogel polymerization process and the discovery of bacteriophage-induced DNAzyme signal amplification, 101 CFU mL-1 E. coli is detected within real-world lake water samples. Subsequent pairing with an artificial intelligence model removed ambiguity in sensor readout, offering 96% true positive and 100% true negative accuracy. Finally, high sensor specificity and stability results supported clinical use, where 100% of urine samples collected from patients with E. coli urinary tract infections are accurately identified. No false positives are observed when testing healthy samples. Ultimately, this platform stands to significantly improve population health by substantially increasing pathogen testing accessibility.

Identification of Pseudopestalotiopsis ampullacea as a new pathogen causing tea gray blight in India and its management strategies

Gray blight is a serious threat to the tea [Camellia sinensis (L.) O. Kuntze] production in major tea cultivating countries including India. The disease is caused by Pestalotiopsis-like species. In this study, five isolates of Pseudopestalotiopsis species isolated from symptomatic tea leaf samples in North Bengal, India were investigated. Based on the multi-locus phylogenetic analysis using concatenated sequences of three (ITS, tef-1 alpha, and tub-2) loci, cultural and micromorphological characters, and host association, the fungal isolates were identified as Pseudopestalotiopsis ampullacea F. Liu & L. Cai. The morphological analysis also revealed that the fungal isolates were evidently differentiated from other Pseudopestalotiopsis species. To date, P. ampullacea has not been reported on tea plants in India. Among the five isolates studied, isolate NKT0P03 was randomly selected for pathogenicity tests and its sensitivity to fungicides and microbial antagonists. In pathogenicity test, the isolate showed weak to high virulence reactions on 25 different tea cultivars. The pathogen showed an avirulent reaction on the cultivar TV11. In order to identify an effective management strategy against this new pathogen, synthetic fungicides and microbial biocontrol agents were evaluated in the laboratory. Results revealed that carbendazim + mancozeb, hexaconazole, propiconazole, and valextra were effective fungicides with an 85.1% to 89.8% range of inhibitory activity against P. ampullacea NKT0P03. Among microbial agents, Trichoderma harzianum, T. reesei, T. hamatum, Bacillus subtilis, and Microbacterium barkeri were efficient bioagents against P. ampullacea NKT0P03 with antagonistic activity ranging between 66.6% and 84.2%. Thus, these fungicides and microbial bioagents can be recommended as effective agents for the management of P. ampullacea causing tea gray blight after their field evaluations.

Significance of respiratory virus coinfection in children with Mycoplasma pneumoniae pneumonia

ObjectiveMycoplasma pneumoniae is a major causative pathogen in community-acquired pneumonia. Respiratory viral coinfections in children with Mycoplasma pneumoniae pneumonia (MPP) are not uncommon and cause severe clinical manifestations. This study aims to investigate the impacts of viral coinfection in MPP patients and hopes to offer novel insights for discriminating between MPP and MPP coinfection.MethodsThis study recruited 748 children hospitalized for MP pneumonia between January 2021 and October 2023. Patients were classified into two groups: MPP coinfected with respiratory virus group and MPP group. All children underwent polymerase chain reaction testing for respiratory pathogens. Baseline clinical features and demographic data were obtained retrospectively through medical records.ResultsThe retrospective study included 748 patients, with a viral coinfection rate of 38.75%. Patients in the MPP coinfected with respiratory virus group have a higher disease burden than those in the non-coinfection group. Our findings indicate that patients with Mycoplasma pneumonia co-infected with respiratory viruses had longer hospital stays and prolonged fever post-admission, as well as more severe conditions and a higher incidence of extrapulmonary complications. MPP coinfection was associated with the following factors: patients with extrapulmonary complications of gastroenteritis (OR = 4.474, 95%CI = 1.733–11.554, P = 0.002), longer hospital stay (OR = 1.109, 95%CI = 1.012–1.217, P = 0.027), longer days of fever after admission (OR = 1.215 95%CI = 1.006–1.469, P = 0.043), elevated white blood cell count (OR = 1.332 95%CI = 1.082–1.640, P = 0.007), decreased neutrophil count (OR = 0.768 95%CI = 0.602–0.981, P = 0.035), higher fibrinogen levels (OR = 1.652 95%CI = 1.138–2.398, P = 0.008), and raised lactate dehydrogenase levels (OR = 1.007 95%CI = 1.003–1.011, P = 0.001).ConclusionsWe determined the clinical significance of respiratory viral coinfection in children with MPP. Timely identification of MPP coinfection and provision of early and comprehensive therapeutic measures are vital in shortening the disease severity and improving prognosis.

The Phyllosphere Microbial Community Structure of Three Camellia Species upon Anthracnose

Anthracnose of Camellia plants is caused by the Colletotrichum species. The fungal pathogens mainly infect the leaves of plants and lead to serious economic losses. However, knowledge of Camellia phyllosphere microbial community after Colletotrichum infection has not been explored which limited our understanding of the relationship between the Camellia anthracnose outbreak and interacting microorganisms. In this study, three economically and ecologically important Camellia species with anthracnose symptoms were collected and subjected to bacterial and fungal composition analysis, diversity, co-occurrence characteristics, isolation of key strains, and tie-back pathogenicity test. The results indicated that Sphingomonas and Methylobacterium were the dominant bacterial genera over the three Camellia species and Pallidocercospora, Colletotrichum, and Pichia were the dominant fungal genera. The co-occurrence analysis showed that Methylobacterium, Sphingomonas, Massilia, and Allorhizobium were the key bacterial taxa and Colletotrichum, Pallidocercospora, Pichia, Septophoma, and Septoria were the key fungal taxa over the three infected plants. The hub taxa, including the species significantly associated with the Colletotrichum abundance, were mostly beneficial bacteria over the three Camellia species. Further co-culture and tie-back pathogenicity tests verified that the hub taxa associated with pathogenic Colletotrichum in the microbial networks may play promoting/inhibiting roles on Colletotrichum infection. The results highlight the importance of phytopathological conditions for the interactions between microbial members of foliar fungal and bacterial communities.

Identification and characterization of Epicoccum sorghinum causing leaf spot on sorghum in China

Sorghum[Sorghum bicolor (L.)Moench] is one of the most important food crops and also used as forage, brewing and industrial raw materials in China. In recent years, during field surveys a new leaf spot was detected on sorghum plants in Liaoning Province, China. The pathogen was isolated used a conventional pathogen isolation method and identified by its morphology cultural characters and phylogenetic analysis of the combined genes including the internal transcribed spacer (ITS), β-tubulin (TUB) and Large subunit (LSU) sequences, respectively. Based on morphological characteristics and phylogenetic analyses, the pathogenic fungus was identified as Epicoccum sorghinum. Pathogenicity test was confirmed by inoculating sorghum cultivars under greenhouse conditions. This study is the first time to confirmed E. sorghinum as the causal agent of the disease in China, and this disease represents a potential threat to sorghum productivity in China.

Spatial and temporal distribution of Ixodes scapularis and tick-borne pathogens across the northeastern United States

BackgroundThe incidence of tick-borne diseases is increasing across the USA, with cases concentrated in the northeastern and midwestern regions of the country. Ixodes scapularis is one of the most important tick-borne disease vectors and has spread throughout the northeastern USA over the past four decades, with established populations in all states of the region.MethodsTo better understand the rapid expansion of I. scapularis and the pathogens they transmit, we aggregated and analyzed I. scapularis abundance and pathogen prevalence data from across the northeastern USA, including the states of Connecticut, Maine, New Hampshire, New York and Vermont, from 1989 to 2021. Maine was the only state to collect data during the entire time period, with the other states collecting data during a subset of this time period starting in 2008 or later. We harmonized I. scapularis abundance by county and tick season, where the nymph season is defined as May to September and the adult season is October to December, and calculated I. scapularis pathogen infection prevalence as the percentage of ticks that tested positive for Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, and Borrelia miyamotoi. We then explored temporal trends in I. scapularis abundance and pathogen prevalence data using linear models.ResultsThe resulting dataset is one of the most spatially and temporally comprehensive records of tick abundance and pathogen prevalence in the USA. Using linear models, we found small or insignificant changes in the abundance of nymphs and adults over time; however, A. phagocytophilum, B. microti and B. burgdorferi prevalence in both nymphs and adults has increased over time. For the period 2017–2021, the statewide average prevalence of B. burgdorferi ranged from 19% to 25% in I. scapularis nymphs and from to 49% to 54% in I. scapularis adults. The statewide average prevalence of all other pathogens in I. scapularis for 2017–2021, including A. phagocytophilum (4–6% for nymphs, 4–9% for adults), B. microti (4–8% for nymphs, 2–13% for adults) and B. miyamotoi (1–2% for nymphs, 1–2% for adults), was considerably less.ConclusionsOur efforts revealed the complications of creating a comprehensive dataset of tick abundance and pathogen prevalence across time and space due to variations in tick collection and pathogen testing methods. Although tick abundance has not changed along the more southern latitudes in our study over this time period, and only gradually changed in the more northern latitudes of our study, human risk for exposure to tick-borne pathogens has increased due to increased pathogen prevalence in I. scapularis. This dataset can be used in future studies of I. scapularis and pathogen prevalence across the northeastern USA and to evaluate models of I. scapularis ecology and population dynamics.Graphical

First Report of heart rot disease on Pineapple Caused by Phytophthora nicotianae in Guangxi province, China.

Pineapple (Ananas comosus) is a perennial economically important tropical fruit in Guangxi Province, China. Between September to November 2022, the rot of heart leaves of pineapple was found on 6.7 hectares of pineapple, NaLang village Jiangxi Town, Jiangnan District, Nanning City and on 200 hectare Liutian Village, Wangmao Town, Bobai County, Yulin City. The disease incidence of the investigated 10500 plants was from 20% to 30%. The basal heart leaves near the soil surface showed symptoms of water-soaked lesions and soft rot. The infected heart leaves were easily pulled out. As the disease developed, 800 plants of the investigated diseased plants collapsed and died. The diseased leaves from each district were surface-disinfected for 3 min with 1.0% hypochlorite solution, then rinsed three times in sterile water, and plated to carrot agar medium (CA media) (Luo et al. 2012). Inoculated dishes were incubated at 28°C in the dark. After 5 days, Eight mycelium colonies were recovered from diseased tissue. Colonies developing on CA exhibited white cottony mycelium which was aseptate. Numerous sporangia and chlamydospores were visible after 10-15 d later. The chlamydospores were spherical in shape and measured an average of 25.8 to 37.5 μm. The shape of the sporangia was papillate, oval or spherical, and 34.5 to 58.2 μm. These characteristics were consistent with the description of Phytophthora (Stamps et al. 1990). For molecular identification, the direct colony PCR method (Lu et al. 2012) was used to amplify the ITS of rDNA, β-tubulin (Tu), and elongation factor (EF) locus of the 2 strains NNPN1 from Nanning city and YLPN1 from Yulin city using the primer pairs of ITS4/ITS5, T1/T2 (O'Donnell et al. 1997) and EF1/EF2 (O'Donnell et al. 1998). The resulting sequences were deposited in GenBank (OR612028 and OR612029 for ITS; OR620158 and OR620159 for elongation factor; OR620160 and OR620161 for β-tubulin). BLAST searches with the three gene sequences revealed the greatest similarity with the sequences of some Phytophthora sp. The complete genome sequences of highly similar Phytophthora sp. were downloaded and the sequences of the corresponding 3 genes of each genome was extracted by Bioedit software. Phylogenetic trees were constructed in MEGA 11 using the maximum likelihood (ML) method based on the concatenated sequences of ITS, EF1α, and Tubulin (Figure 1). The 2 isolates grouped with P. nicotianae in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolate was P. nicotianae. Tests of pathogenicity were performed according to Koch's postulates using the P. nicotianae strains NNPN1 and YLPN1. Fresh wounds were made symmetrically with a sterile needle on 2 tender basal leaves of pineapple plants (Bali variety) at the 8- to 10-leaf stage and each wound was covered with a piece of mycelium disc from each isolate. Control seedlings were inoculated identically except CA medium disc was used to cover the wound. Plants were placed in pots in a greenhouse at 28°C and 90% relative humidity. After 12 days, soft rot was observed clearly on the base of heart leaves of 9 inoculated plants, while the 9 control plants appeared normal. The pathogenicity tests were conducted three times with similar results. The strains were then reisolated from the infected plants and found to be Phytophthora sp. as those of the inoculum by morphology. P. nicotianae was previously reported as the causal agent of heart rot of pineapple in Guangdong and Hainan Province of China (Luo et al. 2012; Shen et al. 2013). To our knowledge, this is the first report of P. nicotianae on pineapple in Guangxi province in China. Identification of the pathogen of heart rot disease on pineapple is vital to reduce yield losses of the disease using effective control method.

First Report of Pythium dissotocum Causing Root Rot on Nandina in Tennessee and the United States.

Nandina (Nandina domestica) is a popular landscape plant found extensively beyond the southeastern and southwestern parts of the United States (Wilson et al. 2021). In April 2024, one-year-old nandina 'Fire Power' plants grown in containers at a commercial nursery in Warren County, Tennessee, exhibited severe leaf yellowing, stunted growth, and brown to black root rot lesions. Disease severity affected 65% of the root area, with an incidence of 80% of 450 plants. Symptomatic eight plants' root tissues were surface-sterilized with 70% ethanol and washed twice with distilled water. Small sections of root tissues were placed in Petri dishes containing V8 agar (V8A) and incubated at 24°C in an 8-hour photoperiod cycle. White, radial mycelial growth were observed within 3-4 days of incubation. The aseptate hyphae ranged from 1.81 to 7.21 μm in diameter. The filamentous sporangia formed dendroid-like branches. Oogonia were subglobose, measured 15.06 ± 0.65 μm (n=50) in diameter. Oospores were globose and measured 4.58 ± 0.44 μm (n=50). The isolates (FBG7211 and FBG7216) were identified as Pythium dissotocum based on morphological characterization (Van der Plaats-Niterink 1981). For molecular identification, total DNA was extracted using the DNeasy PowerLyzer Microbial Kit (Qiagen, MA) from 7-day-old cultures of the isolates FBG7211 and FBG7216 grown on V8A. The primer pairs ITS1/ITS4 (White et al. 1990), OomCoxI-Levup/Fm85mod (Robideau et al. 2011), Cox2F/Cox2RC4 (Choi et al. 2015) and Oom-Btub-up415/Oom-Btub-lo1401 (Bilodeau et al., 2007) were used to amplify and sequence isolates using four genetic markers, including the ribosomal internal transcribed spacer (ITS), the mitochondrial cytochrome c oxidase subunit I (COX1), cytochrome c oxidase subunit II (COX2), and nuclear beta-tubulin (BT), respectively. The 100% coverage of sequences (GenBank accession nos. PQ329213 and PQ284875 for ITS; PQ349316 and PQ349317 for COX1; PQ349318 and PQ349319 for COX2; PQ349314 and PQ349315 for BT) were 100%, 99.43%, 100%, and 99.56% Identical to P. dissotocum sequences of ITS, COX1, COX2, and BT genetic markers in the GenBank using core nt BLAST search (ITS: MT348554; COX1: MZ027313; COX2: MG719859; BT: KJ595479). Pathogenicity tests were performed on 1-year-old nandina 'Fire Power' seedlings grown in 1-gal containers to fulfill Koch's postulates. Each seedling was drench-inoculated with a 150 mL/plant of 2 x 105 zoospore suspension of the isolates FBG7211 and FBG7216 (five plants per isolate) (Fu and Kim 2024). Control plants were drenched with sterile distilled water. The plants were maintained in a greenhouse at 21 to 23°C and 70% relative humidity with a 16-h photoperiod and were irrigated twice daily for 2 min using an overhead irrigation system. Fourteen days after inoculation, dark brown necrotic lesions developed on the root of all inoculated plants, similar to those observed in the original samples. In contrast, no symptoms were observed in the control plants. Isolates resembling the morphological characteristics of P. dissotocum were recovered from inoculated plants, and their identity was confirmed as P. dissotocum using sequencing. P. dissotocum has been reported to cause root rot in nursery plants (Weiland et al. 2013). However, to our knowledge, this is the first report of root rot on nandina caused by P. dissotocum in Tennessee and the United States. This finding is crucial to develop an efficient disease management strategy of Pythium root rot on nandina.

First Report of Pestalotiopsis lushanensis Causing Black Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely grown for forage in Southwest China. In December 2023, black leaf spots were observed on Italian ryegrass in a 10 hm2 field in Hongya County, Sichuan Province, China (29°52'11''°N, 103°16'16''°E). The disease developed sporadically with an incidence rate of < 1%. Leaves showed typical symptoms of small, black spots. For isolation, six samples (about 0.5 cm × 1 cm) were cut with a sterilized blade at the junction of diseased and healthy tissues from two different plants. They were surface disinfected in 75% ethanol for 40s, rinsed thrice in sterilized distilled water, air-dried, plated on potato dextrose agar (PDA), and incubated in the dark at 25°C for 3-7 days. A Pestalotiopsis-like fungus with similar colony characteristics was consistently isolated from all samples. Pure cultures were obtained with the single-spore technique (Cai et al. 2009), and two strains (LJH17 and LJH18) were selected for further study. After 5 days in the dark at 25°C, colonies on PDA were cottony, whitish, 70-78 mm in diameter, with an entire edge. The reverse side of the colonies appeared white to light yellow. Conidia were fusoid, straight to slightly curved, 4-septate, with 2-4 (mostly 3) tubular apical appendages, measuring 17.3 to 25 × 6.1 to 9.9 μm (mean ± SD = 20.7 ± 2.3 × 7.9 ± 0.7 µm; n = 50; LJH17). For further identification, the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA (ITS), partial β-tubulin (tub2) and translation elongation factor 1-α (tef1) genes from the two pure strains GMCC000083 (=LJH17; GMCC, Grassland Microbiome Culture Collection) and LJH18 were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997), and EF1-728F/EF2 (Carbone and Kohn 1999; O'Donnell et al. 1998). Sequences were deposited in GenBank (PP779763, PP781949, and PP781950 for LJH17; and PQ013725, PQ015095, and PQ015096 for LJH18). BLAST analysis showed 100% identity across all three segments with ex-type isolate LC4344 of Pestalotiopsis lushanensis (Liu et al. 2017). A randomized accelerated maximum likelihood (RAxML) phylogenetic tree based on concatenated ITS, tub2, and tef1 alignments showed that our two strains clustered with the previously reported P. lushanensis strains ((Liu et al. 2017; Jiang et al. 2022) (Fig. S1F). Eight-week-old healthy plants were grown from Italian ryegrass seeds in pots, with three plants per pot. Following our previous spray inoculation procedures (Xue et al. 2020): fresh 5-day-old mycelia from each strain were prepared as mycelial suspensions at about 2.5 × 104 CFU/ml, and conidial suspensions were adjusted to about 2 × 105 conidia/ml. For each strain (LJH17 and LJH18), five pots were separately spray-inoculated with conidial and mycelial suspensions (about 2 ml/pot). Five pots served as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23-29°C. After incubation for 15 days, both conidial and mycelial suspensions produced black spots on the plants (Fig. S1), resembling those previously observed in the field. No symptoms were observed in the control plants. Pathogenicity tests were conducted thrice using the same method. The same fungus was reisolated from the lesions and confirmed via the morphological and molecular methods described above, thus fulfilling Koch' s postulates. To our knowledge, this is the first report of P. lushanensis causing black leaf spot on Italian ryegrass in China or worldwide. This finding may aid in diagnosing, identifying the pathogen, and controlling the disease on Italian ryegrass.

First Report of Neocosmospora sp., Causing Stem Rot on Agave potatorum Zucc., in Mexico.

Agave potatorum Zucc., also known as "agave tobalá", is an endemic species in the Mexican states of Oaxaca and Puebla and is one of the most important wild species for producing the Mexican alcoholic distillate called mezcal. In September 2021, symptoms of dry stem rot and subsequent death of plants were observed, in a commercial field of 10 acres located in the municipality of Villa Sola de Vega, Oaxaca, Mexico. The disease incidence was 45%. Twenty symptomatic agave stem plants were sampled to isolate the pathogen. Pieces (5 × 5 mm) of the symptomatic tissues from diseased plants were surface disinfested with 1% NaOCl (60 s) and 70% ethanol (1 min), rinsed with sterile water (2 × 60 s), placed on potato dextrose agar (PDA Bioxon®, Mexico), and incubated at 25°C for 6 days with natural light. Next, we prepared single-spore derived cultures on PDA and carnation leaf-piece agar (CLA). Fusarium-like colonies were consistently isolated on potato dextrose agar (PDA) and 14 monoconidial isolates were obtained. On PDA, aerial mycelium was white to beige with diffuse pink pigment in the center of the culture and colonies were appressed with a regular shape after 7 days of incubation at 25℃. Macroconidia (n=100) were falcate, almost straight, 1 to 4-septate, 18.5 to 47.9 × 3.9-5.7 μm. Microconidia (n=100) were ellipsoidal to subcylindrical, slightly curved, hyaline, 8 to 16 × 3.5 to 4.5 μm, 0 to 1-septate, and grouped in false heads. Chlamydospores were globose to subglobose, terminal or intercalary in hyphae, and were solitary or in chains. Morphological features were consistent with the description of Neocosmospora spp. (Crous et al. 2021; Sandoval-Denis et al. 2019). Three isolates were selected for multilocus phylogenetic analysis and pathogenicity tests. Isolates were deposited as IPN-AP1, IPN-AP2, and IPN-AP3 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. For molecular identification, genomic DNA was extracted, and partial sequences of translation elongation factor 1-alpha (EF1-α) and the second largest subunit of RNA polymerase II (rpb2) genes were amplified and sequenced with the primer sets EF1-728F/EF1-986R (Carbone and Kohn 1999) and RBP2-5F/RPB2-7R (Liu et al. 1999), respectively. A phylogenetic tree using concatenated EF1-α and rpb2 sequences was constructed for Neocosmospora spp. based on Maximum Likelihood. The phylogenetic analysis suggested that the three isolates belong to a new species, closely related to N. noneumartii, as they formed a sister clade with this species. The sequences were deposited in GenBank with accession nos. OP902594 to OP902596 for EF1-α, and OP902591 to OP902593 for rpb2. A pathogenicity test for each isolate was performed by inoculating five healthy A. potatorum plants (1-year-old) grown in pots. Conidia were cultured on PDA for 7 days and the conidial solution was prepared with sterile water and adjusted to 1×105 spores/ml. A total of 1 ml of the conidial suspension was placed on the basal stem of each plant. Five plants mock-inoculated with sterilize water served as the controls. All plants were kept in a greenhouse for 45 days at temperatures ranging from 20 to 30°C. At 35 days after inoculation, dry stem rot was visible on the inoculated plants, whereas the control plants remained asymptomatic. The pathogenicity test was repeated twice with similar results. The fungi were reisolated from the infected plants and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Neocosmospora spp., has been previously associated with dry root rot in Citrus sinensis in South Africa (Guarnaccia et al. 2021) as well as rot of Cactaceae and other succulent plants in Iran (Kamali-Sarvestani et al. 2022). To our knowledge, this is the first report of Neocosmospora sp. causing stem rot of A. potatorum in Mexico and worldwide. Further studies should focus on determining the distribution, impact, and management of this disease in the production areas of agave tobalá in Mexico.

First Report of Leaf Spot on Hydrangea macrophylla Caused by Boeremia exigua in Korea.

Hydragea (Hydrangea macrophylla) is a garden plant commonly used in parks and landscapes globally. In late May 2023, brown necrotic spots surrounded by chlorotic halos were observed on the leaves of H. macrophylla (cv. Renate Steiniger) at the Suncheonman National Garden in Suncheon (34°55'45.6" N 127°30'26.5" E), South Korea. The affected area covered approximately 20 m2, with the disease incidence ranging from 2% to 5%. Concentric rings with light brown centers measuring 0.5 to 6 cm were observed on the upper sides of the leaves. To isolate the pathogens, each infected leaf from three different plants was cut into 5 × 5 mm pieces, surface-sterilized with 70% ethanol for 1 min, and rinsed three times with sterile distilled water. The samples were cultured on 1.5% water agar amended with 100 μg/mL of streptomycin in the dark at 25 °C. Single-spore isolation from the grown hyphae was performed on the obtained isolates (SYP-1229 to 1231). The fungal colony on potato dextrose agar was olivaceous gray with floccose aerial mycelia, and black pycnidia developed on the medium after 14 days. The conidia observed were hyaline, cylindrical to ellipsoidal-oblong, and aseptate, measuring 4.9 to 9.1 μm × 1.9 to 4.2 μm (avg. 6.3 × 2.7 μm, n = 100) (length × width). The isolates were identified as Boeremia sp. based on their morphological characteristics (Boerema, 1976; Boerema, 2004). For molecular identification, the internal transcribed spacer (GenBank accession nos.: OR682193-OR682195), actin (OR689851- OR689853), β-tubulin (OR689854- OR689856) and translation elongation factor-1 alpha (OR689857- OR689859) sequences from the isolates SYP-1229 to 1231 exhibited 100% (558/558 bp), 100% (263/263), 100% (314/314), and 100% (316/316) similarity, respectively, with Boeremia exigua (EU167567, EU880846, OP611549, and KY484684, respectively) in GenBank BLAST search. Phylogenetics based on concatenated ITS, ACT, TUB2, and TEF sequences, using the maximum likelihood method of MEGA X (Kumar et al., 2018), showed that sequences of SYP-1229 to 1231 were clustered in the same clade as B. exigua. For pathogenicity tests conducted in pots, 10 mL conidial suspension (1 × 106 conidia/mL) derived from isolate SYP-1229, which had been cultured on PDA for 14 days, was sprayed onto two leaves of each of three independent healthy 1-year-old hydrangeas (cv. Renate Steiniger). The leaves of the control plants were inoculated with sterile distilled water, which was also applied to two leaves of each of the three independent healthy plants. The inoculated plants were placed in clear plastic boxes to maintain high humidity and incubated at 25 °C under 18 h of light. Brown spot lesions were observed on the inoculated two leaves seven days after inoculation, whereas the control were asymptomatic. Similar pathogenicity results were obtained for all three independent biological replicates. The fungus isolated from the lesion was identified as B. exigua through morphological characterization and ITS sequencing, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of B. exigua causing brown-spot disease on H. macrophylla in South Korea. The pathogen has been previously reported H. macrophylla in Italy and the United States (Farr and Rossman 2017; Garibaldi et al. 2006), and H. paniculata in Italy (Garibaldi et al. 2018). This study will help in future detection and control to enhance the ornamental value of hydrangeas in Korea.