Drought stress-mediated differences in phyllosphere microbiome and associated pathogen resistance between male and female poplars.

Phyllosphere-associated microbes play a crucial role in plant-pathogen interactions while their composition and diversity are strongly influenced by drought stress. As dioecious plant species exhibited secondary dimorphism between the two sexes in response to drought stress, whether such difference will lead to sex-specific differences in phyllosphere microbiome and associated pathogen resistance between male and female conspecifics is still unknown. In this study, we subjected female and male full siblings of a dioecious poplar species to a short period of drought treatment followed by artificial infection of a leaf pathogenic fungus. Our results showed that male plants grew better than females with or without drought stress. Female control plants had more leaf lesion area than males after pathogen infection, whereas drought stress reversed such a difference. Further correlation and in vitro toxicity tests suggested that drought-mediated sexual differences in pathogen resistance between the two plant sexes could be attributed to the shifts in structure and function of phyllosphere-associated microbiome rather than the amount of leaf main defensive chemicals contained in plant leaves. Supportively, the microbiome analysis through high-throughput sequencing indicated that female phyllosphere enriched a higher abundance of ecologically beneficial microbes that serve as biological plant protectants, while males harbored abundant phytopathogens under drought-stressed conditions. The results could provide potential implications for the selection of suitable poplar sex to plants in drought or semi-drought habitats.

Soil cadmium pollution facilitated the invasion of alligator weed through enhanced herbivore resistance and competitive ability over a congeneric species.

A number of invasive plant species, such as Alternanthera philoxeroides, have been documented to be able to accumulate trace metal elements in their tissues. Since metal accumulation in plants can serve as a defence against herbivores, we hypothesized that metal pollution will increase herbivore resistance of metal-accumulating invasive plant species and such a benefit will grant them a competitive advantage over local co-occurring plants. In this study, we compared the differences in plant growth and herbivore feeding preference between A. philoxeroides and its native congener Alternanthera sessilis in single and mixed cultures with and without soil cadmium (Cd) pollution. The results showed that A. philoxeroides plants were more tolerant to Cd stress and accumulated more Cd in the leaves than A. sessilis. Cd exposure increased the resistance of A. philoxeroides against a specialist and a generalist herbivore compared with A. sessilis. Competition experiments indicated that Cd stress largely increased the competitive advantage of A. philoxeroides over A. sessilis with or without herbivore pressures. The differences in herbivore resistance between the two plant species under soil Cd stress are most likely due to the deterring effect of Cd accumulation and Cd-enhanced mechanical defences rather than changes in leaf specialized metabolites.

Combined effects of cadmium and simulated acid rain on soil microbial communities in the early cultivation of Populus beijingensis seedlings.

The combined cadmium (Cd) and acid rain pollution poses a significant threat to the global ecological environment. Previous studies on the combined adverse effects have predominantly focused on the aboveground plant physiological responses, with limited reports on the microbial response in the rhizosphere soil. This study employed Populus beijingensis seedlings and potting experiments to simulate the impacts of combined mild acid rain (pH=4.5, MA) or highly strong acid rain (pH=3.0, HA), and soil Cd pollution on the composition and diversity of microbial communities, as well as the physiochemical properties in the rhizosphere soil. The results showed that Cd decreased the content of inorganic nitrogen, resulting in an overall decrease of 49.10 % and 46.67 % in ammonium nitrogen and nitrate nitrogen, respectively. Conversely, acid rain was found to elevate the content of total potassium and soil organic carbon by 4.68 %-6.18 % and 8.64-19.16 %, respectively. Additionally, simulated acid rain was observed to decrease the pH level by 0.29-0.35, while Cd increased the pH level by 0.11. Moreover, Cd alone reduced the rhizosphere bacterial diversity, however, when combined with acid rain, regardless of its intensity, Cd was observed to increase the diversity. Fungal diversity was not influenced by the acid rain, but Cd increased fungal diversity to some extend under HA as observed in bacterial diversity. In addition, composition of the rhizosphere bacterial community was primarily influenced by the inorganic nitrogen components, while the fungal community was driven mainly by soil pH. Furthermore, "Metabolism" was emerged as the most significant bacterial function, which was markedly affected by the combined pollution, while Cd pollution led to a shift from symbiotroph to other trophic types for fungi. These findings suggest that simulated acid rain has a mitigating effect on the diversity of rhizosphere bacteria affected by Cd pollution, and also alters the trophic type of these microorganisms. This can be attributed to the acid rain-induced direct acidic environment, as well as the indirect changes in the availability or sources of carbon, nitrogen, or potassium.

Soil cadmium stress affects the phyllosphere microbiome and associated pathogen resistance differently in male and female poplars.

Microorganisms associated with the phyllosphere play a crucial role in protecting plants from diseases, and their composition and diversity are strongly influenced by heavy metal contaminants. Dioecious plants are known to exhibit sexual dimorphism in metal accumulation and tolerance between male and female individuals. Hence, in this study we used male and female full-siblings of Populus deltoides to investigate whether the two sexes present differences in their phyllosphere microbiome structures and in their associated resistance to the leaf pathogenic fungus Pestalotiopsis microspora after exposure to excess soil cadmium (Cd). We found that Cd-treated male plants grew better and accumulated more leaf Cd than females. Cd stress reduced the lesion areas on leaves of both sexes after pathogen infection, but male plants exhibited better resistance than females. More importantly, Cd exposure differentially altered the structure and function of the phyllosphere microbiomes between the male and female plants, with more abundant ecologically beneficial microbes and decreased pathogenic fungal taxa harbored by male plants. In vitro toxicity tests suggested that the sexual difference in pathogen resistance could be attribute to both direct Cd toxicity and indirect shifts in the phyllosphere microbiome. This study provides new information relevant for understanding the underlying mechanisms of the effects of heavy metals involved in plant-pathogen interactions.

ß-sitosterol targets glucocorticoid receptor to reduce airway inflammation and remodeling in allergic asthma.

INTRODUCTION: In most asthma patients, symptoms are controlled by treatment with glucocorticoid, but long-term or high-dose use can produce adverse effects. Therefore, it is crucial to find new therapeutic strategies. ß-sitosterol could suppress type Ⅱ inflammation in ovalbumin (OVA)-induced mice, but its mechanisms have remained unclear. METHODS: A binding activity of ß-sitosterol with glucocorticoid receptor (GR) was analyzed by molecular docking. Human bronchial epithelial cells (BEAS-2B) and human bronchial smooth muscle cells (HBSMC) were treated with different concentrations (0, 1, 5, 10, 20, and 50 µg/mL) of ß-sitosterol for suitable concentration selection. In transforming growth factor (TGF)-ß1 treated BEAS-2B and HBSMC, cells were treated with 20 µg/mL ß-sitosterol or dexamethasone (Dex) to analyze its possible mechanism. In OVA-induced mice, 2.5 mg/kg ß-sitosterol or Dex administration was performed to analyze the therapeutic mechanism of ß-sitosterol. A GR antagonist RU486 was used to confirm the mechanism of ß-sitosterol in the treatment of asthma. RESULTS: A good binding of ß-sitosterol to GR (score = -8.2 kcal/mol) was found, and the GR expression was upregulated with ß-sitosterol dose increase in BEAS-2B and HBSMC. Interleukin (IL)-25 and IL-33 secretion was significantly decreased by ß-sitosterol in the TGF-ß1-induced BEAS-2B, and the levels of collagen 1A and α-smooth muscle actin (SMA) were reduced in the TGF-ß1-induced HBSMC. In the OVA-challenged mice, ß-sitosterol treatment improved airway inflammation and remodeling through suppressing type Ⅱ immune response and collagen deposition. The therapeutic effects of ß-sitosterol were similar to Dex treatment in vitro and in vivo. RU486 treatment clearly hampered the therapeutic effects of ß-sitosterol in the TGF-ß1-induced cells and OVA-induced mice. CONCLUSION: This study identified that ß-sitosterol binds GR to perform its functions in asthma treatment. ß-sitosterol represent a potential therapeutic drug for allergic asthma.

Encapsulation and stabilization of lactoferrin in polyelectrolyte ternary complexes.

Effective delivery of the bioactive protein, lactoferrin (LF), remains a challenge as it is sensitive to environmental changes and easily denatured during heating, restricting its application in functional food products. To overcome these challenges, we formulated novel polyelectrolyte ternary complexes of LF with gelatin (G) and negatively charged polysaccharides, to improve the thermal stability of LF with retained antibacterial activity. Linear, highly charged polysaccharides were able to form interpolymeric complexes with LF and G, while coacervates were formed with branched polysaccharides. A unique multiphase coacervate was observed in the gum Arabic GA-LF-G complex, where a special coacervate-in-coacervate structure was found. The ternary complexes made with GA, soy soluble polysaccharide (SSP), or high methoxyl pectin (HMP) preserved the protein structures and demonstrated enhanced thermal stability of LF. The GA-LF-G complex was especially stable with >90% retention of the native LF after treatment at 90 °C for 2 min in a water bath or at 145 °C for 30 s, while the LF control had only ~ 7% undenatured LF under both conditions. In comparison to untreated LF, LF in ternary complex retained significant antibacterial activity on both Gram-positive and Gram-negative bacteria, even after heat treatment. These ternary complexes of LF maintain the desired functionality of LF, thermal stability and antibacterial activity, in the final products. The ternary complex structure, particularly the multiphase coacervate, may serve as a template for the encapsulation and stabilization of other bioactives and peptides.

Thermal Degradation of Thaumatin at Low pH and Its Prevention Using Alkyl Gallates.

Thaumatin, a potent sweet tasting protein extracted from the Katemfe Plant, is emerging as a natural alternative to synthetic non-nutritive sweeteners and flavor enhancer. As a food additive, its stability within the food matrix during thermal processing is of great interest to the food industry. When heated under neutral or basic conditions, thaumatin was found to lose its sweetness due to protein aggregation caused by sulfhydryl catalyzed disulfide bond interchange. At lower pH, while thaumatin was also found to lose sweetness after heating, it does so at a slower rate and shows more resistance to sweetness loss. SDS-PAGE indicated that thaumatin fragmented into multiple smaller pieces under heating in acidic pH. Using BEMPO-3, a lipophilic spin trap, we were able to detect the presence of a free-radical within the hydrophobic region of the protein during heating. Protein carbonyl content, a byproduct of protein oxidation, also increased upon heating, providing additional evidence for protein cleavage by a radical pathway. Hexyl gallate successfully inhibited the radical generation as well as protein carbonyl formation of thaumatin during heating.

Root rot Caused by Setophoma terrestris on Illicium verum in Yunnan Province, China.

Star anise (Illicium verum Hook. f.), a genus of star anise in the family Magnoliaceae, is an important cash crop of "medicinal and food" origin, mainly from China. In August 2021, root rot of I. verum was first observed on more than 80% of the plants grown within a 500 hectares area in Wenshan city, Yunnan Province. At the early stage of the disease, the phloem of the root was dark yellow-brown, and the leaves turn yellow. With further disease development, the whole root became black (Fig. 1a, 1b), and the leaves gradually fall off, affecting the growth, yield and eventually caused death of the whole plant. A total of 20 root samples were collected from typical symptomatic plant roots with 20 years old in Wenshan City (23°18'12″N, 103°56'98″E) and were cut into 2 × 2 mm pieces at the junction of infected and healthy tissue. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before rinsing three times with distilled water. The sterile filter paper (5×5 cm) was used to dry the tissue, and samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark in the incubator. From 9 isolates obtained in culture, 7 exhibited the morphology described by Boerema et al. (Boerema et al. 2004) for Setophoma sp. The hyphae were hyaline and septate (Fig.1c). After 14 days of culture on V8 juice agar, white round colonies are formed, but there is no groove in the middle of the colonies (Fig.1d), and transparent, oval, or cylindrical conidia were produced, 6.0-8.0 x 2.5 to 4.0 um (Fig.1e). DNA was extracted from a representative isolate BJGF-04 for molecular identification using a fungal genomic DNA extraction kit (Solarbio, Beijing, China). Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990) and primers T1/ß-Sandy-R for the ß-tubulin gene (TUB) region (Yang et al. 2017) and primers NL3/ LR5 for 28S large subunit rDNA (LSU) region (Hu et al. 2021) and NS1/ NS4 for 5.8S large subunit rDNA (SSU) region (Mahesha et al. 2021). Newly generated representative sequences were deposited in GenBank: ITS sequence (ON645256), TUB sequence (ON854484), and LSU sequence (ON644445), SSU sequence (ON644451). were sequenced and blasted, showing 99 to 100% sequence homology with known S. terrestris. Pathogenicity was performed using one-year asymptomatic plants of I. verum. A conidial suspension (1 x 106 conidia/ml) collected from V8 juice cultures with 0.05% Tween buffer was poured at a volume of 10 ml/plant. Three individual seedlings were used as replicates for each treatment, and sterile water was used as the negative control. All plants were placed in an artificial climate incubator at 25°C under 90% relative humidity. After 20 days, all inoculated plants showed symptoms identical to those described above, whereas controls remained healthy. Setophoma terrestris was reisolated from the infected roots, which was confirmed by morphological and molecular identification, which completed Koch's postulates. To our knowledge, this is the first report of S. terrestris as a causal agent of root rot on I. verum in China.

Colletotrichum fructicola Causal agent of Shot-Hole Symptoms on Leaves of Prunus sibirica in China.

Prunus sibirica L. (Siberian apricot) is a member of the Rosaceae family and an ecologically important tree species in China (Buer et al., 2022). Shot hole symptoms on the leaves were observed in five Siberian apricot groves in Chengdu (103.81 E, 30.97 N), Sichuan province in July 2020. The symptoms first appeared as small purplish-brown spots with yellow rings around them. As the disease progressed, the damaged area (diameter 1.5-3.0 cm) became necrotic and fell off. The disease incidence was about 60% and the disease index was 28.6 of leaves in the grove. in most severe cases. Fifteen symptomatic leaves were collected from 5 different trees in an orchard. Pathogen isolation was performed from symptomatic leaf tissue (5 × 5 mm) though surface disinfection (in 70% ethanol and 2% NaClO) and incubation on Potato Dextrose Agar (PDA) at 28℃ for 3 days. Overall 10 isolates with similar colony morphology were obtained from the 15 infected tissue pieces, and three representative isolates (XCK 2-4) were selected for further study. Colonies of the isolates on PDA were initially cottony, pale white to grayish-green with abundant aerial hyphae and produced conidial masses after 7 days. Conidiogenous cells were clavate and aggregated in acervuli. Conidia were smooth-walled, single-celled, straight, and slightly obtusely rounded at both ends, 12.8 to 18.7 × 4.3 to 5.7 µm in size (Fig. 1). The morphological characteristics of the three isolates were consistent with the description of species in the Colletotrichum gloeosporioides complex. DNA was amplified using the following primers pairs for the internal transcribed spacer (ITS) region of rDNA and partial sequences of beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and translation elongation factor (TEF-1), respectively: ITS1/ITS4, T1/Bt2b, GDF/GDR, CHS-F/CHS-R, and EF-F/EF-R (Vieira et al., 2014). Accession numbers (MW228049, MW284974, MW284976, MW284975 and MW284977, respectively) were obtained afterepositing all the resulting sequences in GenBank. Nucleotide blast showed 99 to 100% identities with Colletotrichum fructicola (GenBank accessions nos. MZ961683, MW284974, MN525881, MN525860, MF627961). Phylogenetic analysis of combined ITS-TUB-GAPDH genes using the Mrbayes inference method showed that the three isolates clustered with three reference isolates of C. fructicola as a distinct clade (Fig. 2). To verify Koch's postulates, ten 3-year-old healthy potted plants of P. sibirica were inoculated by spraying a conidial suspension (6 × 105 conidia/mL) of isolate XCK2 on both sides of leaves, and the control leaves were sprayed with sterile water. Then, all treatments were placed in a moist environment (25±2°C, 80% relative humidity, natural light). The inoculated plants showed typical symptoms of plants with natural infections, while the controls remained asymptomatic after 14 days. The pathogen C. fructicola was re-isolated from all inoculated plants, and the culture and fungus characteristics were the same as those of the original isolate. Colletotrichum fructicola was not isolated from the control plants. The results indicated that C. fructicola is the causal agent of the disease. Colletotrichum fructicola was reported as a leaf pathogen on Camellia chrysantha in China (Zhao et al., 2021). This is the first report of C. fructicola causing P. sibirica leaf shot-hole in the world. The identification of C. fructicola could provide relevant information for applying management strategies and research on the Siberian apricot disease.

Nigrospora musae Causing Taxus chinensis var. mairei stem blight in Sichuan, China.

Taxus chinensis var. mairei is the endemic, endangered, and first-class protected tree species in China. This species is considered as an important resource plant because it can produce Taxol which is an effective medicinal compound against various cancers (Zhang et al., 2010). Stem blight was observed in two plant nurseries in Ya'an (102°44'E,30°42'N), Sichuan province in April 2021. The symptoms first appeared as round brown spots on the stem. As the disease progressed, the damaged area gradually expanded into an oval or irregular shape, which was dark brown. About 800 square meters of planting area were investigated and the disease incidence was up to approximately 64.8%. Twenty obviously symptomatic stems which exhibited the same symptoms as above were collected from 5 different trees in the nursery. To isolate the pathogen, the symptom margin was cut into small blocks (5 x 5 mm), and the blocks were surface sterilized in 75% ethanol for 90 s and 3% NaClO solution for 60 s . Finally incubated on Potato Dextrose Agar (PDA) at 28℃ for 5 days. Ten pure cultures were isolated by transferring hyphal and the three strains (HDS06, HDS07 and HDS08) were selected as representative isolates for further study. Initially, colonies on the PDA of three isolates were white and cotton-like, and then gradually turned gray-black from the center. After 21 days, conidia were produced and were smooth-walled, single-celled, black, oblate, or spherical, measuring 9.3 to 13.6 × 10.1 to 14.5 µm in size (n = 50). Conidia were present at the tip of conidiophores on hyaline vesicles. These morphological features were generally consistent with those of N. musae (Wang et al., 2017). To validate the identification, DNA were extracted from the three isolates, followed by the amplification of transcribed spacer region of rDNA (ITS), the translation elongation factor EF-1 (TEF-1), and the Beta-tubulin (TUB2) sequences with the respective primer pairs ITS1/ITS4 (White et al., 1990), EF-728F/EF-986R (Vieira et al., 2014) and Bt2a/Bt2b (O'Donnell et al., 1997) .The sequences were deposited in GenBank with the accession numbers ON965533, OP028064, OP028068, OP060349, OP060353, OP060354, OP060350, OP060351 and OP060352, respectively. Phylogenetic analysis of combined ITS, TUB2, and TEF genes using the Mrbayes inference method showed that the three isolates clustered with Nigrospora musae as a distinct clade (Fig. 2). Combine with morphological characteristics and phylogenetic analysis, three isolates were identified as N. musae. 30 2-year-old healthy potted plants of T. chinensis were used for pathogenicity test. 25 of these plants were inoculated by injecting 10 µL of the conidia suspension (1 × 106 conidia/mL) into stems and then wrap around the seal to moisturize. The remaining 5 plants were injected with the same amount of sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 25°C and 80% relative humidity. After 2 weeks, the inoculated stems developed lesions similar to those observed in the field, whereas controls were asymptomatic. N. musae was re-isolated from the infected stem and identified by both morphological characteristics and DNA sequence analysis. The experiments repeated three times showed similar results. As far as we know, this is the first report of N. musae causing T. chinensis stem blight in the world. The identification of N. musae could provide a certain theoretical basis for field management and further research of T. chinensis.

Integrated Transcriptome and Metabolome Analysis Reveals the Molecular Mechanism of Rust Resistance in Resistant (Youkang) and Susceptive (Tengjiao) Zanthoxylum armatum Cultivars.

Chinese pepper rust is a live parasitic fungal disease caused by Coleosporium zanthoxyli, which seriously affects the cultivation and industrial development of Z. armatum. Cultivating and planting resistant cultivars is considered the most economical and environmentally friendly strategy to control this disease. Therefore, the mining of excellent genes for rust resistance and the analysis of the mechanism of rust resistance are the key strategies to achieve the targeted breeding of rust resistance. However, there is no relevant report on pepper rust resistance at present. The aim of the present study was to further explore the resistance mechanism of pepper by screening the rust-resistant germplasm resources in the early stage. Combined with the analysis of plant pathology, transcriptomics, and metabolomics, we found that compared with susceptible cultivar TJ, resistant cultivar YK had 2752 differentially expressed genes (DEGs, 1253 up-, and 1499 downregulated) and 321 differentially accumulated metabolites (DAMs, 133 up- and 188 down-accumulated) after pathogen infection. And the genes and metabolites related to phenylpropanoid metabolism were highly enriched in resistant varieties, which indicated that phenylpropanoid metabolism might mediate the resistance of Z. armatum. This finding was further confirmed by a real-time quantitative polymerase chain reaction analysis, which revealed that the expression levels of core genes involved in phenylpropane metabolism in disease-resistant varieties were high. In addition, the difference in flavonoid and MeJA contents in the leaves between resistant and susceptible varieties further supported the conclusion that the flavonoid pathway and methyl jasmonate may be involved in the formation of Chinese pepper resistance. Our research results not only help to better understand the resistance mechanism of Z. armatum rust but also contribute to the breeding and utilization of resistant varieties.

Changes in the Histology of Walnut (Juglans regia L.) Infected with Phomopsis capsici and Transcriptome and Metabolome Analysis.

Phomopsis capsici (P. capsici) causes branch blight of walnuts, which leads to significant economic loss. The molecular mechanism behind the response of walnuts remains unknown. Paraffin sectioning and transcriptome and metabolome analyses were performed to explore the changes in tissue structure, gene expression, and metabolic processes in walnut after infection with P. capsici. We found that P. capsici caused serious damage to xylem vessels during the infestation of walnut branches, destroying the structure and function of the vessels and creating obstacles to the transport of nutrients and water to the branches. The transcriptome results showed that differentially expressed genes (DEGs) were mainly annotated in carbon metabolism and ribosomes. Further metabolome analyses verified the specific induction of carbohydrate and amino acid biosynthesis by P. capsici. Finally, association analysis was performed for DEGs and differentially expressed metabolites (DEMs), which focused on the synthesis and metabolic pathways of amino acids, carbon metabolism, and secondary metabolites and cofactors. Three significant metabolites were identified: succinic semialdehyde acid, fumaric acid, and phosphoenolpyruvic acid. In conclusion, this study provides data reference on the pathogenesis of walnut branch blight and direction for breeding walnut to enhance its disease resistance.

Human epididymis protein 4 as a new diagnostic biomarker for rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: This study aimed to evaluate the role of human epididymis protein 4 (HE4) in the diagnosis and determination of the severity of interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients. METHODS: HE4 levels in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) samples were determined via electrochemiluminescence immunoassays in 102 RA patients (46 patients with ILD and 56 patients without ILD) and 51 healthy controls (HCs). RESULTS: Serum HE4 levels were significantly higher in RA-ILD patients (141.8±65.92 pmol/l) than those in the RA-no ILD patients (82.67±26.17 pmol/l) and healthy controls (35.72±7.6 pmol/l) (p<0.0001). Consistent with serum HE4 levels, BALF HE4 levels were significantly higher in RA-ILD patients (637.6±154.9 pmol/l) than those in the RA-no ILD patients (427.3±111.2 pmol/l) and healthy controls (206.9±30.46 pmol/l) (p<0.0001). In RA-ILD patients, HE4 levels were positively correlated with HRCT (high-resolution computed tomography) fibrosis scores, whereas a significant inverse relationship was found between HE4 levels and lung function parameters (such as, diffusion capacity of the lung for carbon monoxide (DLCO)). The logistic regression analysis showed that high levels of BALF HE4 (≥595 pmol/l) were associated with RA-ILD (odds ratio [OR] =8.09; 95% confidence interval [CI] =1.317-49.682; p=0.024). CONCLUSIONS: Serum and BALF HE4 levels were elevated in RA-ILD patients and strongly associated with the severity of ILD, thus supporting their potential clinical value as a new diagnostic aid for patients with RA-ILD.

Two cases of fetal hyperechogenic kidneys who had HNF1-ß gene variation.

We report two cases of HNF1-ß gene variation diagnosed in infancy, in whom fetal ultrasonography revealed enhanced echogenicity and multiple cysts in the renal parenchyma of both patients. They were initially diagnosed as autosomal recessive polycystic kidney disease. Gene testing showed a variation of HNF1-ß gene, one showed chromosome 17q12 deletion including HNF1-ß, the other was a de novo nonsense mutation in the HNF1-ß gene. The two children showed different renal function states. Extrarenal phenotypes also vary widely according to HNF1-ß gene variation including early-onset diabetes, autism spectrum, cognitive disorders, liver function abnormalities, and genital malformations, etc. We emphasize the importance of performing gene detection in order to make an accurate diagnosis, especially in those with fetal hyperechogenic kidneys, and so as to carry out reasonable multidisciplinary management. Early intervention for diabetes and neurodevelopmental disorders are especially important.

Design, synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists.

A series of novel dual A2A/A2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A2B AR (IC50 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A2A/A2B AR antagonists for cancer immunotherapy.

Brown leaf spot of Jacaranda mimosifolia Caused by Colletotrichum karstii in Sichuan, China.

Jacaranda mimosifolia D. Don is widely cultivated in southwest China (Yunnan, Sichuan, and other regions). It is widely applied in papermaking, medicine, environmental monitoring, timber, urban and rural afforestation, and soil and water conservation. In October 2020, a new brown leaf spot disease of J. mimosifolia was discovered in Xichang City (27°49' to 27°56'N, 102°16' to 102°11'E), with approximately 66.23% disease incidence. Firstly, the typical symptoms showed deep yellow necrotic lesions in the center or on the margin of the leaves. Gradually, the necrotic lesions expanded and developed into brown spots. Under humid conditions, the edges of necrotic lesions turned dark brown progressively. Finally, the leaves withered, died, and fell off. Infected tissues from ten samples were cut into small pieces of 2.5 × 2.5 mm. The surfaces of infected tissues were sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, and rinsed three times in sterile water. They were then blot-dried with autoclaved paper towels and cultured on potato dextrose agar (PDA) at 25℃ for 3 to 8 days. After culturing for 8 days at 25℃ and 12 h/12 h light/dark on PDA, the colony diameter reached 78.2 to 82.7 mm. The colonies were light orange, turned pale pink with light orange beneath. The conidia were single-celled, aseptate, cylindrical, smooth-walled, straight, hyaline with both ends bluntly rounded, measuring 12.3 to 16.8 × 4.3 to 5.6 µm (n = 100; average=14.5 × 5.1µm). These morphological characteristics were consistent with the description of C. karstii (Zhao et al. 2021). For molecular identification, the genomic DNA of the representative isolate JM202010 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). The internal transcribed spacer (ITS) [ITS1/ITS4 (White et al., 1990)], calmodulin (CAL) [CL1C/CL2C (Weir et al., 2012)], actin (ACT) [ACT512F/ACT-783R (Carbone & Kohn, 1999)], chitin synthase (CHS-1) [CHS-79F/CHS-345R (Carbone & Kohn, 1999)], ß-tubulin (TUB2) [BT2A/BT2B (O'Donnell et al., 1997)], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GDF/GDR (Templeton et al. 1992)] were amplified. Sequences were deposited in GenBank (ITS: OL454787, CAL: OL518966, ACT: OL518967, CHS-1: OL518968, TUB2: OL518969, and GAPDH: OL518970). BLAST results indicated that the ITS, CAL, ACT, CHS-1, TUB2 and GAPDH sequences showed >99% identity with Colletotrichum karstii sequences at NCBI (GenBank MW494453.1, MW495036.1, MG387951.1, MW495038.1, MW495042.1, and MG602034.1). The conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old J. mimosifolia plants (10 plants) and inoculated for pathogenicity test. Fifteen leaves of each plant (10 pots in total) were inoculated with spore suspensions on both sides of the leaves. An equal number of control leaves was sprayed with sterilized distilled water as a control. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 60 to 68% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, but the controls remained asymptomatic. Colletotrichum karstii was re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of brown leaf spot on J. mimosifolia caused by C. karstii in China. C. karstii was previously reported as the causal agent of anthracnose on Fatsia japonica (Xu et al. 2020) and Nandina domestica (Li et al. 2017) in China. This finding provides an important basis for further research on the control of this disease.

First report of Coniella koreana causing leaf blight on Loropetalum chinense var. rubrum (Chinese Fringe Flower) in Sichuan, China.

Loropetalum chinense var. rubrum (Chinese Fringe Flower) is widely distributed in the middle and lower reaches of the Yangtze River, as well as northern India. It is a popular landscape plant for its red evergreen foliage and its showy red flowers in the spring. In July 2020, This leaf blight was discovered in Chengdu city (30°42＇41＂N, 103°51＇58＂E). In June 2021, the disease incidence rate at two places in Wenjiang District of Chengdu was 76% and 64%, respectively. The symptoms began to appear from May to June, worsened from July to August, and then disappeared gradually in November. Initially, brown-edged irregular necrotic patches appeared at the leaf margins. Progressively, the patches increased in number, expanded to leaf middle, and turned grayish-white. The scattered black fruiting bodies (conidia) were appeared at patches under humid conditions. Eventually, the leaves tended to dry up and fall off. Infected tissues from five samples and collected were cut into small pieces 2×2 mm, surface sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25℃ in the dark. A total of eight isolates were collected, five isolates exhibited similar culture characteristics while two were Nigrospora sp. and one was a Fusarium sp.. The five similar isolates produced sparse, grayish-withe mycelia with a flat elevation and curled margin. Abundant globose and yellow pycnidia were formed on the PDA surface and arranged in irregular concentric zones. Conidia were 18.20 to 22.36 × 2.64 to 3.05 µm (average 20.36 × 2.82 µm, n=50) in size, fusiform, sickle-shaped, aseptate. DNA was extracted from the representative strain (HMcj B03), and the internal transcribed spacer (ITS) region, the large subunit of the nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2), were amplified by polymerase chain reaction and sequenced with primers ITS1/ITS4 (White et al. 1990), LR0R/LR7 (Rehner and Samuels 1994; Vilaglys and Hester 1990), 728F/986R (Carbome and Kohn 1999), and 5F2/7cR (Alvarez et al. 2016), respectively. The sequences were deposited in GenBank, viz. OL468959, OL469170, OL489770, and OL855833, respectively. BLAST analysis showed >98.7% identity with several reference sequences of Coniella koreana strain CBS 143.97 and Coniella quercicola strain CBS 904.69, deposited in GenBank. A conidial suspension (1 × 107 conidia/mL) having 0.05% Tween 80 buffer was used for foliar inoculation of 6-year-old Loropetalum chinense var. rubrum plants for pathogenicity test. Ten leaves of each plant (10 pots in total) were inoculated with spore suspensions (20 µL onto the wounded sites). An equal number of control leaves were sprayed with 0.05% Tween 80 buffer to serve as a control. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH > 80%). Twenty days later, all the inoculated leaves showed similar symptoms as the original diseased plants, however, the controls remained asymptomatic. The C. koreana was re-isolated from the infected leaves. To our knowledge, this is the first report of L. chinense var. rubrum caused by C. koreana in China. The discovery of this new disease will provide useful information for developing effective control strategies, and prove beneficial in reducing economic losses in floral product.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

Bambusa pervariabilis × Dendrocalamopsis grandis is the main cultivated bamboo species used for ecological construction in the Yangtze River basin. This species has the advantages of easy reproduction, wide adaptability and strong resistance and has high economic, ecological and social benefits (Peng et al. 2020). One area of B. pervariabilis × D. grandis with basal rot disease was discovered in Renshou County, Sichuan Province, China (29°41'N, 104°11'E) in June 2020. The disease occurrence area was 68 hm2 in Renshou County, with an incidence rate of 34.8%, and 5% of the B. pervariabilis × D. grandis with basal rot disease died. The pathogen initially invaded from the first section of the base of the bamboo stalk, appearing as black to yellowish brown strips or lumps of disease spots, and rapidly developed horizontally and vertically, which caused the whole plant to wither in severe cases. Diseased tissues were collected from the base of a 4-year-old bamboo stalk with a sterile blade. 100 pieces (5 × 5 × 2 mm) of diseased tissues were sterilized with 3% NaClO for 30 s and in 75% ethanol for 90 s, rinsed three times with sterile distilled water, dried with sterile surface water on sterile filter paper, plated onto potato dextrose agar amended with streptomycin sulfate (Solarbio, 50 µg/ml), and incubated at 25 °C for 7 days with light. A total of five isolates were obtained, of which four isolates were similar in morphology. Using the method of monospore isolation (Leslie and Summerell 2006) and culturing it on PDA, the fungus produced round colonies with a diameter of approximately 8.4 mm and a surface color ranging from white to purple within 7 days at 25 °C. For identification by typical spores, the fungus was cultured on carnation leaf agar (CLA) medium at 25 °C for 7 days. The microconidia by the isolates BD2002, BD2004, BD2008 and BD2010 cultured on CLA medium were elliptical, ovoid, without septum, and measured 4.56 to 15.53 µm long × 1.36 to 6.98 µm wide (n=100). The macroconidia were rod-shaped or slightly curved, tapering apically with three to five septa, and measured 18.86 to 52.99 × 1.56 to 6.42 µm in size (n=100). According to the morphological characteristics of macroconidia and microconidia, the isolates were identified as Fusarium sp. (Leslie and Summerell 2006). For molecular identification, fungal DNA of isolates BD2002, BD2004, BD2008 and BD2010 was extracted by a fungal genomic DNA extraction kit. Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) rDNA region (White et al. 1990), primers Bt2a/Bt2b for the ß-tubulin (TUB) region (Glass and Donaldson 1995), primers EF1F/EF2R for the translation elongation factor 1α (TEF) region (Carbone et al. 1999), primers 5f2/7cr for the RNA polymerase II genes (RPB2) region (O'Donnell et al. 2010), primers H3-1a/H3-1b for the histone H3 (HIS) region (Jacobs et al. 2010), and primers NMS1/NMS2 for the mitochondrial small subunit (mtSSU) rDNA region (Stenglein et al. 2010). Using BLASTn to search GenBank for ITS, TUB, TEF, RPB2, HIS and mtSSU sequences, all isolates showed the highest similarity with Fusarium proliferatum (Matsushima) Nirenberg. The representative isolate BD2010 showed that ITS had 99.61% similarity to F. proliferatum Z23-28 (FJ648201.1); HIS had 99.57% similarity to F. proliferatum M06A_4G_4 (KX681532.1); and the TUB, TEF, RPB2, and mtSSU sequences showed 99.67%, 99.10%, 99.06%, and 99.57% similarity, respectively, to F. proliferatum ITEM2287 (accession numbers LT841243.1, LT841245.1, LT841252.1, and LT841247.1 in GenBank). The GenBank numbers of the representative isolate BD2010 were ITS, OK325614; TUB, OK377026; TEF, OK377027; RPB2, OK377028; HIS, OK377029; and mtSSU, OK338638. To confirm the pathogenicity, thirty 4-year-old healthy bamboo plants were grown in 30 pots. Each five plants were inoculated with one isolate, and a total of twenty-five plants were inoculated with five isolates. A conidia suspension (1 × 106 conidia/ml) of the fungus was inoculated (100 µl each) into plants that had been acupunctured at the base by a sterile syringe. Five control plants were inoculated only with the same amount of sterile distilled water. The inoculation site was wrapped with wet gauze to maintain moisture. All bamboo plants were watered every seven days. The illumination conditions were 12 h light and 12 h dark. All plants were cultured in a greenhouse at 25-28 °C and 70-80% relative humidity. One month later, twenty plants inoculated with conidial suspensions of BD2002, BD2004, BD2008 and BD2010 showed the same symptoms as those observed in the field, whereas plants inoculated with the other fungus and the control treatment remained asymptomatic. The pathogenicity test was conducted three times, and the experimental results were consistent. Furthermore, the fungi were reisolated from the diseased part and were identified as F. proliferatum by morphological and molecular comparison. To our knowledge, this is the first report of basal rot disease caused by F. proliferatum on B. pervariabilis × D. grandis in China. This research is conducive to laying the foundation for the development of effective control strategies for basal rot disease in this species.

Branch blight of Juglans regia caused by Palmiascoma qujingense in China.

Juglans regia L. is one of the major cultivated walnut species in China for nuts and wood (Pollegioni et al. 2012). In June 2020, branches with blight symptoms were observed in an orchard at Chongzhou City (30°33'34″N, 103°38'35″E). In an orchard of 30 hectares, disease incidence was around 50%. A total of 15 plants were sampled and 40% of their branches were affected by this disease. Firstly, brown and irregular spots appeared, then the spots gradually expanded and encircled the branch, which eventually killed the branch. Five samples of diseased branches from different trees were collected and a single fungal isolate was obtained from each of the five samples using the single ascospore isolation (Chomnunti et al. 2014). Colonies of the five isolates on potato dextrose agar (PDA) were identical that initially appeared white on the top, becoming light to dark brown with age. On the host, ascostroma were black, globose to subglobose, short-papillate, ostiolate, 260 - 410 × 210 - 320 µm (x = 335 × 265 µm, n = 20). Asci were 8-spored, bitunicate, cylindrical, short pedicellate, 55 - 78 × 8 - 12 µm (x = 67.5 × 10 µm, n = 40). Ascospores were 1-septate, fusiform to ellipsoidal, slightly curved, guttulate, 12 - 17 × 3 - 5 µm (x = 14.5 × 4 µm, n = 40). These sexual morphological characteristics are consistent with the Palmiascoma qujingense Phook. & K.D. Hyde (Monkai et al. 2021). Asexual morphs were formed on PDA in incubator after 17 days (25℃, 90% relative humidity, 12-h photoperiod). Conidiomata were black, globose to subglobose, 220 - 300 × 240 - 380 µm (x = 270 × 310 µm, n = 20). Conidia were oblong to ellipsoidal, aseptate and smooth-walled, 3 - 7 × 2 - 4 µm (x = 4.9 × 3 µm, n = 50). The genomic DNA of a representative isolate SICAUCC 21-0013 was extracted, and the internal transcribed spacers (ITS) region, large subunit rDNA (LSU) region, small subunit rDNA (SSU) region, and the largest subunit of RNA polymerase II (rpb2) gene were amplified and sequenced with primers ITS5/ITS4 (White et al. 1990), LR0R/LR5 (Rehner et al. 1994), NS1/NS4 (White et al. 1990), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. The sequences were deposited in NCBI with accession numbers MZ983549, MZ959419, MZ951112, and MZ818772, respectively, which showed 100%, 100%, 99.14%, and 99.59% identities with P. qujingense KUMCC 19-0201 (holotype) (accession numbers MT477185, MT477186, MT477183, MT495782respectively). Phylogenetic analysis (maximum likelihood) based on a concatenated dataset showed 93% bootstrap support values with P. qujingense. To verify Koch's postulates, 9 healthy branches from three 1-year-old seedlings were inoculated with conidial suspension (106 conidia/ml) from 4-week-old cultures via pin-prick inoculation (Desai et al. 2019), and the same number of seedlings and branches were inoculated with sterile water as controls. Plants were placed in a greenhouse at 25℃ and 90% RH on a 12-h fluorescent light/dark regime. After 28 days, brown spots were formed on P. qujingense-inoculated branches and similar to those observed in the field, while the controls remained asymptomatic. The pathogen was re-isolated from the lesions and identified by morphology and phylogeny. To our knowledge, this is the first report of P. qujingense causing branch blight on J. regia in the world. This disease potentially impacts the growth and yield of J. regia, and control measures should be made.

Branch blight of Juglans sigillata caused by Juglanconis appendiculata in China.

Juglans sigillata Dode, an endemic walnut species native in southwest China, is mainly used as nuts in Sichuan Province (Jin et al., 2019). In May 2021, symptoms of branches blight were observed in an orchard measuring 10 hectares located in Mianyang City, Sichuan Province (31°5' 25″N, 105°27'36″E, 365 m above sea level). About 40% of plants were diseased in the quadrat consisting of twenty walnut trees, and 20% of branches were dead on each affected tree. Initially, light brown spots appeared; then, the spots expanded to surround the whole branches; finally, the branches changed from brown to reddish-brown and died. Four symptomatic branches were sampled randomly from different trees. Next, four fungal isolates were obtained from the acervuli of each branch using the single-conidium isolation (Chomnunti et al. 2014) and cultured on potato dextrose agar (PDA). The Petri dishes were placed in an incubator and cultured at 25 °C under a 12-h photoperiod. Colonies were initially white with thin aerial mycelia and gradually turned dark grey with irregular margins. Conidiomata were acervular, black and scattered, with a diameter of 0.3 - 0.7 mm. Conidiophores were narrowly cylindrical, simple or branched at the base, 30 - 43 × 3 - 8 µm (x = 36.5 × 5.5 µm, n = 40). Conidiogenous cells were annellidic with distinct annellations. Conidia were unicellular, brown when mature, narrowly ellipsoid with gelatinous sheaths and truncate scars at the base, 17 - 32 × 7 - 12 µm (x = 27 × 9 µm, n = 40). The genomic DNA of a representative isolate SICAUCC 22-0064 was extracted, and the internal transcribed spacer (ITS) region, guanine nucleotide-binding protein subunit beta gene (ms204), translation elongation factor 1-alpha (tef1-α), and partial sequences of ß-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers V9G/LR5 (de Hoog & van den Ende 1998), MS-E1F1/MS-E5R1 (Walker et al. 2012), EF1-728F (Carbone & Kohn 1999)/TEF1LLErev (Jaklitsch et al. 2005), and T1/BtHV2r (Voglmayr et al. 2017), respectively. The sequences of ITS, ms204, tef1-α, and tub2 were deposited in NCBI with accession numbers ON000068, ON112376, ON112374, and ON112375, respectively. With the consideration of the sequence lack of ms204 and tub2 in the ex-type strain (D96) of Juglanconis appendiculata Voglmayr & Jaklitsch, the isolate D140 was used for nucleotide blast. The results showed 99.68%, 100%, 100%, and 100% identities of ITS, ms204, tef1-α, and tub2 with D140 (accession numbers KY427138, KY427157, KY427207, KY427226). Phylogenetic analysis based on a combined dataset showed 100% bootstrap with J. appendiculata, and the morphology was consistent with the asexual stage of J. appendiculata (Voglmayr et al., 2017). To verify Koch's postulates, five branches wounded by pin-prick were sprayed with conidial suspension (1 × 105 conidia/mL) in each plant, and three repetitions were performed on healthy 2-year-old potted plants. The same number of branches were sprayed with sterile distilled water as controls. The plants were placed in a greenhouse at 25 ℃ under 90% relative humidity and a 12-h fluorescent light/dark regime. After five weeks, all the inoculated branches showed brown necrosis similar to that observed in the field, and no symptoms occurred on the controls. The pathogens were re-isolated from the necrotic lesions and identified by morphology and phylogeny. J. appendiculata has been reported on Juglans nigra and J. regia in Austria, France, Spain and Greece (Farr & Rossman 2022). This paper is the first report of branch blight on Juglans sigillata caused by J. appendiculata in China. This result may develop the understanding of walnut diseases and lay a foundation for further management.