Metabolism-Based Herbicide Resistance to Mesosulfuron-methyl and Identification of Candidate Genes in Bromus japonicus.

The evolved resistance of Bromus japonicus Houtt. to ALS-inhibiting herbicides is well established. Previous studies have primarily focused on target-site resistance; however, non-target-site resistance has not been well characterized. This investigation demonstrated that ALS gene sequencing did not detect any previously known resistance mutations in a mesosulfuron-methyl-resistant (MR) population, and notably, treatment with the P450 monooxygenase (P450) inhibitor malathion markedly heightened susceptibility to mesosulfuron-methyl. Utilizing UPLC-MS/MS analysis confirmed elevated mesosulfuron-methyl metabolism in MR plants. The integration of Isoform Sequencing (Iso-Seq) and RNA Sequencing (RNA-Seq) facilitated the identification of candidate genes associated with non-target sites in a subpopulation with two generations of herbicide selection. Through qRT-PCR analysis, 21 differentially expressed genes were characterized, and among these, 10 genes (comprising three P450s, two glutathione S-transferases, one glycosyltransferase, two ATP-binding cassette transporters, one oxidase, and one hydrolase) exhibited constitutive upregulation in resistant plants. Our findings substantiated that increased herbicide metabolism is a driving force behind mesosulfuron-methyl resistance in this B. japonicus population.

Biochar Loaded with a Bacterial Strain N33 Facilitates Pecan Seedling Growth and Shapes Rhizosphere Microbial Community.

Biochar and beneficial microorganisms have been widely used in ecological agriculture. However, the impact of biochar loaded with microbes (BM) on plant growth remains to be understood. In this study, BM was produced by incubating pecan biochar with the bacterial strain N33, and the effects of BM on pecan growth and the microbial community in the rhizosphere were explored. BM application significantly enhanced the biomass and height of pecan plants. Meanwhile, BM treatment improved nutrient uptake in plants and significantly increased the chlorophyll, soluble sugars, and soluble proteins of plants. Furthermore, BM treatment improved the soil texture and environment. Finally, BM application substantially enhanced the diversity of soil fungi and bacteria as well as the relative abundances of the phyla Firmicutes and Chloroflexi, and families Bacillaceae and Paenibacillaceae, as shown by high-throughput sequencing. Together, this study clarified the growth-promotive effects of BM on pecan plants and suggested an alternative to synthetic fertilizers in their production.

Electrocatalytic Generation of Singlet Oxygen via ROS-Mediated Redox Chain Reaction for Efficient Disinfection.

The risk of harmful microorganisms to ecosystems and human health has stimulated exploration of singlet oxygen (1O2)-based disinfection. It can be potentially generated via an electrocatalytic process, but is limited by the low production yield and unclear intermediate-mediated mechanism. Herein, we designed a two-site catalyst (Fe/Mo-N/C) for the selective 1O2 generation. The Mo sites enhance the generation of 1O2 precursors (H2O2), accompanied by the generation of intermediate •HO2/•O2-. The Fe site facilitates activation of H2O2 into •OH, which accelerates the •HO2/•O2- into 1O2. A possible mechanism for promoting 1O2 production through the ROS-mediated chain reaction is reported. The as-developed electrochemical disinfection system can kill 1 × 107 CFU mL-1 of E. coli within 8 min, leading to cell membrane damage and DNA degradation. It can be effectively applied for the disinfection of medical wastewater. This work provides a general strategy for promoting the production of 1O2 through electrocatalysis and for efficient electrochemical disinfection.

Validation of numerical simulations and experiments on impulse characteristics induced by self-excited oscillation.

The high-frequency pulse flow, equivalent to the natural frequency of rocks, is generated by a self-excited oscillating cavity to achieve resonance rock-breaking. The flow field and oscillating mechanism of the self-excited oscillating cavity were simulated using the large eddy simulation method of Computational Fluid Dynamics (CFD). A field-scale testing apparatus was developed to investigate the impulse characteristics and verify the simulation results. The results show that the fluid at the outlet at the tool is deflected due to the pulse oscillation of the fluid. The size and shape of low-pressure vortices constantly change, leading to periodic changes in fluid impedance within the oscillating cavity. The impulse frequency reaches its highest point when the length-diameter ratio is 0.67. As the length-diameter ratio increases, the tool pressure loss also increases. Regarding the cavity thickness, the impulse frequency of the oscillating cavity initially decreases, then increases, and finally decreases again. Moreover, both the impulse frequency and pressure loss increase with an increase in displacement. The numerical simulation findings align with the experimental results, thus confirming the validity of the theoretical model. This research provides theoretical guidance for the practical application of resonance rock-breaking technology.

Highly selective nanozyme-based glucose sensing platform via construction of artificial recognition sites on gold nanospheres.

Nanozymes have been regarded as the ideal alternatives to natural enzymes in bioassays due to their good stability and low cost. However, their applications in sensing usually suffer from poor selectivity. For example, Au-based nanozymes, as a kind of classical glucose oxidase mimic enzyme, could catalyze diverse monosaccharides. Therefore, it is of great necessity and urgency to endow the Au-based nanozymes with enhanced selectivity for the construction of specific glucose sensing platform. In our study, easily recyclable polydopamine (PDA)-supported Au-based nanozymes (PDA@Au NPs) were successfully prepared and could catalyze diverse monosaccharides including glucose, xylose, mannose, and sucrose. To enhance the selectivity of PDA@Au NPs, molecularly imprinted polymers (MIPs) were constructed on the surface of PDA@Au NPs using glucose and boronic acid derivatives as template and functional monomer. Impressively, the catalytic activity of the obtained molecularly imprinted nanozyme (PDA@Au NPs-MIPs) only shows a slight decrease (6.3%) while their selectivity is obviously enhanced (≥230%). Accordingly, the as-prepared sensor achieved the sensitive and selective detection of glucose in the concentration range of 10 µM-1 mM and a low detection limit (LOD) of 0.227 µM (S/N = 3), avoiding the influence of other monosaccharides exited in the sensing solutions to a great extent. As expected, the as-prepared sensors also showed good recovery, and long-term stability.

Identification and Characterization of a Plant Endophytic Fungus Paraphaosphaeria sp. JRF11 and Its Growth-Promoting Effects.

Endophytic fungi establish mutualistic relationships with host plants and can promote the growth and development of plants. In this study, the endophytic fungus JRF11 was isolated from Carya illinoinensis. Sequence analysis of the internal transcribed spacer (ITS) region and 18S rRNA gene combined with colonial and conidial morphology identified JRF11 as a Paraphaosphaeria strain. Plant-fungus interaction assays revealed that JRF11 showed significant growth-promoting effects on plants. In particular, JRF11 significantly increased the root biomass and soluble sugar content of plants. Furthermore, transcriptome analysis demonstrated that JRF11 treatment reprogrammed a variety of genes involved in plant mitogen-activated protein kinase (MAPK) signaling and starch and sucrose metabolism pathways through Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Our research indicates that beneficial endophytic fungi are able to interact with plants and exhibit outstanding plant growth-promoting activities.

Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to plant volatiles.

Entomopathogenic nematodes (EPNs) use the chemical cues emitted by insects and insect-damaged plants to locate their hosts. Steinernema carpocapsae, a species of EPN, is an established biocontrol agent used against insect pests. Despite its promising potential, the molecular mechanisms underlying its ability to detect plant volatiles remain poorly understood. In this study, we investigated the response of S. carpocapsae infective juveniles (IJs) to 8 different plant volatiles. Among these, carvone was found to be the most attractive volatile compound. To understand the molecular basis of the response of IJs to carvone, we used RNA-Seq technology to identify gene expression changes in response to carvone treatment. Transcriptome analysis revealed 721 differentially expressed genes (DEGs) between carvone-treated and control groups, with 403 genes being significantly upregulated and 318 genes downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the responsive DEGs to carvone attraction were mainly involved in locomotion, localization, behavior, response to stimulus, and olfactory transduction. We also identified four upregulated genes of chemoreceptor and response to stimulus that were involved in the response of IJs to carvone attraction. Our results provide insights into the potential transcriptional mechanisms underlying the response of S. carpocapsae to carvone, which can be utilized to develop environmentally friendly strategies for attracting EPNs.

Nanozyme with tailored selectivity and highly catalytic activity for efficient sensing of endotoxin and sourcing gram-negative bacteria.

Endotoxin detection is important for determining bacterial contamination and infection in fields of food, pharmaceutical and clinical disease diagnosis. The horseshoe crab deformed cell lysate analysis is regarded as the gold-standard method, but the endangered and high-cost horseshoe crab animals required in sensing process further raise animal ethical issues and hinder their applications. The colorimetric methods based on nanozymes are simple and economical, but the low selectivity and sensitivity are still the bottleneck for their further application. Herein, we successfully developed a phenylboronic acid functionalized iron-based nanozyme with higher selectivity and highly catalytic activity for endotoxin sensing. The as-prepared colorimetric sensor using the obtained nanozyme as sensing probes shows a good linear relationship for endotoxin sensing in the range of 1-20 µg mL-1, with a LOD = 0.42 µg mL-1, along with good selectivity and reproducibility. The sensor can also be well applied to detecting endotoxin in practical samples such as beer and serum. Moreover, the parameters including time and temperature which could affect the endotoxin release from E. coli were also studied and optimized, based on the relationship between endotoxin and Gram-negative bacteria, the as-prepared sensor achieves the qualitative and quantification of E. coli.

B. subtilis CNBG-PGPR-1 induces methionine to regulate ethylene pathway and ROS scavenging for improving salt tolerance of tomato.

Soil salinity severely threatens plant growth and crop yields. The utilization of PGPR is an effective strategy for enhancing plant salt tolerance, but the mechanisms involved in this process have rarely been reported. In this study, we investigated the effects of Bacillus subtilis CNBG-PGPR-1 on improving plant salt tolerance and elucidated the molecular pathways involved. The results showed that CNBG-PGPR-1 significantly improved the cellular homeostasis and photosynthetic efficiency of leaves and reduced ion toxicity and osmotic stress caused by salt in tomato. Transcriptome analysis uncovered that CNBG-PGPR-1 enhanced plant salt tolerance through the activation of complex molecular pathways, with plant hormone signal transduction playing an important role. Comparative analysis and pharmacological experiments confirmed that the ethylene pathway was closely related to the beneficial effect of CNBG-PGPR-1 on improving plant salt tolerance. Furthermore, we found that methionine, a precursor of ethylene synthesis, significantly accumulated in response to CNBG-PGPR-1 in tomato. Exogenous L-methionine largely mimicked the beneficial effects of CNBG-PGPR-1 and activated the expression of ethylene pathway-related genes, indicating CNBG-PGPR-1 induces methionine accumulation to regulate the ethylene pathway in tomato. Finally, CNBG-PGPR-1 reduced salt-induced ROS by activating ROS scavenger-encoding genes, mainly involved in GSH metabolism and POD-related genes, which were also closely linked to methionine metabolism. Overall, our studies demonstrate that CNBG-PGPR-1-induced methionine is a key regulator in enhancing plant salt tolerance through the ethylene pathway and ROS scavenging, providing a novel understanding of the mechanism by which beneficial microbes improve plant salt tolerance.

Effective automatic detection of anterior cruciate ligament injury using convolutional neural network with two attention mechanism modules.

BACKGROUND: To develop a fully automated CNN detection system based on magnetic resonance imaging (MRI) for ACL injury, and to explore the feasibility of CNN for ACL injury detection on MRI images. METHODS: Including 313 patients aged 16 - 65 years old, the raw data are 368 pieces with injured ACL and 100 pieces with intact ACL. By adding flipping, rotation, scaling and other methods to expand the data, the final data set is 630 pieces including 355 pieces of injured ACL and 275 pieces of intact ACL. Using the proposed CNN model with two attention mechanism modules, data sets are trained and tested with fivefold cross-validation. RESULTS: The performance is evaluated using accuracy, precision, sensitivity, specificity and F1 score of our proposed CNN model, with results of 0.8063, 0.7741, 0.9268, 0.6509 and 0.8436. The average accuracy in the fivefold cross-validation is 0.8064. For our model, the average area under curves (AUC) for detecting injured ACL has results of 0.8886. CONCLUSION: We propose an effective and automatic CNN model to detect ACL injury from MRI of human knees. This model can effectively help clinicians diagnose ACL injury, improving diagnostic efficiency and reducing misdiagnosis and missed diagnosis.

First Report of Colletotrichum aenigma Causing Anthracnose on Bromus japonicus in Tianjin, China.

Japanese brome (Bromus japonicus Thunb.) is a weed commonly found in roadsides, floodplain wetlands, and farmlands. During September 2020 and 2021, a leaf spot disease was observed on B. japonicus in greenhouses of Baodi district, Tianjin, China (117°15'E, 39°47'N). More than 10% of the weeds were infected. Initial irregular brown spots on leaf apices continued to expand until adjacent spots coalesced. Eventually, severely infected leaves became yellow, thinner, drier and withered. Small patches (3×3 mm) were cut from symptomatic leaves, sterilized with 75% ethanol for 30s, rinsed three times with sterile water and incubated on Petri dishes with potato dextrose agar at 25°C in darkness for 7 days. Three isolates, with uniform morphology were selected for further analysis. Colonies were cottony with entire edges and aerial white mycelia; and average growth rate of 4.5 mm/day. The upper side was pale white, and the reverse side was grayish-green. Conidia were aseptate, hyaline, subcylindrical with rounded ends, 8.6 to 18.7×4.4 to 8.3 µm (n = 50). Appressoria were dark brown, oval or irregular shaped with a few lobes, 5.7 to 9.4×4.5 to 7.8 µm (n = 50). Total genomic DNA of isolates was extracted with Fungal DNA Kit (GBCBIO, Guangzhou, China). Primers for sequences of internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), and calmodulin (CAL) genes were amplified and sequenced (Weir et al., 2012). After aligned and trimmed, the sequences of TJBDA1, TJBDA2, and TJBDA3 were identical. TJBDA1 representative isolate sequences were deposited in GenBank ITS OP247554 with 99.83% (576/577) similarity to MT476809, GAPDH OP414834 with 99.59% (244/245) similarity to MT501009, TUB2 OP414836 with 100% (703/703) to MT501053, and CAL OP414835 with 100% (601/601) to MT500921. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from B. japonicus were closely related to C. aenigma with 99% bootstrap support. Pathogenicity tests were conducted on 3-leaf stage B. japonicus seedlings. Conidial suspension of TJBDA1 (1×106 conidia/ml) brushed from a 7-day-old culture of the fungus were sprayed on 9 B. japonicus seedlings. Control plants were sprayed with sterile water. All treatments were replicated four times. The treatment plants were placed in an incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms resembling ones in the fields were observed on inoculated leaves after 7 days, but control leaves remained symptomless. The fungi reisolated from diseased leaves were morphologically and molecularly identical to the inoculated isolatescompleting Koch's postulates. According to morphological, pathological characteristics and multilocus phylogenetic analysis, the isolated strains from B. japonicus were identified as C. aenigma. To our knowledge, this is a new host record for C. aenigma causing anthracnose on B. japonicus in China. Currently, B. japonicus has evolved a high level of resistance to herbicides in some regions of China (Li et al, 2022) and C. aenigma caused serious disease to B. japonicus. We hope to discover a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. aenigma.

First Report of Botrytis californica Causing Gray Mold on Blueberry in China.

Blueberry (Vaccinium corymbosum) is a perennial shrub which grows in acid soil. Its cultivation area has recently expanded rapidly due to its unique flavor and high nutritional value (Silver and Allen 2012). In June 2021, gray mold symptoms (8 to 12% incidence) were observed on harvested fruits of the blueberry cultivar 'Lanmei 1' during storage in Jiangning (31°50'N, 118°40'E), Nanjing, China. The infection started with wrinkles, atrophy, and depressed spots on the fruit surfaces, ultimately resulting in fruit rot. To determine the causal agent, diseased fruits were sampled and rinsed with sterile water (Gao et al. 2021). Small fragments (5 × 5 × 3 mm) of decayed tissues were excised and plated onto acidified potato dextrose agar (PDA) (containing 4 ml of 25% lactic acid per liter). Plates were incubated at 25°C for 3 to 5 days, and edges of fresh culture were transferred onto new plates. This procedure was repeated thrice to obtain pure cultures. Two isolates (BcB-1 and BcB-2) were obtained. Their colonies appeared whitish to gray, with an average daily growth rate of 11.3 ± 0.6 mm (no. of plates = 30). Conidiophores were long and erect, 2560.9 to 4885.3 µm × 10.7 to 13.0 µm in size. Conidia were one-celled, elliptical to ovoid, nearly hyaline, and 9.6 to 12.5 µm × 6.7 to 8.9 µm in size. Sclerotia were gray to black in color and round or irregular in shape. These morphological features were identical to those of Botrytis spp. (Amiri et al. 2018). To further identify the isolates, we amplified four genetic markers including internal transcribed spacer region (ITS), heat-shock protein 60 (HSP60), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), and DNA-dependent RNA polymerase subunit II (RPBII) (Saito et al. 2014; Walker et al. 2011). Sequences of BcB-1 and BCB-2 were deposited in GenBank under accession nos. OP721062 and OP721063 for ITS, OP737384 and OP737385 for HSP60, OP746062 and OP746063 for G3PDH, and OP746064 and OP746065 for RPBII, respectively. BLAST analysis indicated that these sequences shared high identities (99 to 100%) with those of other B. californica isolates. Phylogenetic analysis showed that BcB-1 and BcB-2 clustered with several reference isolates in the B. californica clade. To confirm their pathogenicity, fresh blueberry fruits were surface sterilized with 0.5% sodium hypochlorite, rinsed with sterile water, air-dried, then wounded thrice per fruit using a sterile needle at the equator. Twenty wounded fruits were sprayed with 10 ml conidial suspension (1 × 105 conidia/ml) of each isolate onto the fruit surface. Twenty fruits treated with sterile water were used as controls. Inoculated or non-inoculated fruits were incubated at 25°C with 90% relative humidity. The pathogenicity test was performed twice. After 5 to 7 days, all inoculated fruits developed disease symptoms similar to those observed on the original fruits, while non-inoculated control fruits were asymptomatic. Re-isolated pathogens from the inoculated fruits exhibited identical morphological characteristics to those of BcB-1 and BcB-2. Their identity as B. californica was also verified based on their ITS sequences. Previously, B. californica has been reported to cause gray mold on blueberry in the Central Valley of California (Saito et al. 2016). To our knowledge, this is the first report of B. californica causing gray mold on post-harvest blueberry fruits in China. These results can provide the basis for future studies on the occurrence, prevention, and control of this disease.

N4-acetylation of cytidine in mRNA plays essential roles in plants.

The biological function of RNA can be modulated by base modifications. Here, we unveiled the occurrence of N4-acetylation of cytidine in plant RNA, including mRNA, by employing LC-MS/MS and acRIP-seq. We identified 325 acetylated transcripts from the leaves of 4-week-old Arabidopsis (Arabidopsis thaliana) plants and determined that 2 partially redundant N-ACETYLTRANSFERASEs FOR CYTIDINE IN RNA (ACYR1 and ACYR2), which are homologous to mammalian NAT10, are required for acetylating RNA in vivo. A double-null mutant was embryo lethal, while eliminating 3 of the 4 ACYR alleles led to defects in leaf development. These phenotypes could be traced back to the reduced acetylation and concomitant destabilization of the transcript of TOUGH, which is required for miRNA processing. These findings indicate that N4-acetylation of cytidine is a modulator of RNA function with a critical role in plant development and likely many other processes.

Visual Cascaded-Progressive Convolutional Neural Network (C-PCNN) for Diagnosis of Meniscus Injury.

OBJECTIVE: The objective of this study is to develop a novel automatic convolutional neural network (CNN) that aids in the diagnosis of meniscus injury, while enabling the visualization of lesion characteristics. This will improve the accuracy and reduce diagnosis times. METHODS: We presented a cascaded-progressive convolutional neural network (C-PCNN) method for diagnosing meniscus injuries using magnetic resonance imaging (MRI). A total of 1396 images collected in the hospital were used for training and testing. The method used for training and testing was 5-fold cross validation. Using intraoperative arthroscopic diagnosis and MRI diagnosis as criteria, the C-PCNN was evaluated based on accuracy, sensitivity, specificity, receiver operating characteristic (ROC), and evaluation performance. At the same time, the diagnostic accuracy of doctors with the assistance of cascade- progressive convolutional neural networks was evaluated. The diagnostic accuracy of a C-PCNN assistant with an attending doctor and chief doctor was compared to evaluate the clinical significance. RESULTS: C-PCNN showed 85.6% accuracy in diagnosing and identifying anterior horn injury, and 92% accuracy in diagnosing and identifying posterior horn injury. The average accuracy of C-PCNN was 89.8%, AUC = 0.86. The diagnosis accuracy of the attending physician with the aid of the C-PCNN was comparable to that of the chief physician. CONCLUSION: The C-PCNN-based MRI technique for diagnosing knee meniscus injuries has significant practical value in clinical practice. With a high rate of accuracy, clinical auxiliary physicians can increase the speed and accuracy of diagnosis and decrease the number of incorrect diagnoses.

A plant growth-promoting bacteria Priestia megaterium JR48 induces plant resistance to the crucifer black rot via a salicylic acid-dependent signaling pathway.

Xanthomonas campestris pv. campestris (Xcc)-induced black rot is one of the most serious diseases in cruciferous plants. Using beneficial microbes to control this disease is promising. In our preliminary work, we isolated a bacterial strain (JR48) from a vegetable field. Here, we confirmed the plant-growth-promoting (PGP) effects of JR48 in planta, and identified JR48 as a Priestia megaterium strain. We found that JR48 was able to induce plant resistance to Xcc and prime plant defense responses including hydrogen peroxide (H2O2) accumulation and callose deposition with elevated expression of defense-related genes. Further, JR48 promoted lignin biosynthesis and raised accumulation of frees salicylic acid (SA) as well as expression of pathogenesis-related (PR) genes. Finally, we confirmed that JR48-induced plant resistance and defense responses requires SA signaling pathway. Together, our results revealed that JR48 promotes plant growth and induces plant resistance to the crucifer black rot probably through reinforcing SA accumulation and response, highlighting its potential as a novel biocontrol agent in the future.

First Report of Colletotrichum truncatum Causing Anthracnose of Green Foxtail (Setaria viridis) in China.

Green foxtail (Setaria viridis (L.) Beauv.), belonging to the family Gramineae, is a monocotyledonous plant that is distributed in tropical and subtropical regions of the world. S. viridis is one of the most abundant weeds in corn, soybean, rice and other major crops in China, which competes with crops for light, moisture and nutrients, leading to yield losses. In September 2021, an unknown leaf spot disease was observed on the leaves of S. viridis in many greenhouses of Xinkou town, Xiqing district, Tianjin, China (116.95729, 39.09088), under cloudy and high humid conditions after a week of rain. Over 60% of the weeds were observed with leaf spots in 28 greenhouses of XinKou town. The characteristics of the disease were observed and investigated. Initial symptoms were brown spots of 1 to 5 mms, longitudinal elliptic, round, or spindle-shaped lesions on leaves of S. viridis. These spots continued to spread shortly after the onset of the symptoms. At the late-stage disease, the spots' edges were dark brown and irregular. Eventually, the center of the spots turned grayish-white and became thinner and drier until fracture. To investigate the disease, symptomatic weed leaves were separated and small patches with infected spots were cut out. Diseased tissues (3×3 mm) were disinfected with 75% alcohol for 30s  35s, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25°C with a 12-h photoperiod for 7 days in incubators (RXZ-280C, Ningbo, China). With the pathogen growing on the PDA, three mycelia with uniform morphology were observed, which were named SVCT-01, SVCT-02, and SVCT-03, respectively. These mycelia were transferred and cultured for daily observation. The color of these mycelia on PDA appeared gray at first, which eventually turned to grayish black with numerous black microsclerotia, setae, and a few aerial mycelia after 7 days. The setae were 75 to 120 ×3.5 to 5 µm, with elliptic to claviform appressoria. Conidia were hyaline, falcate, unicellular, 16 to 25 × 2.6 to 3.8 µm (n=50). All characteristics of isolates were consistent with the description of Colletotrichum truncatum (Sutton, 1992). Pathogenicity testing was conducted on 3-leaves-stage S. viridis seedlings. Conidial suspension (106 conidia/mL) of isolates were sprayed on 20 S. viridis seedlings with the suspension of each isolate was sprayed on 10 seedlings. Ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. The treatment plants were maintained in the incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms were observed on inoculated leaves after 7 days, the control leaves remained symptomless. The fungus reisolated from the lesions of diseased leaves were morphological and molecularly identical to the inoculated isolates. The results echo with Koch's postulates，suggesting that the obtained isolates SVCT-01, SVCT-02 and SVCT-03 are potential pathogen in Setaria viridis. To confirm the species' identity, total genomic DNA of isolates were extracted using a Fungal DNA Kit (GBCBIO, Guangzhou, China). Sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and ß-tubulin (TUB2) regions were identified via PCR (Guerber et al, 2003; Weir et al, 2012). The sequences of SVCT-01, SVCT-02 and SVCT-03 showed more than 99% homology with Colletotrichum truncatum strains CBS:151.35 (GenBank Accession No. GU227862, GU228254, GU227960, GU228156) (Damm, 2009). The sequences of SVCT-01 were deposited in GenBank as a representative isolate under the accession numbers OL629177, OL627527, OM040388, OM040389. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from Setaria viridis were closely related to Colletotrichum truncatum with 100% bootstrap support. According to morphological, pathological characteristics, and multilocus phylogenetic analysis, the isolated strains (SVCT-01, SVCT-02 and SVCT-03) from S. viridis were identified as Colletotrichum truncatum (Weir et al, 2012). Colletotrichum sp. is a significant plant pathogen that was previously reported causing anthracnose on Setaria sp. Up to now, it has been reported that C. graminicola has infected nine species of Setaria sp. Such as Setaria glauca in New Zealand (Pennycook, 1989) and Setaria pumila in Zimbabwe (Lenne, 1990). In 1979s, C.graminicola was obtained from Setaria lutescens in China (Tai, 1979). To our knowledge, this is a new host record for Colletotrichum truncatum causing anthracnose on S. viridis in China. Colletotrichum truncatum spread rapidly and caused serious disease to Setaria viridis. We hope to discovery a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. truncatum.

Crucial role of Ca2+ /CN signalling pathway in the antifungal activity of disenecioyl-cis-khellactone against Botrytis cinerea.

BACKGROUND: Botrytis cinerea causes grey mould and is one of the most destructive fungal pathogens affecting important fruit and vegetable crops. In preliminary studies, we found that disenecioyl-cis-khellactone (DK) had strong antifungal activity against several fungi species including B. cinerea [half maximal effective concentration (EC50 ) = 11.0 µg mL-1 ]. In this study, we aimed to further evaluate the antifungal activity of DK against B. cinerea and determine the role of calcium ion/calcineurin (Ca2+ /CN) signalling pathway on its antifungal effect. RESULTS: DK was effective against B. cinerea in both in vitro and in vivo assays. Exogenous Ca2+ reduced the antifungal activity of DK. The combination of DK and cyclosporine A (CsA) did not exhibit an additive effect against B. cinerea. In contrast to CsA, DK reduced the intracellular Ca2+ concentration in B. cinerea. DK bound to calcineurin A (cnA) and up-regulated the expression of PMC1 and PMR1 genes. Moreover, DK sensitivity of △bccnA significantly decreased compared with that of Bc05.10 strain. CONCLUSION: DK is a promising lead compound for developing fungicides against B. cinerea. The Ca2+ /CN signalling pathway plays a crucial role in the DK antifungal activity, and cnA is one of the targets of DK against B. cinerea. DK directly reacts with cnA, which up-regulates the transcription of Ca2+ /CN-dependent target genes PMC1 and PMR1, decreasing the intracellular Ca2+ concentration and disturbing the intracellular Ca2+ balance, leading to cell death. © 2022 Society of Chemical Industry.

Antifungal Activity and Possible Mechanism of Bacillus amyloliquefaciens FX2 Against the Postharvest Apple Ring Rot Pathogen.

Botryosphaeria dothidea-induced apple ring rot is one of the most serious postharvest diseases in apple production. In our preliminary work, we isolated a bacterial strain (FX2) from an infested apple orchard. Here, we confirmed the strong antifungal activity of FX2 on B. dothidea. Through phylogenetic analysis and morphological observations, we identified FX2 as a Bacillus amyloliquefaciens strain. We also found that 10% cell-free supernatant (CFS) of FX2 significantly affected mycelial growth and morphology and almost completely inhibited spore germination and germ tube elongation in B. dothidea. Furthermore, 10% CFS damaged the cell ultrastructure, resulting in a remarkable increase in cellular leakage in B. dothidea mycelia. Thus, CFS has the potential to effectively reduce in vivo B. dothidea infection, reduced lesion diameters to 64.7% compared with the control group, and reduced disease incidence by 15%. Finally, ultrafiltration, desalting chromatography, and anion exchange chromatography showed that the antifungal constituents in CFS are composed mainly of antifungal proteins. We further characterized these potential antifungal proteins via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Herein, we provide novel insights into the antifungal mechanisms of B. amyloliquefaciens FX2, and we highlight its potential as a novel biocontrol agent for controlling postharvest apple ring rot.

First report of Leaf Spot Caused by Paramyrothecium foliicola on Tomato (Solanum lycopersicum) in China.

Tomato (Solanum lycopersicum) is a staple vegetable across the world. In October 2019, leaf spots were observed on tomato (cv. Tianmi) in a greenhouse in JiZhou District Tianjin, China(117°10 'E; 39°55 'N). Symptoms initially appeared as small brown spots, which gradually expanded and turned into circular, oval or irregular spots (some spots with distinct concentric zones). In severe cases, some spots coalesced and eventually covered the whole leaf. Disease incidence ranged between 12 and 18%. Twenty symptomatic leaves from five plants were collected and cut into small pieces, surface disinfested in 2% NaClO for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark for 7 days. A total of 102 isolates were obtained and 92 isolates had the same morphology. Colonies were initially white with abundant aerial mycelia and formed sporodochia with conidial masses in olivaceous green concentric rings. All isolates formed single-celled, hyaline, and rod-shaped conidia were 4.91 to 7.43 (avg. 6.53±0.72) × 1.41 to 2.45 （avg. 2.11±0.30）µm with rounded ends (n=50). Conidiophores were highly branched. These characteristics resembled a Paramyrothecium-like fungus (Lombard et al. 2016). The genomic DNA of three representative single-spored isolates TJJXPF1-3 were extracted and the internal transcribed spacer (ITS) region, ß-tubulin (tub2), large subunit ribosomal RNA (LSU), calmodulin (cmdA) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LR0R/LR5 (Rehner and Samuels 1995; Vilgalys and Hester 1990), CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013) and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. All sequences were deposited in GenBank (ITS: MW463444, OM368178, OM368179; tub2: MW269542，OM714930，OM714931; LSU: OM349050, OM397398, OM390582; cmdA: MW280443, OM350474, OM350476; tef1: MW560083, OM350475, OM350477). BLASTN analysis showed 99.3-100% similarity with reference isolate QB1 of P. foliicola (MK335967, MT415353, MT415362, MT415356 and MT415359). Multilocus phylogenetic analysis showed that TJJXPF1-3 best grouped with the P. foliicola clade, which was identified by morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, pathogenicity tests were conducted by spray-inoculation with a conidial suspension of isolate TJJXPF1 prepared with distilled water (1×105 conidia/mL) on five 45-day old tomato plants. Three healthy plants were sprayed with sterile water as control. All treatments were incubated in an artificial climate chamber (25°C, 80% RH, 12h light/12h dark ). After two weeks, leaf spots were observed on all inoculated plants, which were similar to those in the greenhouse of JiZhou District, while control plants remained asymptomatic. Additionally, the pathogens were reisolated from symptomatic leaves and three representative isolates TJJXPF4-6 were identified as P. foliicola. The pathogenicity tests were repeated thrice. To our knowledge, this is the first report of leaf spot caused by P. foliicola on tomato in China. This disease could be a serious threat to tomato production in the future. Our findings will help to differentiate this disease from other leaf spot-like diseases and develop disease control strategies.