Anatomical Changes during Chestnut (Castanea mollissima BL.) Gall Development Stages Induced by the Gall Wasp Dryocosmus kuriphilus (Hymenoptera: Cynipidae).

This study delved into the larval development and the morphological and anatomical transformations that occur in the galls of chestnut trees (Castanea mollissima BL.) and are induced by the chestnut gall wasp Dryocosmus kuriphilus Yasumatsu (GWDK) across various stages: initial, growth, differentiation, maturity, and lignification. Chestnut galls in the five development stages were collected. Gall structural characteristics were observed with an anatomical stereomicroscope, and anatomical changes in galls were analyzed with staining and scanning electron microscope techniques. The chestnut gall wasp laid its eggs on young leaves and buds. Chestnut gall wasp parasitism caused plant tissues to form a gall chamber, with parenchyma, protective, and epidermal layers. The development of the gall structure caused by the infestation of the GWDK gall led to the weakening of the reactive oxygen species (ROS) elimination ability of the host. The accumulation of ROS led to cell wall peroxidation, resulting in structural damage and diminished host resistance, and the parenchyma layer exhibited significant nutrient supply and thickening. The thickness of the protective and epidermal layers varied notably across different growth stages. The oviposition of the chestnut gall wasp induced modifications in the original plant tissues, with gall formation being most favorable in young tissues, correlating with the maturity level of the host plant tissues. Variances in the internal structures of the galls primarily stemmed from nutrient supplementation, while those in the external structure were attributed to defensive characteristics. This research contributes a foundational understanding of gall development induced by the chestnut gall wasp in Chinese chestnut, offering valuable insights into the intricate interplay between insect infestation and plant physiology.

Microbial Diversity Associated with the Cabernet Sauvignon Carposphere (Fruit Surface) from Eight Vineyards in Henan Province, China.

The microbial diversity on the carposphere (berry) surface of the grape cultivar Cabernet Sauvignon grown in eight different locations/vineyards of Henan Province was determined by high-throughput sequencing of the bacterial 16S rRNA gene and fungal 18S rRNA gene. The structure of bacterial and fungal communities varied according to the sampling sites, but with some common phyla. Proteobacteria and Ascomycota were dominant/common phyla for bacteria and fungi, respectively. A total of 27 and 20 bacterial and fungal families, respectively, and 39 and 20 bacterial and fungal genera, respectively, with statistically significant differences, were found among different sampling sites. The difference for metabolic pathways of bacteria among the sampling sites existed. In addition, various abundances of enzymes from different sites might indicate that different function patterns exist in microbiota from different sites. The results revealed that locations of grape vineyards might play a significant role in shaping the microbiome, as well as the fact that vineyards can be distinguished based on the abundance of several key bacterial and fungal taxa. Overall, these findings extend our understanding of the similarities and differences in microbial community and their metabolic function on Cabernet Sauvignon grape surfaces from different geographic locations.

A novel xylanase from a myxobacterium triggers a plant immune response in Nicotiana benthamiana.

Xylanases derived from fungi, including phytopathogenic and nonpathogenic fungi, are commonly known to trigger plant immune responses. However, there is limited research on the ability of bacterial-derived xylanases to trigger plant immunity. Here, a novel xylanase named CcXyn was identified from the myxobacterium Cystobacter sp. 0969, which displays broad-spectrum activity against both phytopathogenic fungi and bacteria. CcXyn belongs to the glycoside hydrolases (GH) 11 family and shares a sequence identity of approximately 32.0%-45.0% with fungal xylanases known to trigger plant immune responses. Treatment of Nicotiana benthamiana with purified CcXyn resulted in the induction of hypersensitive response (HR) and defence responses, such as the production of reactive oxygen species (ROS) and upregulation of defence gene expression, ultimately enhancing the resistance of N. benthamiana to Phytophthora nicotianae. These findings indicated that CcXyn functions as a microbe-associated molecular pattern (MAMP) elicitor for plant immune responses, independent of its enzymatic activity. Similar to fungal xylanases, CcXyn was recognized by the NbRXEGL1 receptor on the cell membrane of N. benthamiana. Downstream signalling was shown to be independent of the BAK1 and SOBIR1 co-receptors, indicating the involvement of other co-receptors in signal transduction following CcXyn recognition in N. benthamiana. Moreover, xylanases from other myxobacteria also demonstrated the capacity to trigger plant immune responses in N. benthamiana, indicating that xylanases in myxobacteria are ubiquitous in triggering plant immune functions. This study expands the understanding of xylanases with plant immune response-inducing properties and provides a theoretical basis for potential applications of myxobacteria in biocontrol strategies against phytopathogens.

Chickpea: Its Origin, Distribution, Nutrition, Benefits, Breeding, and Symbiotic Relationship with Mesorhizobium Species.

Chickpea (Cicer arietinum L.), encompassing the desi and kabuli varieties, is a beloved pulse crop globally. Its cultivation spans over fifty countries, from the Indian subcontinent and southern Europe to the Middle East, North Africa, the Americas, Australia, and China. With a rich composition of carbohydrates and protein, constituting 80% of its dry seed mass, chickpea is also touted for its numerous health benefits, earning it the title of a 'functional food'. In the past two decades, research has extensively explored the rhizobial diversity associated with chickpea and its breeding in various countries across Europe, Asia, and Oceania, aiming to understand its impact on the sustainable yield and quality of chickpea crops. To date, four notable species of Mesorhizobium-M. ciceri, M. mediterraneum, M. muleiense, and M. wenxiniae-have been reported, originally isolated from chickpea root nodules. Other species, such as M. amorphae, M. loti, M. tianshanense, M. oportunistum, M. abyssinicae, and M. shonense, have been identified as potential symbionts of chickpea, possibly acquiring symbiotic genes through lateral gene transfer. While M. ciceri and M. mediterraneum are widely distributed and studied across chickpea-growing regions, they remain absent in China, where M. muleiense and M. wenxiniae are the sole rhizobial species associated with chickpea. The geographic distribution of chickpea rhizobia is believed to be influenced by factors such as genetic characteristics, competitiveness, evolutionary adaptation to local soil conditions, and compatibility with native soil microbes. Inoculating chickpea with suitable rhizobial strains is crucial when introducing the crop to new regions lacking indigenous chickpea rhizobia. The introduction of a novel chickpea variety, coupled with the effective use of rhizobia for inoculation, offers the potential not only to boost the yield and seed quality of chickpeas, but also to enhance crop productivity within rotation and intercropped systems involving chickpea and other crops. Consequently, this advancement holds the promise to drive forward the cause of sustainable agriculture on a global scale.

Terpene Synthase Gene Family in Chinese Chestnut (Castanea mollissima BL.) Harbors Two Sesquiterpene Synthase Genes Implicated in Defense against Gall Wasp Dryocosmus kuriphilus.

Chinese chestnut (Castanea mollissima BL.) is a well-known fruit tree that has been cultivated in East Asia for millennia. Leaves and buds of the plant can become seriously infested by the gall wasp Dryocosmus kuriphilus (GWDK), which results in gall formation and associated significant losses in fruit production. Herbivore-induced terpenes have been reported to play an important role in plant-herbivory interactions, and in this study, we show that upon herbivory by GWDK, four terpene-related compounds were significantly induced, while the concentrations of these four compounds in intact buds were relatively low. Among these compounds, (E)-nerolidol and (E, E)-α-farnesene have frequently been reported to be involved in plant herbivory defenses, which suggests direct and/or indirect functions in chestnut GWDK defenses. Candidate terpene synthase (TPS) genes that may account for (E)-nerolidol and (E, E)-α-farnesene terpene biosynthesis were characterized by transcriptomics and phylogenetic approaches, which revealed altered transcript levels for two TPSs: CmAFS, a TPS-g subfamily member, and CmNES/AFS, a TPS-b clade member. Both genes were dramatically upregulated in gene expression upon GWDK infestation. Furthermore, Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana showed that CmAFS catalyzed the formation of (E, E)-α-farnesene, while CmNES/AFS showed dual (E)-nerolidol and (E, E)-α-farnesene synthase activity. Biochemical assays of the recombinant CmAFS and CmNES/AFS proteins confirmed their catalytic activity in vitro, and the enzymatic products were consistent with two of the major volatile compounds released upon GWDK-infested chestnut buds. Subcellular localization demonstrated that CmAFS and CmNES/AFS were both localized in the cytoplasm, the primary compartment for sesquiterpene synthesis. In summary, we show that two novel sesquiterpene synthase genes CmAFS and CmNES/AFS are inducible by herbivory and can account for the elevated accumulation of (E, E)-α-farnesene and (E)-nerolidol upon GWDK infestation and may be implicated in chestnut defense against GWDK herbivores.

Extraction of DNA from trace forensic samples with a modified lysis buffer and chitosan coated magnetic beads.

The trace amounts of human tissue cells or body fluids left at the crime scene are often mixed with inhibitors such as rust, pigments, and humic acid. The extraction of the DNA from the trace cells is crucial for the investigation of cases. Usually, specially designed magnetic nanoparticles were chosen by the case investigators to enrich and elute DNA, which was then used for polymerase chain reaction (PCR) and short tandem repeat (STR) analysis. The traditional approach often had the following problems, such as low DNA enrichment efficiency, possible DNA breakage, and complex operations. Here, the 1%(w/v) of chitosan (75% deacetylation degree) was used to modify the 50 nm magnetic nanoparticles to gain the Chitosan@Beads, which theoretically carried positively charged in the pH = 5 of lysis buffer so as to adsorb negatively charged DNA through electrostatic interactions. The XPS and FT-IR results demonstrated that chitosan was successfully attached to the surface of magnetic nanoparticles. A set of simulated samples, containing 20 mg/µL of humic acid, pigments, iron ions (Fe2+, Fe3+), and the coexistence of the above three substances, were prepared to simulate the case scene. Human bronchial epithelial cells were mixed with the 200 µL of the above simulated samples for DNA extraction. 400 µL of lysis buffer, 20 µL of proteinase K (10 mg/mL) and 20 µL of Chitosan@Beads solution (20 mg/mL) was used for cell disruption and DNA enrichment. The extraction sensitivity of Chitosan@Beads was confirmed to be 10 cells, superior to commercial reagent kits. The Chitosan@Beads@DNA can directly use for "In-situ PCR" with elution-free operations. The STR loci rate of DNA extracted by Chitosan@Beads was around 97.9%, higher than the commercial kit (66.7%). In short, we foresee here developed novel Chitosan@Beads and modified lysis buffer could provide a new model for the DNA extraction of forensic trace evidence.

First Report of Colletotrichum aenigma causing Anthracnose on Pecan in China.

Pecan (Carya illinoinensis) is an important economic forest crops widely cultivated in China. From June to September in both 2021 and 2022, severe leaf disease resembling anthracnose was observed in 6.6-ha pecan orchard in Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 15 to 25% with 5 to 12% incidence on 100 surveyed trees of the orchard in 2022. Symptoms initially appeared as small gray-bark sunken lesions, which gradually developed to big sunken lesions with brown edges and irregular-shaped. Small fragments (4 × 4 mm) from the necrotic borders of infected leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained by monosporic isolation. Twenty-one isolates with similar characteristics were obtained from the infected leaves (isolation frequency about 90%). The upper side of colonies on the PDA plates was milky, and the reverse side was pale yellow at the center and pale white at the margin. After 10 days of growth on the PDA medium, these isolates produced spores separately. . Through electron microscopic observation, conidia were smooth walled, hyaline, aseptate, guttulate, cylindrical with rounded ends with 15 to 20.5 × 5.3 to 6.7 µm (mean 18.5 × 5.8 µm, n = 50) in size. These morphological characteristics were similar to those of the species of Colletotrichumspp (Weir et al. 2012, Fu et al. 2019). To further identify the isolates, the regions of internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHSI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (TUB2) loci of the three representative isolates (JSJT-1, JSJT-2, and JSJT-3) were amplified and sequenced with the primer pairs ITS-1F/ITS-4, ACT-512F/ACT-783R, CL1/CL2A, CHS-79F/CHS-345R, GDF/GDR and T1/T2 primers, respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OR214960 to OR214962 (ITS), OR228543 to OR228545 (ACT)，OR228546 to OR228548 (CAL), OR228549 to OR228551 (CHSI), OR228552 to OR228554 (GAPDH), and OR228555 to OR228557 (TUB2). Multilocus phylogenetic analysis revealed that the three isolates and C. aenigma were clustered in the same clade. Based on the results of morphological and molecular analysis, these isolates were identified as C. aenigma. The pathogenicity of three isolates was tested on leaves of pecan seedlings. Suspensions of conidia were obtained by scraping the surface of a 10-day-old sporulated petri dish PDA cultures into sterile water. Suspensions were adjusted to a density of 2 × 106 conidia/ml with a hemocytometer.The conidial suspension of each isolate was sprayed evenly on the surface of leaves from three healthy pecan seedlings. Sterilized distilled water was used for negative controls. The pathogenicity experiment was repeated three times. Finally, all inoculated plants were kept in a light-incubator at 28°C under 100% relative humidity and 12 h photoperiod. Two weeks after inoculation, the inoculated plants developed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. aenigma were re-isolated from from inoculated leaves. C. aenigma has been reported as the causal agent of anthracnose on several economically important plants, such as grape ( Kim et al. 2021), tree peonies (Wang et al.2023), chili (Diao et al. 2017), and pear (Fu et al. 2019), but this is the first report of C. aenigma causing anthracnose on pecan in China. Identification of C. aenigma as a pathogen of pecan is important for implementing control management strategies for pecan disease. References: Diao, Y. Z., et al. 2017. Persoonia. 38:20. Fu, M., et al. 2019. Persoonia. 42:1. Kim, J. S., et al. 2021. Plant Dis. 105:2729. Weir, B. S., et al. 2012. Stud. Mycol.. 73:115. Wang, Y. L., et al. 2023. Plant Dis. 107(4):1242. The author(s) declare no conflict of interest. Keywords: Colletotrichum aenigma, Anthracnose, Carya illinoinensis, Pathogenicity.

First Report of Diaporthe pseudophoenicicola causing Leaf Blight on Pecan in China.

Pecan (Carya illinoinensis) is one of the important economic forest crops widely cultivated in Jiangsu Provinces, China. From August to September in both 2021 and 2022, a foliar blight was observed in 7-ha and 6-ha pecan orchards in Changzhou (31°58'9.6″ N, 119°48'33.84″ E), and Jurong (31°52'15.46″ N, 119°9'24.62″ E), Jiangsu Province. The disease severity was about 32% with 8% incidence on 120 surveyed trees of the two orchards. Typical symptoms were lesions with a dark-brown color, which later became brown. We collected eighteen pecan leaves with typical symptoms in the surveyed pecan orchards and took them back to the laboratory for identification. Small fragments (approximately 9 mm2) from the necrotic borders of infected leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C. Pure cultures were obtained by single-spore culture. Thirty-three isolates with similar characteristics were obtained from the infected leaves (isolation frequency 85%), and the colonies surface on PDA was ochreous with patchs of olivaceous-yellow and sparse aerial mycelium. Observing from the back of the plate, the colonies were cream-yellow. Two types of single-cell conidia were produced on PDA. Alpha-conidia were 7.4 (range, 5.9 to 8.8) × 2.1 (range, 1.6 to 2.8) µm (n = 100), aseptate, smooth, fusiform, straight and tapering towards both ends. Beta-conidia were 25.1 (range, 19.1 to 36.2) × 1.3 (range, 1.0 to 2) µm (n = 100), filiform, hyaline, aseptate and curved at one end. The morphological features of these isolates agreed with those of Diaporthe sp. (Gomes et al. 2013; Gao et al. 2017). To further identify the isolates, the regions of internal transcribed spacer (ITS, OR214967 to OR214969), calmodulin (CAL, OR228558 to OR228560), translation elongation factor 1-α (EF1a, OR228561 to OR228563), histone H3 (HIS, OR228564 to OR228566), and beta-tubulin 2 (TUB2, OR228567 to OR228569) were amplified and sequenced from genomic DNA for the three representative isolates (LSM1, LSM2 and LSM3), respectively (Gomes et al. 2013). Multilocus phylogenetic analysis revealed that the three isolates and D. pseudophoenicicola were clustered in the same clade. Based on the results of morphological and molecular analysis, these isolates were identified as D. pseudophoenicicola. The pathogenicity of three isolates were tested on leaves of pecan seedlings. The conidial suspension (1 × 105 conidia/ml) of each isolate was sprayed evenly on the surface of leaves of three healthy seedlings. Sterilized distilled water was used for negative controls. Finally, all inoculated plants were kept in a greenhouse at 28°C under 100% relative humidity. Two weeks after inoculation, the inoculated plants developed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. D. pseudophoenicicola were re-isolated from from inoculated plants. The pathogenicity experiment was repeated three times. Previously, D. pseudophoenicicola has been reported to cause stem-end browning disease in ripe mango (Takushi et al. 2016; Xu et al 2022). To our knowledge, this is the first report of D. pseudophoenicicola causing leaf blight on pecan . This study provides important information for developing effective pecan disease management practices.

First report of leaf spot on Chaste-Tree Caused by Alternaria alternata in China.

Chaste-tree (Vitex agnus-castus Linn.) is a perennial ornamental shrub that is native to Europe, which has been widely distributed in China. Since 2021, a serious leaf spot on chaste-tree leaves was observed in Nanjing Botanical Garden, Jiangsu Province, China (31°14'6″N, 118°22'12″E). The disease incidence on the leaves ranged from 20 to 40%. The disease symptom initially appeared as irregular small gray spots on leaves that gradually coalesced into larger lesions with diseased leaves turning black and withering. From August of 2021 to 2022, small pieces of leaf tissues (5×5mm) from the necrotic borders of five typical symptomatic infected leaves were collected and surface sterilized (with 75% ethanol), then incubated in darkness at 25°C for 7 days. A total of fifteen isolates were obtained by monosporic isolation (isolation frequency of 76%). The fungal colonies were initially grayish-white and turned into dark gray with abundant cotton-like aerial hyphae. Microscopic observations revealed light-brown conidia that were obclavate or obpyriform (inversely pear-shaped) with length between 10 and 20 µm (mean 13.3 ± 2.4 µm) and widths between 5 and 10 µm (mean 7.8 ± 1.2 µm), 2 to 4 transverse septa and 0 to 2 longitudinal septa per conidium (n=30) were observed. The fungus was identified as Alternaria alternata based on the colony characteristics (Simmons 2007) and the representative isolate Aa1 was used for further studies. To further identify Aa1, the region of internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (EF-1a) and RNA polymerase second largest subunit (RPB2) genes were amplified from genomic DNA and sequenced with the primer pairs ITS1/ITS4 (Jayawardena et al. 2019), EF-728F/EF-986R (Carbone and Kohn 1999), Gpd1/Gpd2 (Berbee et al. 1999) and RPB2-5F/RPB2-7cR (Liu et al. 1999) respectively. Sequences were deposited into GenBank (Accession No. OQ626644 and OQ630494-OQ630496), which showed 99.2 to 100% sequence homology with those A. alternata strains in GeneBank (ITS, MN394880; GAPDH, MN410920; EF-1a, MN410916; RPB2, MN410918). The multigenes phylogenetic analysis revealed that isolate Aa1 and Alternaria alternata TCS3002 + CBS 916.96 clustered within the same clade with 99% bootstrap support. To test pathogenicity, conidial suspension (1×106 spores/ml) of Aa1 was sprayed uniformly across the leaves of three 1-year-old healthy chaste-tree seedlings; sterilized distilled water sprayed on other trees were used as negative control and the experiment was repeated three times. All inoculated plants were kept in same condition (25°C, under a 16 h/8 h photoperiod and 70% relative humidity). One week later, black/gray spots were observed on the leaves of inoculated plants, similar to the symptoms that were observed on the original diseased plants, while controls remained asymptomatic. Cultures were re-isolated from the infected leaves and were again identified as Aa1 by both morphological characteristics and DNA sequence analysis. The pathogen reported here has a broad host range, and has also been reported on Magnolia grandiflora L. (Liu et al. 2019), Kalanchoe pinnata (Sanahuja et al. 2018) and Kadsura coccinea (Zhang et al. 2020) to cause leaf spot. To our knowledge, this is the first report of A. alternata causing leaf spot disease on chaste-tree and provides an important reference for further biology and epidemiology research.

Genetic Relationships of 118 Castanea Specific Germplasms and Construction of Their Molecular ID Based on Morphological Characteristics and SSR Markers.

To understand the genetic relationships of Castanea species, 16 phenotypic traits were measured, simple sequence repeat (SSR) markers were analyzed, and molecular identity cards (IDs) were constructed for 118 Castanea materials using fluorescent capillary electrophoresis. The coefficient of variation values of the 16 morphological traits of the test materials ranged from 11.11% to 60.38%. A total of 58 alleles were detected using six pairs of SSR core primers, with an average number of 9.7 alleles per locus. The average number of valid alleles per locus was 3.9419 and the proportion of valid alleles was 40.78%. A total of 105 genotypes were detected, and the number of genotypic species that could be amplified per primer pair ranged from 8 to 26. The mean value of the observed heterozygosity was 0.4986. The variation in the He, H, and PIC values was similar; the size of I value was approximately 2.21 times larger, and its mean number of variations was 0.7390, 0.7359, 0.6985, and 1.6015, respectively. The classification of 118 Castanea species was performed using three analytical methods: structure analysis, neighbor-joining (NJ) cluster analysis, and principal coordinate analysis (PCoA), and the results of the three methods were in high agreement. Six pairs of SSR core primers with high polymorphism and strong discriminatory properties were used to identify 118 Castanea plants, and a unique molecular ID card was constructed for each material. These results provide insight into the genetic diversity and population structure of Castanea plants and a theoretical basis for improving the phenomenon of mixed varieties and substandard plants in the Castanea plant market.

Mkit: A mobile nucleic acid assay based on a chitosan-modified minimalistic microfluidic chip (CM3-chip) and smartphone.

Mobile sensing enabled by MS2 technology, which integrates microfluidic and smartphone components, has seen many applications in recent years. In this direction, we developed an MS2 platform (an integrated kit) for nucleic acid assay, which included a chitosan-modified minimalistic microfluidic chip (CM3-chip), a smartphone-based fluorescence detector (SF-detector), an APP for imaging and analysis, reagents, and accessories. Once the lysed sample was loaded into the CM3-chip modified by 1% concentration and 200-260 kDa molecular weight of chitosan, the following assay can be completed in approximately 1 h. The Mkit can detect 3 × 10° copies µL-1 of plasmid DNA and its polymerase chain reaction (PCR) efficiency was 96.8%. The CM3-chip equipped for the Mkit can enrich nucleic acid from the pH = 5 of lysis buffer, instead of using conventional adsorption mediums such as the magnetic beads and silica gel membranes, which could result in unexpected impurity residuals and tedious cleaning operations. In addition, the performance of the Mkit equipped with the pristine chip was demonstrated to perform poorer than that coupled with the CM3-chip in which the enriched nucleic acid can be all used for "in-situ PCR". The universality, selectivity, and user-friendliness of the Mkit were also validated. We finally demonstrated the feasibility of the Mkit for testing artificially prepared infected samples. H5N6 and IAV-infected saliva samples provided the limits of detection of 5 × 102 copies mL-1 and 3.24 × 102 copies mL-1 per chamber, respectively. The streamlined assay and compact device should enable the great potential of the Mkit in research and potential diagnostic uses.

Genome-wide identification and expression analysis of the SAUR gene family in foxtail millet (Setaria italica L.).

BACKGROUND: Auxin performs important functions in plant growth and development processes, as well as abiotic stress. Small auxin-up RNA (SAUR) is the largest gene family of auxin-responsive factors. However, the knowledge of the SAUR gene family in foxtail millet is largely obscure. RESULTS: In the current study, 72 SiSAUR genes were identified and renamed according to their chromosomal distribution in the foxtail millet genome. These SiSAUR genes were unevenly distributed on nine chromosomes and were classified into three groups through phylogenetic tree analysis. Most of the SiSAUR members from the same group showed similar gene structure and motif composition characteristics. Analysis of cis-acting elements showed that many hormone and stress response elements were identified in the promoter region of SiSAURs. Gene replication analysis revealed that many SiSAUR genes were derived from gene duplication events. We also found that the expression of 10 SiSAURs was induced by abiotic stress and exogenous hormones, which indicated that SiSAUR genes may participated in complex physiological processes. CONCLUSIONS: Overall, these results will be valuable for further studies on the biological role of SAUR genes in foxtail development and response to stress conditions and may shed light on the improvement of the genetic breeding of foxtail millet.

First Report of Colletotrichum siamense causing Anthracnose on Pecan in China.

Pecan (Carya illinoinensis) is one of the important economic forest crops which has been widely cultivated in Anhui and Jiangsu Provinces, China. Since 2019, symptoms resembling anthracnose disease had been observed in 5-ha and 6.6-ha pecan orchards in Quanjiao ( 32°5'7.08″ N, 118°16'2.91″ E), Anhui Province, and Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 20 to 30% with 5 to 15% (about 500 trees) incidence. In May, symptoms of leaf initially appeared as small dark lesions, which gradually developed to irregular-shaped, sunken lesions (Figure S1, A). From August to October, similar symptoms were also observed on the fruits. Infected fruits appeared irregularly, dark and depressed necrotic lesions on which orange spore masses could be occasionally observed (Figure S1, B). As the disease progressed, the necrotic lesions gradually expanded and merged, resulting in abscission of the fruits. Small fragments (4 × 4 mm) from the necrotic borders of infected fruits or leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained from individual conidia by recovering single spores. On the PDA plate, the colonies surface was white and cottony. Observing from the back of the plate, the colonies were pale yellow at the centre and pale white at the margin (Figure S1, E). Spores were produced over PDA plates after 7 days growth. Conidia were hyaline, smooth walls, aseptate, guttulate, cylindrical with rounded ends with 14.8 to 17.5 × 3.3 to 4.7 µm (mean 16.5 × 4.1µm, n = 50) in size (Figure S1, F). These morphological characteristics were similar to those of the species of Colletotrichum siamense (Prihastuti et al. 2009; Weir et al. 2012; Fu et al. 2019). Thirty-two isolates Colletotrichum sp. were obtained from the infected leaves and fruits (isolation frequency about 80%). To further identify the isolates, the regions of internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHSI), and beta-tubulin 2 (TUB2) were amplified and sequenced from genomic DNA for the four representative isolates (JS1 and AH1 from infected fruits; JS2 and AH2 from infected leaves), respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OP389224 to OP389227 (ITS), OP413765 to OP413768 (CAL), OP413761 to OP413764 (ACT), OP413773 to OP413776 (GAPDH), OP413769 to OP413772 (CHSI), and OP413777 to OP413780 (TUB2). Blast analysis showed these sequences shared high identity with C. siamense (100% with ITS, CAL, CHSI, and TUB2; 98.94% with ACT; 98.19% with GAPDH). Multilocus phylogenetic analysis revealed that the four isolates and C. siamense were clustered in the same clade (Figure S2). Based on the results of morphological and molecular analysis, these isolates were identified as C. siamense. The pathogenicity of four isolates was tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 106 conidia/ml was sprayed evenly on the surface of leaves of a healthy seedling, and each isolate inoculated three pecan seedlings. The pathogenicity experiment was repeated three times. For negative controls, pecan seedlings were sprayed with sterilized distilled water. Finally, all inoculated plants were kept in a greenhouse at 25°C under a 16 h/8 h photoperiod and 70% relative humidity. Three weeks after inoculation, the inoculated plants showed symptoms similar to those of the original diseased plants (Figure S1, C), while controls remained asymptomatic (Figure S1, D). Cultures were re-isolated from the infected leaves and were identified as C. siamense by both morphological characteristics and DNA sequence analysis. Previously, C. nymphaeae, C. siamense, C. fructicola and C. viniferum have been reported to cause anthracnose of Pecan worldwide (Zhang et al. 2019; Oh et al. 2021; Poletto et al. 2019; Zhao et al. 2022 ). To our knowledge, this is the first report of C. siamense causing anthracnose on pecan in China. The identification of this pathogen will facilitate the development of strategies for managing the disease in China. References: Oh, J. Y., et al. 2021. Plant disease. 105(10):3296. Poletto, T., et al. 2019. Plant disease. 103(12):3277. Prihastuti, H., et al. 2009. Fungal Divers. 39:89. Fu, M., et al. 2019. Persoonia-Molecular Phylogeny and Evolution of Fungi. 42(1):1-35. Weir, B. S., et al. 2012. Studies in Mycology. 73:115. Zhao, et al. 2022, Acta Phytopathologica Sinica, doi:10.13926/j.cnki.apps.000648 Zhang, Y. B., et al. 2019. Plant disease. 103(6):1432. The author(s) declare no conflict of interest. Keywords: Colletotrichum siamense, Anthracnose, Carya illinoinensis, Pathogenicity †Indicates the corresponding author. Y. Q. Zhao; zhaoyuqiang123@126.com.

Transcriptomic Analysis to Unravel Potential Pathways and Genes Involved in Pecan (Carya illinoinensis) Resistance to Pestalotiopsis microspora.

Fruit black spot (FBS), a fungal disease of pecan (Carya illinoinensis (Wangenh) K. Koch) caused by the pathogen Pestalotiopsis microspora, is a serious disease and poses a critical threat to pecan yield and quality. However, the details of pecan responses to FBS infection at the transcriptional level remain to be elucidated. In present study, we used RNA-Seq to analyze differential gene expression in three pecan cultivars with varied resistance to FBS infection: Xinxuan-4 (X4), Mahan (M), and Wichita (W), which were categorized as having low, mild, and high susceptibility to FBS, respectively. Nine RNA-Seq libraries were constructed, comprising a total of 58.56 Gb of high-quality bases, and 2420, 4380, and 8754 differentially expressed genes (DEGs) with |log2Fold change| ≥ 1 and p-value < 0.05 were identified between M vs. X4, W vs. M, and W vs. X4, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses were performed to further annotate DEGs that were part of specific pathways, which revealed that out of 134 total pathways, MAPK signaling pathway, plant−pathogen interaction, and plant hormone signal transduction were highly enriched. Transcriptomic profiling analysis revealed that 1681 pathogen-related genes (PRGs), including 24 genes encoding WRKY transcription factors, potentially participate in the process of defense against Pestalotiopsis microspora infection in pecan. The correlation of WRKY TFs and PRGs was also performed to reveal the potential interaction networks among disease-resistance/pathogenesis-related genes and WRKY TFs. Expression profiling of nine genes annotated as TIFY, WRKY TF, and disease-resistance protein-related genes was performed using qRT-PCR, and the results were correlated with RNA-Seq data. This study provides valuable information on the molecular basis of pecan−Pestalotiopsis microspora interaction mechanisms and offers a repertoire of candidate genes related to pecan fruit response to FBS infection.

Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus.

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus.

First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease. References: Hwang, M. S., et al. 2005. Applied and Environmental Microbiology, 71:5182-5191. Lelliott, R. A., and Stead, D. E. 1988. Blackwell Scientific, Sussex, UK. Sarkar, S. F., and Guttman, D. S. 2004. Applied and Environmental Microbiology, 70:1999. Weisburg, W. G., et al. 1991. Journal of Bacteriology, 173: 697. Zhang, R., et al. 2015. Scientia Horticulturae, 197: 719-727. The author(s) declare no conflict of interest. Keywords: Carya illinoinensis, Pseudomonas syringae, Canker, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

First Report of Scab Disease Caused by Venturia effusa on Pecan in Anhui Province of China.

Pecan (Carya illinoinensis) is a world-famous nut tree which widely cultivated in China. Quanjiao County, located in Anhui province, is reputed to be the capital of pecan production in China. Since 2019, typical scab symptoms were observed on most pecan cultivars in orchards located in the regions of Quanjiao (32°5'7.08″ N, 118°16'2.91″ E). In April, dark brown to black lesions of scab could be observed on both the abaxial and adaxial surface of the lamina, and were often associated with the veins or midrib. In July, small, brownish, and circular lesions ranging from 1 to 2 mm in diameter were observed at the end of stems and shoulder of the fruit. In the surveyed orchards, disease incidence on the leaves reached more than 35%. While, according to the number of infected nut clusters, disease incidence ranged from 40 to 60% on the infected fruits. Using a sterilized scalpel, conidia were scraped from the surface of a single lesion from the infected leaves or fruits, and a dilute spore suspension was prepared in sterile distilled water, of which 100 microliters was spread on 1% water-agar plate (Bock et al. 2014). The conidia were incubated at 25°C for 48 h under fluorescent lights with a 12-hphotoperiod. Single germinated conidia were selected and transferred into potato dextrose agar (PDA) plate to obtain monospore isolates. From 2019 to 2020, more than 20 isolates were obtained from the infected leaves and fruits. Incubated at 24°C for 6 weeks in darkness on PDA, the colonies were gray-black with circular morphology and floccose texture, which were consistent with the characteristics of Venturia effusa described previously (Gottwald 1982). The conidia were pyriform to ellipsoid, zero to one septate, smooth, attenuated towards apex and base, base truncate, pale brown and 10.08 to 18.14 × 4.86 to 9.56 µm (n = 50) in size. To further identify the isolates, the regions of internal transcribed spacer (ITS), beta-tubulin 2 (TUB2) and translation elongation factor 1 alpha (EF1-a) were amplified and sequenced from genomic DNA for the three representative isolates (AH-81 and AH-82 from the infected leaves, and AH-41 from the infected fruits), respectively (White et al. 1990; Young et al. 2018; Bensch et al. 2006). Sequences of them were deposited in GenBank under nos. OP199056 to OP199058 (ITS), OP566581 to OP566583 (TUB2) and OP566578 to OP566580 (EF1-a). Multilocus phylogenetic analysis revealed that three isolates and V. effusa were clustered in the same clade, indicating high genetic similarity between these organisms. Their morphological and molecular characteristics were consistent with those for V. effusa. The pathogenicity of three isolates were tested on two-year-old container-grown pecan seedlings, which were grown in the nursery. The conidial suspension with a concentration of 5 × 105 conidia/ml was sprayed evenly on the surface of leaves of a healthy pecan seedling, and each isolate inoculated four pecan seedlings. The pathogenicity experiment was repeated three times. The plants inoculated with sterile water were used a negative control. The inoculated plants were enclosed in plastic bags for 2 days, and kept in the nursery greenhouse. Four weeks after inoculation, a similar symptom of scab was observed on leaves of cultivar Mahan, and V. effusa was isolated again from inoculated leaves with the frequency of 100% by the single-spore isolation, whereas no symptoms were observed on the control plants. To our knowledge, this is the first report of V. effusa as a scab pathogen on pecan in Anhui Province of China and underscores the need for monitoring this disease and developing disease control strategies to prevent severe reduction in the value of fruit. References: Bensch, K., et al. 2006. Studies in Mycology, 55(1): 299-305. Bock, C. H., et al. 2014. Forest Pathology, 44(4): 266-275. Gottwald, T. R. 1982. Taxonomy of the pecan scab fungus Cladosporium caryigenum. Mycologia. 74 (3), 382-390. White, T. T., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Application. Academic Press, San Diego, CA. Young, C. A., et al. 2018. Phytopathology, 108(7): 837-846. The author(s) declare no conflict of interest. Keywords: Venturia effusa, Scab, Pecan, Identification †Indicates the corresponding author.Y. Q. Zhao; zhaoyuqiang123@126.com.

Construction of a ceRNA Network and Comprehensive Analysis of lncRNA in Hepatocellular Carcinoma.

To explore the RNA biomolecular marker associated with hepatocellular carcinoma (HCC) prognosis, we constructed a regulatory network of competitive endogenous RNAs (ceRNAs), which provides favorable conditions for the early diagnosis, prognostic monitoring, and personalized treatment of HCC. In this study, the differentially expressed genes (DEGs) of patients with HCC were obtained from the Gene Expression Omnibus. We identified 574 upregulated genes and 274 downregulated genes relevant to HCC occurrence (p < 0.05). Subsequently, we constructed the protein−protein interaction (PPI) network using these DEGs and identified the hub genes from the PPI. We then determined the expression and prognostic values of the hub genes from the GEPIA and Kaplan−Meier plotter databases. After the upstream microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) were respectively identified by miRTarBase and miRNet, we validated the expression of the key miRNAs in the serum using qPCR experiments. Moreover, we identified a two-lncRNA (LINC01184 and ADORA2A-AS1) signature from the upstream lncRNA that effectively predicted overall survival and had promotive effects for HCC. To verify the clinical significance of the signature, we validated the expression of the lncRNA in HCC tissues. Finally, we discovered and identified four mRNAs, four miRNAs, and five lncRNAs associated with the prognosis of HCC and constructed a new ceRNA regulatory network, which will be beneficial for the accurate diagnosis and treatment of HCC.

Transcriptomics and Antioxidant Analysis of Two Chinese Chestnut (Castanea mollissima BL.) Varieties Provides New Insights Into the Mechanisms of Resistance to Gall Wasp Dryocosmus kuriphilus Infestation.

Chinese chestnut is a popular fruit tree with a high nutritional value of its nuts, which can suffer from infestation by the chestnut gall wasp Dryocosmus kuriphilus (GWDK) that results in gall formation and resultant loss of production and profitability. The physiological and molecular mechanisms of GWDK resistance found in certain genotypes currently remains elusive. To gain new insights into this phenomenon, a series of RNA-Seq integrated with metabolomic profiling experiments were executed to investigate the chemical and transcriptional differences in response to GWDK infestation in two contrasting chestnut varieties grown in China (the susceptible "HongLi," HL and the partially resistant "Shuhe_Wuyingli," SW). Three time points were selected for comparison: The initiation stage (A), growth stage (B), and maturation stage (C). Results showed that concentrations of hydrogen peroxide (H2O2) and the activities of peroxidase (POD) and superoxide dismutase (SOD) enzyme were elevated in the resistant SW leaves compared with those in HL leaves at all three developmental stages, while catalase (CAT) and polyphenol oxidase (PPO) activities were mostly higher in HL leaves. RNA-Seq transcriptomic analyses of HL and SW leaves revealed that various metabolic pathways involved in GWDK stress responses, such as plant hormone signal transduction, MAPK signaling, and the peroxisome pathway, were enriched in the contrasting samples. Moreover, the weighted gene co-expression network analysis (WGCNA) of differentially expressed genes in the POD pathway combined with transcription factors (TFs) indicated that the expression of TF members of bHLH, WRKY, NAC, and MYB family positively correlated with POD pathway gene expression. The TFs CmbHLH130 (EVM0032437), CmWRKY31 (EVM0017000), CmNAC50 (EVM0000033), and CmPHL12 (EVM0007330) were identified as putative TFs that participate in the regulation of insect-induced plant enzyme activities in chestnut, which may contribute to GWDK resistance in SW. Expression levels of 8 random differentially expressed genes (DEGs) were furthermore selected to perform quantitative reverse transcription PCR (qRT-PCR) to validate the accuracy of the RNA-Seq-derived expression patterns. This study guides the functional analyses of further candidate genes and mechanisms important for GWDK resistance in chestnuts in the future as well as can help in identifying the master transcriptional regulators and important enzyme steps that support major insect defense pathways in chestnut.

Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.