Bacillus licheniformis Jrh14-10 enhances alkaline tolerance in Arabidopsis thaliana by regulating crosstalk between ethylene and polyamine pathways.

Plant growth-promoting rhizobacteria (PGPR) are known for their role in ameliorating plant stress, including alkaline stress, yet the mechanisms involved are not fully understood. This study investigates the impact of various inoculum doses of Bacillus licheniformis Jrh14-10 on Arabidopsis growth under alkaline stress and explores the underlying mechanisms of tolerance enhancement. We found that all tested doses improved the growth of NaHCO3-treated seedlings, with 109 cfu/mL being the most effective. Transcriptome analysis indicated downregulation of ethylene-related genes and an upregulation of polyamine biosynthesis genes following Jrh14-10 treatment under alkaline conditions. Further qRT-PCR analysis confirmed the suppression of ethylene biosynthesis and signaling genes, alongside the activation of polyamine biosynthesis genes in NaHCO3-stressed seedlings treated with Jrh14-10. Genetic analysis showed that ethylene signaling-deficient mutants (etr1-3 and ein3-1) exhibited greater tolerance to NaHCO3 than the wild type, and the growth-promoting effect of Jrh14-10 was significantly diminished in these mutants. Additionally, Jrh14-10 was found unable to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indicating it does not reduce the ethylene precursor ACC in Arabidopsis. However, Jrh14-10 treatment increased the levels of polyamines (putrescine, spermidine, and spermine) in stressed seedlings, with spermidine particularly effective in reducing H2O2 levels and enhancing Fv/Fm under NaHCO3 stress. These findings reveal a novel mechanism of PGPR-induced alkaline tolerance, highlighting the crosstalk between ethylene and polyamine pathways, and suggest a strategic redirection of S-adenosylmethionine towards polyamine biosynthesis to combat alkaline stress.

Quorum-quenching enzyme Est816 assisted antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in rats.

Introduction: Quorum-quenching enzyme Est816 hydrolyzes the lactone rings of N-acyl homoserine lactones, effectively blocking the biofilm formation and development of Gram-negative bacteria. However, its applications in the oral field is limited. This study aimed to evaluate the efficacy of enzyme Est816 in combination with antibiotics against periodontitis induced by Aggregatibacter actinomycetemcomitans in vitro and in vivo. Methods: The antimicrobial efficacy of enzyme Est816 in combination with minocycline, metronidazole, and amoxicillin was determined using the minimum inhibitory concentration test. The anti-biofilm effect of enzyme Est816 was assessed using scanning electron microscopy, live/dead bacterial staining, crystal violet staining, and real-time quantitative PCR. Biocompatibility of enzyme Est816 was assessed in human gingival fibroblasts (HGF) by staining. A rat model of periodontitis was established to evaluate the effect of enzyme Est816 combined with minocycline using micro-computed tomography and histological staining. Results: Compared to minocycline, metronidazole, and amoxicillin treatment alone, simultaneous treatment with enzyme Est816 increased the sensitivity of biofilm bacteria to antibiotics. Enzyme Est816 with minocycline exhibited the highest rate of biofilm clearance and high biocompatibility. Moreover, the combination of enzyme Est816 with antibiotics improved the antibiofilm effects of the antibiotics synergistically, reducing the expression of the virulence factor leukotoxin gene (ltxA) and fimbria-associated gene (rcpA). Likewise, the combination of enzyme Est816 with minocycline exhibited a remarkable inhibitory effect on bone resorption and inflammation damage in a rat model of periodontitis. Discussion: The combination of enzyme Est816 with antibiotics represents a prospective anti-biofilm strategy with the potential to treat periodontitis.

Invert global and China's terrestrial carbon fluxes over 2019-2021 based on assimilating richer atmospheric CO2 observations.

With China's commitment to reach carbon peak by 2030 and achieve carbon neutrality by 2060, it is particularly important to obtain terrestrial ecosystem carbon fluxes with low uncertainty both globally and in China. The use of more observation data may help reduce the uncertainty of inverting carbon fluxes. This study uses the observation data from global stations, background stations and provincial stations in China, as well as the OCO-2 satellite, and uses the China Carbon Monitoring, Verification and Supporting System for Global (CCMVS-G) to estimate the carbon fluxes of global and Chinese terrestrial ecosystems from 2019 to 2021. The results revealed that the global terrestrial ecosystem carbon sink was approximately -3.40 Pg C/yr from 2019 to 2021. The carbon sinks in the Northern Hemisphere are large, especially in Asia, North America, and Europe. From 2019 to 2021, the carbon sink of China's terrestrial ecosystem was approximately -0.44 Pg C/yr. Carbon sinks exhibit significant seasonal and interannual variations in China. After assimilating the observation data, the uncertainty of the posterior flux is smaller than that of the prior flux, a more reasonable distribution of carbon sources and sinks can be obtained, and more accurate boundary conditions can be provided for the China Carbon Monitoring, Verification and Supporting System for Regional (CCMVS-R). In the future, it is important to establish a well-designed CO2 ground-based observation network.

Labial inverse dilaceration of bilateral maxillary central incisors: A case report.

BACKGROUND: Dilaceration is a rare dental developmental anomaly characterized by an abrupt deviation along the longitudinal axis of the root in which an angulation forms between the root and the crown. Here, we report on dilacerated bilateral maxillary central incisors in mixed dentition. CASE SUMMARY: A 10-year-old girl presented with a chief complaint of unerupted central incisors. An oral examination and radiography provided the basis for a diagnosis of dilaceration of the maxillary central incisors. After surgical exposure of the impacted teeth, a button with an attached chain was applied to the palatal surface of teeth 11 and 21. After 8 mo, a button was bonded to the labial surface of the crown to fix an elastic chain and move the teeth toward the maxillary arch. Finally, a fixed appliance was applied to tooth alignment to Class 1 malocclusion using a 0.019 × 0.025-inch nickel-titanium wire. After 3 years of follow-up, the clinical findings and radiographic assessment showed that the roots had developed with vital dental pulp and healthy periodontium, were acceptable aesthetically, and showed no resorption. CONCLUSION: The rare occurrences of dilacerated bilateral maxillary central incisors can be successfully treated through surgical exposure and orthodontics.

Unravelling Species Diversity and Pathogenicity of Fusarium spp. Associated with Soybean Leaf and Root in Heilongjiang Province, China.

Soybean (Glycine max L.) holds significant global importance and is extensively cultivated in Heilongjiang Province, China. Soybean can be infected by Fusarium species, causing root rot, seed decay, stem rot, and leaf blight. In 2021 to 2022, a field survey of soybean diseases was carried out in 11 regions of Heilongjiang Province, and 186 soybean leaves with leaf blight symptoms and 123 soybean roots with root rot symptoms were collected. Unexpectedly, a considerable number of Fusarium isolates were obtained not only from root samples but also from leaf samples. A total of 584 Fusarium isolates (416 from leaves and 168 from roots) were obtained and identified as 18 Fusarium species based on morphological features and multilocus phylogenetic analyses with tef1 and rpb2 sequences. Fusarium graminearum and Fusarium sp. 1 in FOSC were the dominant species within soybean leaf and root samples, respectively. Pathogenicity tests were conducted for all Fusarium isolates on both soybean leaves and roots. Results showed that F. graminearum, F. ipomoeae, F. citri, F. compactum, F. flagelliforme, F. acuminatum, and F. sporotrichioides were pathogenic to both soybean leaves and roots. F. solani, F. avenaceum, F. pentaseptatum, F. serpentinum, F. annulatum, and Fusarium sp. 1 in FOSC were pathogenic to soybean roots, not to leaves. To our knowledge, this is the first study to thoroughly investigate soybean-associated Fusarium populations in leaves and roots in Heilongjiang Province.

Herbicidal Activity of Secondary Metabolites Isolated from Streptomyces sp. NEAU-HV44 on Selected Weeds and Crops.

Streptomyces is an effective source of new natural bioherbicides. In this study, a novel isolated strain NEAU-HV44 showed strong inhibitory activity against Amaranthus retroflexus L. and was concluded to the genus Streptomyces. Strain NEAU-HV44 fermentation conditions were optimized to maximize the herbicidal activity. The supernatant of strain NEAU-HV44 could significantly control the growth of weeds (A. retroflexus L., Setaria viridis, Portulaca oleracea L., and Chenopodium album) and crops (maize, soybean, wheat, Chinese cabbage, cucumber, tomato, and romaine lettuce) with dose-dependent in preemergence. Notably, weeds were more sensitive to a low-concentration supernatant extract than crops in preemergence. In postemergence, the 2 mg mL-1 supernatant extract could significantly reduce the height and >50% biomass (fresh weight) of tested weeds. The supernatant extract could cause cell membrane destabilization and the cell death of weeds. In addition, the growth of tomato was also inhibited at a high concentration, but no obvious symptoms were observed on soybean and romaine lettuce after spraying the supernatant extract. Then two novel julichrome monomers, julichromes Q12 (1) and Q13 (2), and two known julichromes, julichrome Q3.3 (3) and julichrome Q3.5 (4), were isolated from the supernatant extract of strain NEAU-HV44 by bioactivity-guided approach. This is the first report of the herbicidal activity of julichromes. These four herbicidal compounds could inhibit the shoot and root growth of weeds at 0.2 mg mL-1, and compound 4 could completely inhibit the growth of P. oleracea L. Thus, julichromes (Q12, Q13, Q3.3, Q3.5 1-4) may be new bioherbicidal candidates.

Enhancement of sulfur metabolism and antioxidant machinery confers Bacillus sp. Jrh14-10-induced alkaline stress tolerance in plant.

Alkaline stress is a major environmental challenge that restricts plant growth and agricultural productivity worldwide. Plant growth-promoting rhizobacteria (PGPR) can be used to effectively enhance plant abiotic stress in an environment-friendly manner. However, PGPR that can enhance alkalinity tolerance are not well-studied and the mechanisms by which they exert beneficial effects remain elusive. In this study, we isolated Jrh14-10 from the rhizosphere soil of halophyte Halerpestes cymbalaria (Pursh) Green and found that it can produce indole-3-acetic acid (IAA) and siderophore. By 16S rRNA gene sequencing, it was classified as Bacillus licheniformis. Inoculation Arabidopsis seedlings with Jrh14-10 significantly increased the total fresh weight (by 148.1%), primary root elongation (by 1121.7%), and lateral root number (by 108.8%) under alkaline stress. RNA-Seq analysis showed that 3389 genes were up-regulated by inoculation under alkaline stress and they were associated with sulfur metabolism, photosynthetic system, and oxidative stress response. Significantly, the levels of Cys and GSH were increased by 144.3% and 48.7%, respectively, in the inoculation group compared to the control under alkaline stress. Furthermore, Jrh14-10 markedly enhanced the activities of antioxidant enzymes, resulting in lower levels of O2•-, H2O2, and MDA as well as higher levels of Fv/Fm in alkaline-treated seedlings. In summary, Jrh14-10 can improve alkaline stress resistance in seedlings which was accompanied by an increase in sulfur metabolism-mediated GSH synthesis and antioxidant enzyme activities. These results provide a mechanistic understanding of the interactions between a beneficial bacterial strain and plants under alkaline stress.

Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus.

MYB transcription factors (TFs) have been extensively studied in plant abiotic stress responses and growth and development. However, the role of MYB TFs in the heat stress response and growth and development of Pleurotus ostreatus remains unclear. To investigate the function of PoMYB12, PoMYB15, and PoMYB20 TFs in P. ostreatus, mutant strains of PoMYB12, PoMYB15, and PoMYB20 were generated using RNA interference (RNAi) and overexpression (OE) techniques. The results indicated that the mycelia of OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains exhibited positive effects under heat stress at 32 °C, 36 °C, and 40 °C. Compared to wild-type strains, the OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains promoted the growth and development of P. ostreatus. These mutant strains also facilitated the recovery of growth and development of P. ostreatus after 24 h of 36 °C heat stress. In conclusion, the expression of PoMYB12 and PoMYB20 supports the mycelium's response to heat stress and enhances the growth and development of P. ostreatus, whereas PoMYB15 produces the opposite effect.

Investigation on Cr(VI)-bioreduction mechanism and reduction products by a novel Microbacterium sp. strain NEAU-W11.

Cr(VI) widely exists in the environment and has highly toxic, carcinogenic and mutagenic effects on all organisms. Physical/chemical methods to remove chromium pollution are economically expensive and have disadvantages like high reagent consumption, energy requirements and so on, while bioremediation is an eco-friendly, simple and cost-effective way. In this study, a novel Cr(VI)-reducing strain, Microbacterium sp. NEAU-W11, was reported, and its reduction mechanism was investigated. Microbacterium sp. NEAU-W11 could effectively degrade Cr(VI) under the conditions of pH 7-10, 15-35 °C, and the coexistence of metal pollutants such as Pb and Ni, etc. In addition, both Fe3+ and Cu2+ could improve the reducing ability of strain NEAU-W11, and glucose and lactose as electron donors also had promoting effect. Heat treatment of resting cells confirmed that chromium removal was not biological sorption but biological reduction. The active reductase of strain NEAU-W11 to chromium(VI) mainly existed in the cell cytoplasm, which is the first report in the genus Microbacterium. Micro-characterization of strain NEAU-W11 and the reduction products identified the reduction products as Cr(III)-ligand complexes bound to extracellular polymeric substances (EPS). Collectively, this study systematically investigated the degradation mechanism of Microbacterium sp. NEAU-W11 and the distribution of degradation product Cr(III), providing a new reduction mechanism for the genus Microbacterium, providing a new perspective for a comprehensive understanding of the degradation and transport of chromium by bacteria, and providing theoretical reference for the migration of metal ions in environmental governance.

Transcriptome Analysis Revealed That Hydrogen Peroxide-Regulated Oxidative Phosphorylation Plays an Important Role in the Formation of Pleurotus ostreatus Cap Color.

Pleurotus ostreatus is widely cultivated in China. H2O2, as a signaling molecule, can regulate the formation of cap color, but its regulatory pathway is still unclear, severely inhibiting the breeding of dark-colored strains. In this study, 614 DEGs specifically regulated by H2O2 were identified by RNA-seq analysis. GO-enrichment analysis shows that DEGs can be significantly enriched in multiple pathways related to ATP synthesis, mainly including proton-transporting ATP synthesis complex, coupling factor F(o), ATP biosynthetic process, nucleoside triphosphate metabolic processes, ATP metabolic process, purine nucleoside triphosphate biosynthetic and metabolic processes, and purine ribonuclease triphosphate biosynthetic metabolic processes. Further KEGG analysis revealed that 23 DEGs were involved in cap color formation through the oxidative phosphorylation pathway. They were enriched in Complexes I, III, IV, and V in the respiratory chain. Further addition of exogenous uncoupling agents and ATP synthase inhibitors clarifies the important role of ATP synthesis in color formation. In summary, H2O2 may upregulate the expression of complex-encoding genes in the respiratory chain and promote ATP synthesis, thereby affecting the formation of cap color. The results of this study lay the foundation for the breeding of dark-colored strains of P. ostreatus and provide a basis for the color-formation mechanism of edible fungi.

Ongoing CO2 monitoring verify CO2 emissions and sinks in China during 2018-2021.

Accurate estimating CO2 emissions and sinks is crucial in achieving carbon neutrality in China. However, CO2 emissions from bottom-up inventories are uncertain at regional scales and lack independent verification from atmospheric perspectives. Here we integrated 39 high-precision CO2 stations in China to top-down invert emission-sink variations at 45 km × 45 km and achieved a full range of inventories verification. The results show that China's CO2 emissions are 15% higher than those of five bottom-up inventories, to an annual total of 3.40 Pg C a-1 for 2018-2021. After deducting human and livestock respiration, the annual CO2 emissions were 3.13 Pg C a-1 (11.48 Pg CO2 a-1). The annual increase in emissions slowed from 3.7% in 2019 to 1.1% in 2020 and resumed growth to 4.0% in 2021, consistent with observed CO2 growth rates in China. China's land CO2 sink, deducting farmland sinks and lateral fluxes, was 0.57 Pg C a-1 (2.09 Pg CO2 a-1) for 2018-2021 (higher than most global inverse models), accounting for ∼16.9% of anthropogenic CO2 emissions. The land sink in China decreased by -19.3% in 2019 due to a weak El Niño event and increased by 3.2% in 2020 and 13.7% in 2021. It is worth noting that inverse CO2 emissions and sinks in western China still face large uncertainty due to limited CO2 monitoring. Overall, our top-down estimates demonstrate spatiotemporal variations in CO2 emissions and sinks from atmospheric perspectives and highlight challenges for different provinces in achieving 2060 carbon neutrality with verified estimates.

Identification, Characterization, and Pathogenicity of Fungi Associated with Strawberry Fruit Rot in Shandong Province, China.

China is the largest strawberry producer and exporter worldwide and has been constantly challenged by fruit rot diseases in recent years. Symptoms of various diseases on strawberry fruits were observed in Huangqiyuan Base, an important strawberry-producing region in Shandong Province, and symptomatic samples were collected from January to April 2021 for follow-up studies. In the present study, 137 isolates were obtained and classified into nine species based on morphological characteristics and multilocus phylogenetic analysis (ITS, GAPDH, HIS3, RPB2, EF-1α, HSP60, G3PDH, and/or TUB2), namely, Botrytis cinerea, B. fabiopsis, Alternaria alternata, A. tenuissima, Fusarium proliferatum, F. graminearum, F. ipomoeae, F. incarnatum, and Colletotrichum siamense. Pathogenicity results suggested that all nine pathogenic species could induce fruits to exhibit symptoms similar to those naturally infected in fields. The symptoms around the inoculation points varied, including dense white mycelia caused by Botrytis spp., fading and depression caused by Fusarium spp., black-brown rot caused by Alternaria spp., and shrinkage and dehydration caused by Colletotrichum spp. Overall, B. cinerea was the dominant pathogen, accounting for 61.3% of the total isolates, and showed significantly higher virulence against strawberry fruits than other species. In addition, this is the first report to identify B. fabiopsis, A. alternata, A. tenuissima, F. proliferatum, F. graminearum, F. ipomoeae, and F. incarnatum as causal agents of strawberry fruit rot in Shandong Province, China.

Description of Jidongwangia harbinensis gen. nov. sp. nov.

During our previous study, strain NEAU-J3T was classified as representing a novel genus 'Wangella' within the family Micromonosporaceae. Nevertheless, it is a great pity the name cannot be validated as the proposed genus name is illegitimate (Principle 2 of the ICNP). In this study, we describe Jidongwangia as a novel genus within the family Micromonosporaceae and a polyphasic approach was used to provide evidence to support the classification. The G+C content of the genomic DNA of the type strain is 71.6 %. Digital DNA-DNA hybridization and average nucleotide identity (ANI) values could be used to differentiate NEAU-J3T from its related type strains. The phenotypic, genetic and chemotaxonomic data also indicated that NEAU-J3T occupies a branch separated from those of known genera in the family Micromonosporaceae. Therefore, NEAU-J3T represents a novel species of a novel genus in the family Micromonosporaceae, for which the name Jidongwangia harbinensis gen. nov., sp. nov. is proposed. The type strain of Jidongwangia harbinensis is NEAU-J3T (= CGMCC 4.7039T = DSM 45747T).

Diversity and Pathogenicity of Fungi Associated with Fruit Rot of Winter Jujube in Shandong Province, China.

Winter jujube originated from China and had an extremely high nutritional value. In 2021, symptomatic winter jujube fruits were collected from eight locations in Zhanhua District of Binzhou City, Shandong Province. In total, 108 fungal isolates were obtained and grouped into 11 species based on morphological characteristics and multilocus phylogenetic analysis, including Nothophoma quercina (43.52%), Fusarium lateritium (20.37%), Alternaria alternata (12.03%), F. proliferatum (7.41%), F. graminearum (4.63%), Botryosphaeria dothidea (3.70%), Fusarium sp. (2.78%), A. tenuissima (2.78%), Diaporthe eres (1.85%), Nigrospora oryzae (0.93%), and Cercospora nicotianae (0.93%). All fungal isolates obtained in this study showed aggressiveness on detached winter jujube fruits except N. oryzae and C. nicotianae isolates, of which F. proliferatum was the most virulent, while A. alternata isolates, which have been considered the major pathogen of winter jujube fruit rot, showed a relatively low-level virulence in this study. Furthermore, D. eres, F. graminearum, F. lateritium, and an unclassified Fusarium species were first reported as causal agents of winter jujube fruit rot. The typical symptoms of winter jujube fruit rot observed in this study could be distinguished into two types. N. quercina, A. alternata, A. tenuissima, Fusarium sp., D. nobilis, and F. lateritium isolates caused reddish brown to dark gray lesions on the peel, while B. dothidea, F. graminearum, and F. proliferatum isolates caused peel and pulp decay, resulting in red to reddish brown and water-soaked lesions. In addition, haplotype analysis of N. quercina isolates obtained in this study and validly published articles showed that there were 11 haplotypes worldwide; the isolates obtained in the current study were grouped into three haplotypes (Hap 1, Hap 2, and Hap 11), and two of them (Hap 2 and Hap 11) were confirmed as new haplotypes.

MAC Family Transcription Factors Enhance the Tolerance of Mycelia to Heat Stress and Promote the Primordial Formation Rate of Pleurotus ostreatus.

Pleurotus ostreatus is a typical tetrapolar heterologous edible mushroom, and its growth and development regulatory mechanism has become a research hotspot in recent years. The MAC1 protein is a transcription factor that perceives copper and can regulate the expression of multiple genes, thereby affecting the growth and development of organisms. However, its function in edible mushrooms is still unknown. In this study, two transcription factor genes, PoMCA1a and PoMAC1b, were identified. Afterwards, PoMAC1 overexpression (OE) and RNA interference (RNAi) strains were constructed to further explore gene function. The results showed that the PoMAC1 mutation had no significant effect on the growth rate of mycelia. Further research has shown that OE-PoMAC1a strains and RNAi-PoMAC1b strains exhibit strong tolerance under 32 °C heat stress. However, under 40 °C heat stress, the OE of PoMAC1a and PoMAC1b promoted the recovery of mycelial growth after heat stress. Second, the OE of PoMAC1a can promote the rapid formation of primordia and shorten the cultivation cycle. In summary, this study indicated that there are functional differences between PoMAC1a and PoMAC1b under different heat stresses during the vegetative growth stage, and PoMAC1a has a positive regulatory effect on the formation of primordia during the reproductive growth stage.

Nucisporomicrobium flavum gen. nov., sp. nov., a new member of the family Micromonosporaceae isolated from saline-alkali soil.

A Gram-stain-positive, aerobic actinobacterium, designated strain NEAU-24T, was isolated from saline-alkali soil collected from Daqing City, Heilongjiang Province, PR China. Strain NEAU-24T was found to produce abundant substrate mycelia but no aerial hyphae. The substrate mycelia formed irregular pseudosporangia consisting of nuciform spores, and the surface of the spores was smooth. 16S rRNA gene sequence analysis showed that strain NEAU-24T clustered with Pseudosporangium ferrugineum 3-44-a(19)T, Couchioplanes caeruleus subsp. azureus DSM 44103T and C. caeruleus subsp. caeruleus DSM 43634T within the family Micromonosporaceae and was most closely related to P. ferrugineum 3-44-a(19)T (99.17 %). The strain contained meso-diaminopimelic acid as the cell-wall diamino acid and MK-9(H6) as the menaquinone. The whole cell sugar profile consisted of glucose, galactose, xylose and arabinose. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, phosphatidylinositol and an unidentified lipid. The major fatty acids were summarized as C16 : 0, C15 : 0, C17 : 0, iso-C16 : 0 and iso-C17 : 0. The low digital DNA-DNA hybridization and average nucleotide identity values could differentiate strain NEAU-24T from its related type strains. The phenotypic, genetic and chemotaxonomic data also indicated that strain NEAU-24T occupied a branch separated from those of known genera in the family Micromonosporaceae. In addition, genomic analysis confirmed that strain NEAU-24T had the potential to produce chitinase. Therefore, strain NEAU-24T represents a novel species of a new genus and species in the family Micromonosporaceae, for which the name Nucisporomicrobium flavum gen. nov., sp. nov. is proposed. The type strain of Nucisporomicrobium flavum is NEAU-24T (=CCTCC AA 2020016T=JCM 33973T).

First Report of Alternaria tenuissima Causing Leaf spot on Luffa cylindrica in China.

In August 2020-2021, symptoms of leaf spot were observed in luffa (Luffa cylindrical) fields in Qingdao city, Shandong Province. In all the 10 fields investigated, leaf spot occurred. The incidence (% luffa plants with symptoms from a defined number of plants assessed) was 35 to 60%. Early symptoms of infected leaves were small and irregular chlorotic lesions which later became irregular brown spots. As the disease progressed, the lesions gradually spread from the edge to the center of leaves to the middle, and became dark brown. The enlarged spots coalesced and eventually led to the withering and death of the leaves. In order to isolate the pathogen, 30 symptomatic leaves were collected from different planting fields. Small pieces of leaf tissues (5×5 mm) were cut from the junction of healthy and diseased tissues, sanitized with 2% NaClO for 1 min, rinsed three times with sterile distilled water. The tissue samples were then placed on potato dextrose agar (PDA) amended with 50 mg/L streptomycin sulfate, and incubated at 28℃ for 5 days in the dark. Ten purified fungal isolates were obtained by single spore isolation method. Colonies of these fungal isolates on the PDA medium were initially grayish-white, and then turned olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these fungi produced light brown and solitary conidiophore with septum. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 13.2 to 49.5 × 9.5 to 21.6 µm (n=50). The morphological characteristics of these isolates were consistent with that of Alternaria spp. (Simmons 2007). The representative isolate NEAU-SG-1 was selected for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α gene (TEF), histone 3 (HIS3), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), H3-1a/H3-1b (Glass and Donaldson 1995), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007), respectively. Sequences of these genes of isolate NEAU-SG-1 were deposited into GenBank database with the accession numbers of OL307719, OL415166, OL415169, OL415167, and OL415168. BLAST analysis of these sequences showed 99-100% homology with sequence homology with Alternaria tenuissima strains (ITS, MH824269; GAPDH, MK683783; TEF, MN056178; HIS3, MH824371; RPB2, LC621694). To fulfill Koch's postulates, ten surface disinfected 30-day-old luffa seedlings were inoculated by spraying conidia suspension (106 conidia/ml) of isolate NEAU-SG-1. The other ten surface disinfected seedlings inoculated with sterile distilled water served as the control group. After inoculation, each plant was covered with plastic bags for three days and cultured in greenhouse at 25℃. One week later, leaves inoculated with conidia suspension were observed with the same symptoms as described above, while the leaves of the control group were asymptomatic. Pathogenicity test was repeated twice. The Alternaria isolates were successfully re-isolated from those infected leaves and identified using the morphological and molecular methods described above. A. tenuissima has a wide host range in the world, and is the pathogen of leaf spot of many crops (Ma et al. 2021). To our knowledge, this is the first report of A. tenuissima causing leaf spot on luffa in China. This report will provide basic information for the diagnosis and prevention and control strategies of luffa leaf spot.

Alternaria spp. Associated with Leaf Blight of Maize in Heilongjiang Province, China.

Maize (Zea mays L.) is a major economic crop worldwide. Maize can be infected by Alternaria species causing leaf blight that can result in significant economic losses. In this study, 168 Alternaria isolates recovered from symptomatic maize leaves were identified based on morphological characteristics, pathogenicity, and multilocus sequence analyses of the genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the internal transcribed spacer of ribosomal DNA (rDNA ITS), the RNA polymerase II second largest subunit (RPB2), and histone3 (HIS3). Maize isolates grouped to four Alternaria species including Alternaria tenuissima, A. alternata, A. burnsii, and Alternaria sp. Notably, A. tenuissima (71.4%) was the most prevalent of the four isolated species, followed by A. alternata (21.5%), Alternaria sp. (4.1%), and A. burnsii (3.0%). Pathogenicity tests showed that all four Alternaria species could produce elliptic to nearly round, or strip, lesions on leaves of maize, gray-white to dry white in the lesion centers and reddish-brown at the edges. The average disease incidence (58.47%) and average disease index (63.55) of maize leaves inoculated with A. alternata were significantly higher than levels resulting from A. tenuissima (55.28% and 58.49), Alternaria sp. (55.34% and 58.75), and A. burnsii (56% and 55). Haplotype analyses indicated that there were 14 haplotypes of A. tenuissima and five haplotypes of A. alternata in Heilongjiang Province and suggested the occurrence of a population expansion. Results of the study showed that Alternaria species associated with maize leaf blight in Heilongjiang Province are more diverse than those that have been previously reported. This is the first report globally of A. tenuissima, A. burnsii, and an unclassified Alternaria species as causal agents of leaf blight on maize.