Early surveillance of rice bakanae disease using deep learning and hyperspectral imaging.

Bakanae disease, caused by Fusarium fujikuroi, poses a significant threat to rice production and has been observed in most rice-growing regions. The disease symptoms caused by different pathogens may vary, including elongated and weak stems, slender and yellow leaves, and dwarfism, as example. Bakanae disease is likely to cause necrosis of diseased seedlings, and it may cause a large area of infection in the field through the transmission of conidia. Therefore, early disease surveillance plays a crucial role in securing rice production. Traditional monitoring methods are both time-consuming and labor-intensive and cannot be broadly applied. In this study, a combination of hyperspectral imaging technology and deep learning algorithms were used to achieve in situ detection of rice seedlings infected with bakanae disease. Phenotypic data were obtained on the 9th, 15th, and 21st day after rice infection to explore the physiological and biochemical performance, which helps to deepen the research on the disease mechanism. Hyperspectral data were obtained over these same periods of infection, and a deep learning model, named Rice Bakanae Disease-Visual Geometry Group (RBD-VGG), was established by leveraging hyperspectral imaging technology and deep learning algorithms. Based on this model, an average accuracy of 92.2% was achieved on the 21st day of infection. It also achieved an accuracy of 79.4% as early as the 9th day. Universal characteristic wavelengths were extracted to increase the feasibility of using portable spectral equipment for field surveillance. Collectively, the model offers an efficient and non-destructive surveillance methodology for monitoring bakanae disease, thereby providing an efficient avenue for disease prevention and control. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00169-1.

Epiphytic bacterial community composition on four submerged macrophytes in different regions of Taihu Lake.

The epiphytic bacteria in aquatic ecosystems, inhabiting a unique ecological niche with significant ecological function, have long been the subject of attention. Habitat characteristics and plant species are believed to be important in controlling the assembly of epiphytic bacteria. However, the underlying principle governing the assembly of the epiphytic bacterial community on macrophytes is far from clear. In this study, we systematically compared the diversity and community composition of epiphytic bacteria both in different habitats and on different species of macrophytes where they were attached. Results suggested that neither the plant species nor the habitat had a significant effect on the diversity and community of epiphytic bacteria independently, indicating that the epiphytic bacterial community composition was correlated to both geographical distance and individual species of macrophytes. Furthermore, almost all of the abundant taxa were shared between different lake regions or macrophyte species, and the most abundant bacteria belonged to Proteobacteria and Firmicutes. Our results demonstrated that the competitive lottery model may explain the pattern of epiphytic bacterial colonization of submerged macrophyte surfaces. This research could provide a new perspective for exploring plant-microbe interaction in aquatic systems and new evidence for the lottery model as the mechanism best explaining the assembly of epiphytic bacteria.

Aspergillus cvjetkovicii protects against phytopathogens through interspecies chemical signalling in the phyllosphere.

Resident microbiota produces small molecules that influence the chemical microenvironments on leaves, but its signalling roles in pathogen defence are not yet well understood. Here we show that Aspergillus cvjetkovicii, enriched in rice leaf microbiota, subverts Rhizoctonia solani infections via small-molecule-mediated interspecies signalling. 2,4-Di-tert-butylphenol (2,4-DTBP), identified as a key signalling molecule within the Aspergillus-enriched microbiota, effectively neutralizes reactive oxygen species-dependent pathogenicity by switching off bZIP-activated AMT1 transcription in R. solani. Exogenous application of A. cvjetkovicii and 2,4-DTBP demonstrated varying degrees of protective effects against R. solani infection in diverse crops, including cucumber, maize, soybean and tomato. In rice field experiments, they reduced the R. solani-caused disease index to 19.7-32.2%, compared with 67.2-82.6% in the control group. Moreover, 2,4-DTBP showed activity against other rice phytopathogens, such as Fusarium fujikuroi. These findings reveal a defensive strategy against phytopathogens in the phyllosphere, highlighting the potential of symbiotic microbiota-driven neutralization of pathogenicity.

Deciphering the impact of organic loading rate and digestate recirculation on the occurrence patterns of antibiotics and antibiotic resistance genes in dry anaerobic digestion of kitchen waste.

Organic loading rate (OLR) is crucial for determining the stability of dry anaerobic digestion (AD). Digestate recirculation contributes to reactor stability and enhances methane production. Nevertheless, the understanding of how OLR and digestate recirculation affect the abundance and diversity of antibiotics and antibiotic resistance genes (ARGs), as well as the mechanisms involved in the dissemination of ARGs, remains limited. This study thoroughly investigated this critical issue through a long-term pilot-scale experiment. The metabolome analyses revealed the enrichment of various antibiotics, such as aminoglycoside, tetracycline, and macrolide, under low OLR conditions (OLR ≤ 4.0 g·VS/L·d) and the reactor instability. Antibiotics abundance decreased by approximately 19.66-31.69 % during high OLR operation (OLR ≥ 6.0 g·VS/L·d) with digestate recirculation. The metagenome analyses demonstrated that although low OLR promoted reactor stability, it facilitated the proliferation of antibiotic-resistant bacteria, such as Pseudomonas, and triggered functional profiles related to ATP generation, oxidative stress response, EPS secretion, and cell membrane permeability, thereby facilitating horizontal gene transfer (HGT) of ARGs. However, under stable operation at an OLR of 6.0 g·VS/L·d, there was a decrease in ARGs abundance but a notable increase in human pathogenic bacteria (HPB) and mobile genetic elements (MGEs). Subsequently, during reactor instability, the abundance of ARGs and HPB increased. Notably, during digestate recirculation at OLR levels of 6.0 and 7.0 g·VS/L·d, the process attenuated the risk of ARGs spread by reducing the diversity of ARGs hosts, minimizing interactions among ARGs hosts, ARGs, and MGEs, and weakening functional profiles associated with HGT of ARGs. Overall, digestate recirculation aids in reducing the abundance of antibiotics and ARGs under high OLR conditions. These findings provide advanced insights into how OLR and digestate recirculation affect the occurrence patterns of antibiotics and ARGs in dry AD.

Evolution of quorum sensing process and their regulatory role on biochemical metabolism during the organic loading rate increase in dry anaerobic digestion.

The organic loading rate (OLR) is a critical parameter affecting the stability of dry anaerobic digestion (AD) of kitchen waste (KW), and significantly impacting the variations in physicochemical parameters and microbial communities. However, the evolution of quorum sensing (QS) and their role on anaerobic biochemical metabolism during the increase in OLR in dry AD remain unknown. Therefore, this study systematically elucidated the matter through multi-omics analysis based on a pilot-scale dry AD of KW. The results demonstrated that fluctuations in the OLR significantly influenced the microbial QS in dry AD. When the OLR ≤4.0 g·VS/L·d, the system operated stably, and methane production increased. The enrichment of Proteobacteria was crucial for sustaining high levels of functional genes associated with various types of QS, including acyl-homoserine lactones (AI-1), autoinducer-2 (AI-2), autoinducer-3 (AI-3), and gamma-aminobutyric acid (GABA). This enabled cooperative communication among microbes under low OLR. Furthermore, most genes associated with these QS processes positively affected hydrolysis, acidogenesis, and methanogenesis. When the OLR increased to 6.0 g·VS/L·d, the fatty acids and hydrogen partial pressure increased significantly. The autoinducing peptides (AIP)-type became the predominant QS and was positively correlated with fatty acids abundance. Syntrophaceticus and Syntrophomonas may promote syntrophic oxidation of acetate at high OLR through AIP-type QS. These findings provided new insights into the QS processes of microbes during dry AD of KW and a theoretical foundation for optimizing biochemical metabolic processes in dry AD through QS.

Gut pathobiome mediates behavioral and developmental disorders in biotoxin-exposed amphibians.

Emerging evidence suggests a link between alterations in the gut microbiome and adverse health outcomes in the hosts exposed to environmental pollutants. Yet, the causal relationships and underlying mechanisms remain largely undefined. Here we show that exposure to biotoxins can affect gut pathobiome assembly in amphibians, which in turn triggers the toxicity of exogenous pollutants. We used Xenopus laevis as a model in this study. Tadpoles exposed to tropolone demonstrated notable developmental impairments and increased locomotor activity, with a reduction in total length by 4.37%-22.48% and an increase in swimming speed by 49.96%-84.83%. Fusobacterium and Cetobacterium are predominant taxa in the gut pathobiome of tropolone-exposed tadpoles. The tropolone-induced developmental and behavioral disorders in the host were mediated by assembly of the gut pathobiome, leading to transcriptome reprogramming. This study not only advances our understanding of the intricate interactions between environmental pollutants, the gut pathobiome, and host health but also emphasizes the potential of the gut pathobiome in mediating the toxicological effects of environmental contaminants.

Unlocking sustainable solutions: controlled Cu2+ dosing enables efficient recovery and reuse of high-purity copper pyrophosphate from electroplating wastewater.

The electroplating process of copper pyrophosphate (Cu2P2O7) results in the production of a large volume of wastewater that contains a high concentration of copper (Cu). Currently, conventional lime precipitation creates a substantial amount of secondary pollution, which adds extra economic and environmental burdens. In this study, we suggest a straightforward method for on-site recovery of Cu from Cu2P2O7 electroplating wastewater. By optimizing various parameters, characterizing the resulting product, assessing its electroplating capabilities, and analyzing the speciation during the reaction, we comprehensively investigated the feasibility and mechanism of this technique. The results demonstrated that, under the optimal conditions (Cu/P molar ratio of 0.96, pH of 5.0, and a reaction time of 5.0 min), the concentration of residual Cu remained stable between 22.2 and 27.7 mg/L, even when the initial Cu concentrations varied. The addition of Cu triggered a series of hydrolysis and ionization reactions, primarily leading to the formation of Cu2P2O7·3H2O. The harvested Cu2P2O7·3H2O proved to be suitable for practical electroplating applications, exhibiting comparable performance to commercially available Cu2P2O7·3H2O. This demonstrates the feasibility of recovering high-purity Cu2P2O7·3H2O from copper electroplating wastewater, offering a promising approach for on-site copper reuse and concurrently reducing the demand for natural copper resources. Furthermore, this approach significantly reduces the generation of solid waste, aligning with the principles of sustainable development.

Phyllosphere microbiome induces host metabolic defence against rice false-smut disease.

Mutualistic interactions between host plants and their microbiota have the potential to provide disease resistance. Most research has focused on the rhizosphere, but it is unclear how the microbiome associated with the aerial surface of plants protects against infection. Here we identify a metabolic defence underlying the mutualistic interaction between the panicle and the resident microbiota in rice to defend against a globally prevalent phytopathogen, Ustilaginoidea virens, which causes false-smut disease. Analysis of the 16S ribosomal RNA gene and internal transcribed spacer sequencing data identified keystone microbial taxa enriched in the disease-suppressive panicle, in particular Lactobacillus spp. and Aspergillus spp. Integration of these data with primary metabolism profiling, host genome editing and microbial isolate transplantation experiments revealed that plants with these taxa could resist U. virens infection in a host branched-chain amino acid (BCAA)-dependent manner. Leucine, a predominant BCAA, suppressed U. virens pathogenicity by inducing apoptosis-like cell death through H2O2 overproduction. Additionally, preliminary field experiments showed that leucine could be used in combination with chemical fungicides with a 50% reduction in dose but similar efficacy to higher fungicide concentrations. These findings may facilitate protection of crops from panicle diseases prevalent at a global scale.

Plant pathogenesis: Toward multidimensional understanding of the microbiome.

Single pathogen-targeted disease management measure has shown drawbacks in field efficacy under the scenario of global change. An in-depth understanding of plant pathogenesis will provide a promising solution but faces the challenges of the emerging paradigm involving the plant microbiome. While the beneficial impact of the plant microbiome is well characterized, their potential role in facilitating pathological processes has so far remained largely overlooked. To address these unsolved controversies and emerging challenges, we hereby highlight the pathobiome, the disease-assisting portion hidden in the plant microbiome, in the plant pathogenesis paradigm. We review the detrimental actions mediated by the pathobiome at multiple scales and further discuss how natural and human triggers result in the prevalence of the plant pathobiome, which would probably provide a clue to the mitigation of plant disease epidemics. Collectively, the article would advance the current insight into plant pathogenesis and also pave a new way to cope with the upward trends of plant disease by designing the pathobiome-targeted measure.

Keystone taxa-mediated bacteriome response shapes the resilience of the paddy ecosystem to fungicide triadimefon contamination.

The increasing input of fungicides has emerged as a global concern for agroecosystem stability and sustainability. Agroecosystem resilience has been linked to microbiome response, however, is not well understood. Focusing on a widespread triazole-class fungicide triadimefon in the paddy ecosystem, we characterized that the soils and sediments were dominant triadimefon reservoirs with the peak level at 195 µg kg-1 and 31.3 µg kg-1, respectively, but essential for the resilience of paddy ecosystem to triadimefon. In paddy simulation models, the half-life of triadimefon in soil-sediment was 8.4-28.9 days, while it was prolonged to 86.6-115.5 days after elimination of resident microbial community. Phospholipid fatty acid profiling and high-throughput sequencing showed that the distinctive bacterial community responses contributed to variable degradation of triadimefon in paddy soils and sediments. Sphingomonas and Xanthomonas were identified as positive responders of the keystone taxa in the responsive bacteriome, whereas Enterobacter were negative responders that declined over time. Synthetic assemblages combined with quantitative polymerase chain reaction further validated that Sphingomonas and Xanthomonas were involved in sustaining soil-sediment resilience to triadimefon contamination. Collectively, our results revealed that the shaping of soil and sediment bacteriomes was responsible for the resilience of the paddy agroecosystem to fungicide contamination.

Specific biotests to assess eco-toxicity of biodegradable polymer materials in soil.

Eco-toxicity investigation of polymer materials was considered extremely necessary for their potential menace, which was widely use as mulching materials in agricultural. In this study, polyethylene (PE), polystyrene (PS) and synthetic biomaterials-Ecoflex and cellulose were applying into soil cultivated with two potential indicator plants species: oat (Avena sativa) and red radish (Raphanus sativum). Variety of chemical, biochemical parameters and enzyme activity in soil were proved as effective approach to evaluate polymers phytotoxicity in plant-soil mesocosm. The F-value of biomass, pH, heavy metal and electoral conductivity of Raphanus behaved significant different from T0. Significant analysis results indicated biodegradation was fast in PE than PS, besides, heavy metals were dramatically decrease in the end implied the plant absorption may help decrease heavy metal toxicity. The increase value at T2 of Dehydrogenase activity (0.84 higher than average value for Avena & 0.91 higher for Raphanus), Metabolic Index (3.12 higher than average value for Avena & 3.81 higher for Raphanus) means during soil enzyme activity was promoted by biodegradation for its heterotrophic organisms' energy transportation was stimulated. Statistics analysis was carried on Biplot PC1 (24.2% of the total variance), PC2 (23.2% of the total variance), versus PC3 (22.8% of the total variance), which indicated phosphatase activity and metabolic index was significant correlated, and high correlation of ammonium and protease activity. Furthermore, the effects were more evident in Raphanus treatments than in Avena, suggesting the higher sensitivity of Raphanus to polymers treatment, which indicate biodegradation of polymers in Raphanus treatment has produced intermediate phytotoxic compounds.

Insights into facile synthesized pomelo biochar adsorbing thallium: potential remediation in agricultural soils.

Little information is available on thallium (Tl) adsorption onto fruit-derived biochar. In this study, pomelo peel and waste pomelo were thus chosen to prepare two kinds of biochars recorded as PPB and WPB. The two produced biochars subsequently evaluated their potential remediation of thallium (Tl) contamination in agricultural soils by their Tl adsorption capacity. Results showed that the two pomelo-derived biochars presented obvious microporous structure and rich oxygen-containing functional group, supported by the observant data of specific surface area, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). Furthermore, Langmuir isothermal adsorption model can better fit the adsorption behavior of thallium onto PPB and WPB, and the subsequent maximum adsorption capacity was 4283.9 µg g-1 and 5286.0 µg g-1, respectively. In addition, the pseudo-second-order kinetic model could well fit the kinetic behavior of thallium adsorption onto PPB and WPB, indicating that the process is accompanied by chemical adsorption. Meanwhile, in agricultural soils, PPB and WPB can be used as environmentally friendly adsorbents to remediate Tl contamination due to their pH increase of the tested soils and their comparable adsorption ability of Tl. The obtained findings can provide insights into comprehensively developed fruit-derived biochar technology to remediate Tl contamination in agricultural soils.

Enhanced solubilization and biochemical methane potential of waste activated sludge by combined free nitrous acid and potassium ferrate pretreatment.

The increasing production of waste activated sludge (WAS) from wastewater treatment plants presents an inherent environmental burden. In this study, Free nitrous acid combined with potassium ferrate (FNA + PF) pretreatment was used to enhance solubilization and biochemical methane potential of WAS. Results indicated that the maximum removal rates of total suspended solid by PF, FNA, and PF + FNA pretreatment were 21.84%, 38.09%, and 56.17%, respectively. The biochemical methane potential of WAS without pretreatment reached 61.22 L CH4/kg VSS added while this value increased to 147.07 L CH4/kg VSS added after FNA + PF pretreatment (0.06 g/g TSS NaNO2 and 0.25 g/g TSS K2FeO4). Shotgun metagenomic analysis revealed that FNA + PF pretreatment could increase the diversity and stability of microbial communities by shifting methanogenic pathways from strictly acetoclastic to acetoclastic/hydrogenotrophic, thereby enhancing methane production. This study suggested that FNA + PF pretreatment is a promising technology to reduce WAS and enhance methane production by pretreated WAS during anaerobic digestion.

Microenvironmental Interplay Predominated by Beneficial Aspergillus Abates Fungal Pathogen Incidence in Paddy Environment.

Rice fungal pathogens, responsible for severe rice yield loss and biotoxin contamination, cause increasing concerns on environmental safety and public health. In the paddy environment, we observed that the asymptomatic rice phyllosphere microenvironment was dominated by an indigenous fungus, Aspergillus cvjetkovicii, which positively correlated with alleviated incidence of Magnaporthe oryzae, one of the most aggressive plant pathogens. Through the comparative metabolic profiling for the rice phyllosphere microenvironment, two metabolites were assigned as exclusively enriched metabolic markers in the asymptomatic phyllosphere and increased remarkably in a population-dependent manner with A. cvjetkovicii. These two metabolites evidenced to be produced by A. cvjetkovicii in either a phyllosphere microenvironment or artificial media were purified and identified as 2(3H)-benzofuranone and azulene, respectively, by gas chromatography coupled to triple quadrupole mass spectrometry and nuclear magnetic resonance analyses. Combining with bioassay analysis in vivo and in vitro, we found that 2(3H)-benzofuranone and azulene exerted dissimilar actions at the stage of infection-related development of M. oryzae. A. cvjetkovicii produced 2(3H)-benzofuranone at the early stage to suppress MoPer1 gene expression, leading to inhibited mycelial growth, while azulene produced lately was involved in blocking of appressorium formation by downregulation of MgRac1. More profoundly, the microenvironmental interplay dominated by A. cvjetkovicii significantly blocked M. oryzae epidemics in the paddy environment from 54.7 to 68.5% (p < 0.05). Our study first demonstrated implication of the microenvironmental interplay dominated by indigenous and beneficial fungus to ecological balance and safety of the paddy environment.

Synthesis of a novel ternary HA/Fe-Mn oxides-loaded biochar composite and its application in cadmium(II) and arsenic(V) adsorption.

Cadmium (Cd) and arsenic (As) are two of the most toxic elements. However, the chemical behaviors of these two elements are different, making it challenging to utilize a single adsorbent with high adsorption capacity for both Cd(II) and As(V) removal. To solve this problem, we synthesized HA/Fe-Mn oxides-loaded biochar (HFMB), a novel ternary material, to perform this task, wherein scanning electron microscopy (SEM) combined with EDS (SEM-EDS) was used to characterize its morphological and physicochemical properties. The maximum adsorption capacity of HFMB was 67.11 mg/g for Cd(II) and 35.59 mg/g for As(V), which is much higher compared to pristine biochar (11.06 mg/g, 0 mg/g for Cd(II) and As(V), respectively). The adsorption characteristics were investigated by adsorption kinetics and the effects of the ionic strength and pH of solutions. X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FT-IR) revealed that chelation and deposition were the adsorption mechanisms that bound Cd(II) to HFMB, while ligand exchange was the adsorption mechanism that bound As(V).

Establishing baseline trace metals in marine bivalves in China and worldwide: Meta-analysis and modeling approach.

Bivalves are extensively employed as biomonitors of coastal pollution, but the comparability among different species and geographic environments needs to be further scrutinized. The present study conducted a meta-analysis of trace metals (Ag, Cd, Cr, Cu, Ni, Pb, Ti, and Zn) in the soft tissues of three groups of marine bivalves (oysters, mussels, and clams) collected from China and worldwide. By conducting cumulative frequency distribution analysis of metal distribution, we modeled the 5% cumulative values as the bio-baseline metal concentrations in these bivalves. We further modeled their potential baseline concentrations using a well-developed biokinetic model. The baseline concentrations of Cd, Cu, and Zn in the tissue-specific Environmental Quality Standards (EQSs) for China were 0.99, 34.5, and 340 µg/g dw in oysters, 0.38, 4.32, and 49.6 µg/g dw in mussels, and 0.35, 3.02, 51.4 µg/g dw in clams, respectively. Of the 8 metals examined in this study, the calculated baseline concentrations of Ag, Cr, Cd, Cu and Zn in the oysters were 3.7-48, 2.7-3.6, 2.6-2.8, 8.0-11.4, 6.6-6.8 times higher than those in the mussels and clams, and only Ti showed comparable baseline concentrations among the three bivalves (8.43-9.67 µg/g dw). These data strongly suggested the inter-group as well as inter-metal difference in the baseline metal concentrations in marine bivalves. Further, the potential baseline concentrations of Cd and Cu predicted by the biokinetic model were comparable to those modeled by the probability frequency distribution. Combined statistical frequency analysis and biokinetic modeling therefore provided an innovative method to establish the baseline metal concentrations in bivalves and the tissue-specific EQSs, which are now urgently needed for coastal management, biomonitoring, and geochemical records in the world.

Radioactive status of seawater and its assessment in the northeast South China Sea and the Luzon Strait and its adjacent areas from 2011 to 2014.

To understand the impact of the Fukushima nuclear accident (FNA), 137Cs, 134Cs, 90Sr, and gross beta were analyzed in the northeast South China Sea (NSCS), the Luzon Strait (LS) and its adjacent areas. 137Cs, 90Sr, and gross beta values in the NSCS were similar to those prior to the FNA. 90Sr and 137Cs in the LS and its adjacent areas were consistent with those in the NSCS. The high 137Cs-peak values occurred at depth of 150 m whereas the high 90Sr-peak values occurred at depth of 0.5 m. The 137Cs and gross beta mean values in Cruise I were higher than those in Cruise II whereas the 90Sr mean value was just the reverse. 134Cs in all seawater were below the minimum detectable activity. The past and present data since the 1970s suggested 137Cs and 90Sr in the study areas still originated from global fallout and the FNA influence were negligible.

Distribution, fractions, and potential release of thallium in acidic soils nearby a waste copper mining site from southern China.

Although thallium (Tl) is a highly toxic element, little information is available on the environmental risks of Tl in agricultural soils with intensive practices, particularly nearby mining sites. Therefore, we investigated the potential release of Tl in acidic soils with intensive cultivation nearby a waste copper mining site from southern China based on its level and chemical fractions as well as simulated release under artificial acid rain. Results showed that the average Tl content was 1.31 mg/kg in the studied area, which significantly exceeds the permissible thallium value of 1 mg/kg for agricultural soil in China. Some vertical increases of soil Tl from different land uses indicate the potential transport of Tl downward to groundwater. High positive correlations between surficial soil Tl and rubidium (Rb) and copper (Cu) indicated that Tl has the lithophile and chalcophile behavior. Tl in soils is mainly entrapped in residual fraction. The exchangeable fraction of Tl in agricultural soils was less than undisturbed natural soils and copper mined soils. Additionally, the percentage of Tl release from undisturbed natural soils and soils of copper ore area was more than that from agricultural soils in simulated acid rain. Furthermore, the releases of Tl from the soils increased with the acidity of artificial acid rain. Thus, more attention must be paid to land management of this similar area to avoid the risk of Tl impact on human health.

An improved method for the molecular identification of single dinoflagellate cysts.

BACKGROUND: Dinoflagellate cysts (i.e., dinocysts) are biologically and ecologically important as they can help dinoflagellate species survive harsh environments, facilitate their dispersal and serve as seeds for harmful algal blooms. In addition, dinocysts derived from some species can produce more toxins than vegetative forms, largely affecting species through their food webs and even human health. Consequently, accurate identification of dinocysts represents the first crucial step in many ecological studies. As dinocysts have limited or even no available taxonomic keys, molecular methods have become the first priority for dinocyst identification. However, molecular identification of dinocysts, particularly when using single cells, poses technical challenges. The most serious is the low success rate of PCR, especially for heterotrophic species. METHODS: In this study, we aim to improve the success rate of single dinocyst identification for the chosen dinocyst species (Gonyaulax spinifera, Polykrikos kofoidii, Lingulodinium polyedrum, Pyrophacus steinii, Protoperidinium leonis and Protoperidinium oblongum) distributed in the South China Sea. We worked on two major technical issues: cleaning possible PCR inhibitors attached on the cyst surface and designing new dinoflagellate-specific PCR primers to improve the success of PCR amplification. RESULTS: For the cleaning of single dinocysts separated from marine sediments, we used ultrasonic wave-based cleaning and optimized cleaning parameters. Our results showed that the optimized ultrasonic wave-based cleaning method largely improved the identification success rate and accuracy of both molecular and morphological identifications. For the molecular identification with the newly designed dinoflagellate-specific primers (18S634F-18S634R), the success ratio was as high as 86.7% for single dinocysts across multiple taxa when using the optimized ultrasonic wave-based cleaning method, and much higher than that (16.7%) based on traditional micropipette-based cleaning. DISCUSSION: The technically simple but robust method improved on in this study is expected to serve as a powerful tool in deep understanding of population dynamics of dinocysts and the causes and consequences of potential negative effects caused by dinocysts.