Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing.

A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host uncharacterized endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector that is able to transmit RTIV and found that RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Genetic Diversity and Pathogenicity of Fusarium fujikuroi Species Complex (FFSC) Causing Sugarcane Pokkah Boeng Disease (PBD) in China.

Pokkah boeng disease (PBD), a sugarcane foliar disease, is caused by various Fusarium spp. within the Fusarium fujikuroi species complex (FFSC). In the current study, we investigated the diversity of Fusarium spp. associated with PBD in China. In total, 320 leaf samples displaying PBD symptoms were collected over 10 consecutive years (2012 to 2021), during winter and summer, from six various sugarcane-growing regions (Guangxi, Yunnan, Guangdong, Zhejiang, Hainan, and Fujian) in China. Phylogenetic analysis of Fusarium spp. was reconstructed using translation elongation factor 1-α, and DNA-directed RNA polymerase II largest subunit and second-largest subunit multigene sequences. Evolutionary studies of these regions categorized the isolates into four FFSC species (F. sacchari, F. proliferatum, F. verticillioides, and F. andiyazi). The identified isolates, which developed irregular necrotic patches and rotting symptoms on the sugarcane plant after approximately 30 days were tested for their pathogenicity. Symptoms that appeared during pathogenicity testing were consistent with those observed under field conditions. Each strain of the pathogenic Fusarium spp. belonged to different vegetative compatibility groups (VCGs), and there was no affinity between VCGs. Our results contribute to understanding FFSC and accurately identifying Fusarium spp. associated with the sugarcane crop.

Gene-coexpression network analysis identifies specific modules and hub genes related to cold stress in rice.

BACKGROUND: When plants are subjected to cold stress, they undergo a series of molecular and physiological changes to protect themselves from injury. Indica cultivars can usually withstand only mild cold stress in a relatively short period. Hormone-mediated defence response plays an important role in cold stress. Weighted gene co-expression network analysis (WGCNA) is a very useful tool for studying the correlation between genes, identifying modules with high phenotype correlation, and identifying Hub genes in different modules. Many studies have elucidated the molecular mechanisms of cold tolerance in different plants, but little information about the recovery process after cold stress is available. RESULTS: To understand the molecular mechanism of cold tolerance in rice, we performed comprehensive transcriptome analyses during cold treatment and recovery stage in two cultivars of near-isogenic lines (9311 and DC907). Twelve transcriptomes in two rice cultivars were determined. A total of 2509 new genes were predicted by fragment splicing and assembly, and 7506 differentially expressed genes were identified by pairwise comparison. A total of 26 modules were obtained by expression-network analysis, 12 of which were highly correlated with cold stress or recovery treatment. We further identified candidate Hub genes associated with specific modules and analysed their regulatory relationships based on coexpression data. Results showed that various plant-hormone regulatory genes acted together to protect plants from physiological damage under short-term low-temperature stress. We speculated that this may be common in rice. Under long-term cold stress, rice improved the tolerance to low-temperature stress by promoting autophagy, sugar synthesis, and metabolism. CONCLUSION: Through WGCNA analysis at the transcriptome level, we provided a potential regulatory mechanism for the cold stress and recovery of rice cultivars and identified candidate central genes. Our findings provided an important reference for the future cultivation of rice strains with good tolerance.

Comparative genome analysis unravels pathogenicity of Xanthomonas albilineans causing sugarcane leaf scald disease.

BACKGROUND: Xanthomonas is a genus of gram-negative bacterium containing more than 35 species. Among these pathogenic species, Xanthomonas albilineans (Xal) is of global interest, responsible for leaf scald disease in sugarcane. Another notable Xanthomonas species is Xanthomonas sachari (Xsa), a sugarcane-associated agent of chlorotic streak disease. RESULT: The virulence of 24 Xanthomonas strains was evaluated by disease index (DI) and Area Under Disease Progress Curve (AUDPC) in the susceptible inoculated plants (GT 46) and clustered into three groups of five highly potent, seven mild virulent, and twelve weak virulent strains. The highly potent strain (X. albilineans, Xal JG43) and its weak virulent related strain (X. sacchari, Xsa DD13) were sequenced, assembled, and annotated in the circular genomes. The genomic size of JG43 was smaller than that of DD13. Both strains (JG43 and DD13) lacked a Type III secretory system (T3SS) and T6SS. However, JG43 possessed Salmonella pathogenicity island-1 (SPI-1). More pathogen-host interaction (PHI) genes and virulent factors in 17 genomic islands (GIs) were detected in JG43, among which six were related to pathogenicity. Albicidin and a two-component system associated with virulence were also detected in JG43. Furthermore, 23 Xanthomonas strains were sequenced and classified into three categories based on Single Nucleotide Polymorphism (SNP) mutation loci and pathogenicity, using JG43 as a reference genome. Transitions were dominant SNP mutations, while structural variation (SV) is frequent intrachromosomal rearrangement (ITX). Two essential genes (rpfC/rpfG) of the two-component system and another gene related to SNP were mutated to understand their virulence effect. The mutation of rpfG resulted in a decrease in pathogenicity. CONCLUSION: These findings revealed virulence of 24 Xanthomonas strains and variations by 23 Xanthomonas strains. We sequenced, assembled, and annotated the circular genomes of Xal JG43 and Xsa DD13, identifying diversity detected by pathogenic factors and systems. Furthermore, complete genomic sequences and sequenced data will provide a theoretical basis for identifying pathogenic factors responsible for sugarcane leaf scald disease.

A novel transcription factor, ScAIL1, modulates plant defense responses by targeting DELLA and regulating gibberellin and jasmonic acid signaling in sugarcane.

DELLA proteins are important repressors of gibberellin signaling, regulating plant development and defense responses through crosstalk with various phytohormones. Sugarcane ScGAI encodes a DELLA protein that regulates culm development. However, it is unclear which transcription factors mediate the transcription of ScGAI. Here, we identified two different ScGAI promoter sequences that cooperatively regulate ScGAI transcription. We also identified a nuclear-localized AP2 family transcription factor, ScAIL1, which inhibits the transcription of ScGAI by directly binding to two ScGAI promoters. ScAIL1 was expressed in all sugarcane tissues tested and was induced by gibberellin and various stressors, including NaCl, polyethylene glycol, and pathogenic fungi and bacteria. Overexpression of ScAIL1 in rice significantly improved resistance to bacterial blight and rice blast, while reducing growth and development. In addition, several genes associated with stress responses were significantly up-regulated in transgenic rice overexpressing ScAIL1. Endogenous phytohormone content and expression analysis further revealed that ScAIL1-overexpressing lines improved resistance to bacterial blight and rice blast instead of promoting growth, and that this response was associated with increased jasmonic acid synthesis and gibberellin inactivation. These results provide molecular evidence that the role of ScAIL1 in the plant defense response is related to jasmonic acid and gibberellin signaling.

ScGAIL, a sugarcane N-terminal truncated DELLA-like protein, participates in gibberellin signaling in Arabidopsis.

The hormone gibberellin (GA) is crucial for internode elongation in sugarcane. DELLA proteins are critical negative regulators of the GA signaling pathway. ScGAI encodes a DELLA protein that was previously implicated in the regulation of sugarcane culm development. Here, we characterized ScGAI-like (ScGAIL) in sugarcane, which lacked the N-terminal region but was otherwise homologous to ScGAI. ScGAIL differed from ScGAI in its chromosomal location, expression patterns, and cellular localization. Although transgenic Arabidopsis overexpressing ScGAIL were insensitive to GAs, GA synthesis was affected in these plants, suggesting that ScGAIL disrupted the GA signaling pathway. After GA treatment, the expression patterns of GA-associated genes differed between ScGAIL-overexpressing and wild-type Arabidopsis, and the degradation of AtDELLA proteins in transgenic lines was significantly inhibited compared with wild-type lines. A sugarcane GID1 gene (ScGID1) encoding a putative GA receptor was isolated and interacted with ScGAIL in a GA-independent manner. Five ScGAIL-interacting proteins were verified by yeast two-hybrid assays, and only one interacted with ScGAI. Therefore, ScGAIL may inhibit plant growth by modulating the GA signaling pathway.

The Autophagy-Related Gene CpAtg4 Is Required for Fungal Phenotypic Traits, Stress Tolerance, and Virulence in Cryphonectria parasitica.

Autophagy is an evolutionarily ancient process wherein cells are able to break down intracellular contents to support normal physiology and development. Autophagosome formation is regulated by several different proteins, including the key cysteine protease Atg4. The contribution of Atg4 protein in the pathogenic fungus Cryphonectria parasitica, which causes blight in chestnut plants, has not been completely understood. In this context, we aimed to investigate the role of Atg4 during autophagy formation and their contribution to nonautophagic events in C. parasitica. By complementation assay, we determined that the CpAtg4 gene from C. parasitica was able to functionally complement the deletion of yeast Atg4. Using a yeast two-hybrid assay system, we confirmed that CpAtg4 and CpAtg8 directly interact with one another, and amino acids 377 to 409 of CpAtg4 were identified as being responsible for its binding with CpAtg8. The deletion mutant of CpAtg4 did not demonstrate positive monodansylcadaverine staining, which indicated that CpAtg4 is required for autophagy in C. parasitica. Moreover, the ΔCpAtg4 strain exhibited a decrease in aerial hyphae formation and sporulation, and reduction in virulence on apple and chestnut stem. The ΔCpAtg4 strains were also more sensitive to H2O2 and Congo red-induced stress. We further determined that amino acids 377 to 409 of CpAtg4 were essential for the function of CpAtg4 in vivo. Together, our findings indicated that CpAtg4 is required for the autophagy formation, fungal phenotypic traits, stress tolerance, and virulence in C. parasitica.

Evaluation of conventional and point-of-care real-time RT-PCR tests for the detection of SARS-CoV-2 through a pooled testing strategy.

BACKGROUND: The rapid identification and isolation of individuals infected with SARS-CoV-2 are fundamental countermeasures for the efficient control of the COVID-19 pandemic, which has affected millions of people around the world. Real-time RT-PCR is one of the most commonly applied reference methods for virus detection, and the use of pooled testing has been proposed as an effective way to increase the throughput of routine diagnostic tests. However, the clinical applicability of different types of real-time RT-PCR tests in a given group size remains inconclusive due to inconsistent regional disease prevalence and test demands. METHODS: In this study, the performance of one dual-target conventional and two point-of-care real-time RT-PCR tests in a 5-specimen pooled testing strategy for the detection of SARS-COV-2 was evaluated. RESULTS: We demonstrated the proof of concept that all of these real-time RT-PCR tests could feasibly detect SARS-CoV-2 from nasopharyngeal and oropharyngeal specimens that contain viral RNA loads in the range of 3.48 × 105 to 3.42 × 102 copies/ml through pooled testing in a group size of 5 with overall positive percent agreement (pooling vs. individual testing) ranging from 100% to 93.75%. Furthermore, the two POC real-time RT-PCR tests exhibited comparable sensitivity to that of the dual-target conventional one when clinical specimens were tested individually. CONCLUSION: Our findings support the feasibility of using real-time RT-PCR tests developed as a variety of platforms in routine laboratory detection of suspected COVID-19 cases through a pooled testing strategy that is beneficial to increasing the daily diagnostic capacity.

Overexpression of Sugarcane ScDIR Genes Enhances Drought Tolerance in Nicotiana benthamiana.

Dirigent proteins (DIRs) are known to function in lignin biogenesis and to be involved in stress resistance in plants. However, the sugarcane DIRs have not been functionally characterized. In this study, we investigated the DIR-protein-encoding genes in Saccharum spp. (ScDIR) by screening collections of sugarcane databases, monitoring the responses of these genes to drought stress by real-time quantitative PCR, and identifying their heterologous expression in tobacco. Of the 64 ScDIRs identified, four belonging to the DIR-b/d (ScDIR5 and ScDIR11) and DIR-c (ScDIR7 and ScDIR40) subfamilies showed a significant transcriptional response when subjected to drought stress. ScDIR5, ScDIR7, and ScDIR11 are localized in the cell membrane, whereas ScDIR40 is found in the cell wall. The overexpression of these ScDIR genes in tobacco generally increased the drought tolerance of the transgenic lines, with ScDIR7 conferring the highest degree of drought tolerance. The characterization of the physiological and biochemical indicators (superoxide dismutase, catalase, malondialdehyde, and H2O2) confirmed that the ScDIR-overexpressing lines outperformed the wild type. These results demonstrated that specific ScDIRs in sugarcane respond and contribute to tolerance of drought stress, shedding light on potential means of improving drought tolerance in this crop.

Comparative Analysis of Chloroplast Genome in Saccharum spp. and Related Members of 'Saccharum Complex'.

High ploids of the sugarcane nuclear genome limit its genomic studies, whereas its chloroplast genome is small and conserved, which is suitable for phylogenetic studies and molecular marker development. Here, we applied whole genome sequencing technology to sequence and assemble chloroplast genomes of eight species of the 'Saccharum Complex', and elucidated their sequence variations. In total, 19 accessions were sequenced, and 23 chloroplast genomes were assembled, including 6 species of Saccharum (among them, S. robustum, S. sinense, and S. barberi firstly reported in this study) and 2 sugarcane relative species, Tripidium arundinaceum and Narenga porphyrocoma. The plastid phylogenetic signal demonstrated that S. officinarum and S. robustum shared a common ancestor, and that the cytoplasmic origins of S. sinense and S. barberi were much more ancient than the S. offcinarum/S. robustum linage. Overall, 14 markers were developed, including 9 InDel markers for distinguishing Saccharum from its relative species, 4 dCAPS markers for distinguishing S. officinarum from S. robustum, and 1 dCAPS marker for distinguishing S. sinense and S. barberi from other species. The results obtained from our studies will contribute to the understanding of the classification and plastome evolution of Saccharinae, and the molecular markers developed have demonstrated their highly discriminatory power in Saccharum and relative species.

High-Quality Genome Sequence Resource for Fusarium andiyazi Causing Pokkah Boeng Disease of Sugarcane in China.

Sugarcane pokkah boeng disease (PBD) is emerging as a prevalent foliar disease in China. This airborne disease is caused by the Fusarium species complex. To investigate the diversity and evolution of Fusarium spp., we performed whole-genome sequencing of Fusarium andiyazi YN28 using a combination of Oxford Nanopore and Illumina technology. The F. andiyazi YN28 genome was sequenced, assembled, and annotated. A high-quality genome was assembled into 24 contigs with an N50 of 2.80 Mb. The genome assembly generated a total size of 44.1 Mb with a GC content of 47.64%. In total, 15,508 genes were predicted, including 794 genes related to the carbohydrate-active enzymes, 397 noncoding RNA, 155 genes associated with transporter classification, 4,550 genes linked to pathogen-host interactions, and 269 genes involved in effector proteins. Collectively, our results will provide insight into the host-pathogen interactions and will facilitate the breeding of new varieties of sugarcane resistant to PBD.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Photosynthetic characterization and expression profiles of sugarcane infected by Sugarcane mosaic virus (SCMV).

Sugarcane mosaic virus (SCMV), belonging to genus Potyvirus, family Potyviridae, is a severe pathogen of several agricultural important crops, mainly sugarcane. Due to complex nature of sugarcane, the effect of SCMV pathogenicity on sugarcane photosynthetic systems remains to be explored. In this study, we investigated the alterations occurring in the photosynthetic system in the sugarcane genotypes at the cytopathological, physiological and biological, transcriptome and proteome level. We generated the transcriptome assembly of two genotypes (susceptible Badila and resistant B-48) using Saccharum spontaneum L. as a reference genome. RNA-sequencing data revealed the significant upregulation of NAD(P)H, RubisCO, oxygen-evolving complex, chlorophyll a and b binding protein, Psb protein family, PSI reaction center subunit II, and IVgenes in B-48, as compared to its counterparts. Upregulated genes in B-48 are associated with various processes such as stability and assembly of photosystem, protection against photoinhibition and antiviral defense. The expression pattern of differentially abundant genes were further verified at the proteomics level. Overall, differentially expressed genes/proteins (DEGs/DEPs) showed the consistency of expression at both transcriptome and proteome level in B-48 genotype. Comprehensively, these data supported the efficiency of B-48 genotype under virus infection conditions and provided a better understanding of the expression pattern of photosynthesis-related genes in sugarcane.

Genetic Dissection of T-DNA Insertional Mutants Reveals Uncoupling of Dikaryotic Filamentation and Virulence in Sugarcane Smut Fungus.

The biotrophic basidiomycetous fungus Sporisorium scitamineum causing smut disease in sugarcane is characterized by a life cycle composed of a yeast-like nonpathogenic haploid basidiosporial stage outside the plant and filamentous pathogenic dikaryotic hyphae within the plant. Under field conditions, dikaryotic hyphae are formed after mating of two opposite mating-type strains. However, the mechanisms underlying genetic regulation of filamentation and its association with pathogenicity and development of teliospores are unclear. This study has focused on the characterization and genetic dissection of haploid filamentous mutants derived from T-DNA insertional mutagenesis. Our results support the existence of at least three genotypes among the six haploid filamentous mutants that differentially contribute to virulence and development of the whip and teliospore, providing a novel foundation for further investigation of the regulatory networks associated with pathogenicity and teliospore development in S. scitamineum.

Incidence, Geographical Distribution, and Genetic Diversity of Sugarcane Striate Virus in Saccharum Species in China.

A novel virus of the genus Mastrevirus, family Geminivirdae, has been reported in sugarcane germplasm collections in Florida, Guadeloupe, and Réunion, and was named sugarcane striate virus (SStrV). Although the full-length sequence of an SStrV isolate from China was obtained in 2015, the incidence, geographical distribution, and genetic diversity of this virus remained unclear. A single leaf sample from 2,368 sugarcane plants from main sugarcane-producing regions of China and germplasm collections were tested for SStrV by PCR. Average virus incidence was 25.1% for field-collected samples, and SStrV was detected in most Saccharum species and two sugarcane-related species, with the highest incidence in Saccharum officinarum (44.1%) followed by Saccharum spp. local varieties (33.3%) grown for chewing cane for a long time. The virus incidence was much lower (6.8%) in modern commercial cultivars (Saccharum spp. hybrids). Phylogenetic trees based on full-length genomes of 157 SStrV isolates revealed that Chinese isolates comprised strains A and B, but not C and D, that were reported in Florida, U.S.A. SStrV strain A was the most prominent (98.7%) and widespread strain in China and was further divided into eight subgroups. Almost half (45.6%) of the SStrV-positive samples from S. officinarum and Saccharum spp. local varieties were coinfected with sugarcane mosaic disease viruses or sugarcane yellow leaf virus. Interestingly, most of the plants infected by strain A of SStrV were asymptomatic. SStrV appears to be widespread in China, and its influence on chewing cane deserves further investigation.

Comparative Analysis of Sugar Metabolites and Their Transporters in Sugarcane Following Sugarcane mosaic virus (SCMV) Infection.

Sugarcane mosaic virus (SCMV) is one of the major pathogens of sugarcane. SCMV infection causes dynamic changes in plant cells, including decreased photosynthetic rate, respiration, and sugar metabolism. To understand the basics of pathogenicity mechanism, we performed transcriptome and proteomics analysis in two sugarcane genotypes (Badila: susceptible to SCMV and B-48: SCMV resistant). Using Saccharum spontaneum L. genome as a reference, we identified the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) that participate in sugar metabolism, transport of their metabolites, and Carbohydrate Activating enZYmes (CAZymes). Sequencing data revealed 287 DEGs directly or indirectly involved in sugar metabolism, transport, and storage, while 323 DEGs are associated with CAZymes. Significant upregulation of glucose, sucrose, fructose, starch, and SWEET-related transcripts was observed in the Badila after infection of SCMV. B-48 showed resistance against SCMV with a limited number of sugar transcripts up-regulation at the post-infection stage. For CAZymes, only glycosyltransferase (GT)1 and glycosyl hydrolase (GH)17 were upregulated in B-48. Regulation of DEGs was analyzed at the proteomics level as well. Starch, fructose, glucose, GT1, and GH17 transcripts were expressed at the post-translational level. We verified our transcriptomic results with proteomics and qPCR data. Comprehensively, this study proved that Badila upregulated sugar metabolizing and transporting transcripts and proteins, which enhance virus multiplication and infectionl.

Genome Sequence of Phoma sorghina var. saccharum That Causes Sugarcane Twisted Leaf Disease in China.

Phoma sorghina var. saccharum is a fungal pathogen that causes sugarcane twisted leaf disease in China. Here, we report complete genome assemblies of the Phoma sorghina var. saccharum isolate BS2-1, generated using single-molecule real-time sequencing. We present a high-quality genome sequence of a Phoma isolate that was assembled into 22 contigs with an N50 length of 1.92 Mb, a total length of 33.12 Mb, and a GC content of 52.12%. A total of 7,870 genes were annotated, using a combination of gene prediction tools, including 281 noncoding RNAs, 515 genes encoding carbohydrate-active enzymes, 2,440 genes associated with pathogen-host interactions, and 583 genes encoding secreted proteins. The complete genome sequence will be useful for understanding host-pathogen interaction and for improving disease management strategies.

Gene expression profiling of reactive oxygen species (ROS) and antioxidant defense system following Sugarcane mosaic virus (SCMV) infection.

BACKGROUND: Viruses are infectious pathogens, and plant virus epidemics can have devastating consequences to crop yield and quality. Sugarcane mosaic virus (SCMV, belonging to family Potyviridae) is one of the leading pathogens that affect the sugarcane crop every year. To combat the pathogens' attack, plants generate reactive oxygen species (ROS) as the first line of defense whose sophisticated balance is achieved through well-organized antioxidant scavenging pathways. RESULTS: In this study, we investigated the changes occurring at the transcriptomic level of ROS associated and ROS detoxification pathways of SCMV resistant (B-48) and susceptible (Badila) sugarcane genotypes, using Saccharum spontaneum L. genome assembly as a reference genome. Transcriptomic data highlighted the significant upregulation of ROS producing genes such as NADH oxidase, malate dehydrogenase and flavin-binding monooxygenase, in Badila genotype after SCMV pathogenicity. To scavenge the ROS, the Badila genotype illustrated a substantial enhancement of antioxidants i.e. glutathione s-transferase (GST), as compared to its resistant counterpart. GST is supposed to be a key indicator of pathogen attacks on the plant. A remarkably lower GST expression in B-48, as compared to Badila, indicated the development of resistance in this genotype. Additionally, we characterized the critical transcription factors (TFs) involved in endowing resistance to B-48. Among these, WRKY, AP2, NAC, bZIP, and bHLH showed enhanced expression in the B-48 genotype. Our results also confirmed the linkage of transcriptomic data with the enzymatic and qPCR data. The estimation of enzymatic activities for superoxide dismutase, catalase, ascorbate peroxidase, and phenylalanine ammonia-lyase supported the transcriptomic data and evinced higher resistance in B-48 genotype. CONCLUSION: The current study supported the efficiency of the B-48 genotype under SCMV infection. Moreover, comparative transcriptomic data has been presented to highlight the role of significant transcription factors conferring resistance to this genotype. This study provides an in-depth knowledge of the expression profiling of defense mechanisms in sugarcane.

Purified meta-Cresol Purple dye perturbation: How it influences spectrophotometric pH measurements.

Ocean acidification, a phenomenon of seawater pH decrease due to increasing atmospheric CO2, has a global effect on seawater chemistry, marine biology, and ecosystems. Ocean acidification is a gradual and global long-term process, the study of which demands high-quality pH data. The spectrophotometric technique is capable of generating accurate and precise pH measurements but requires adding an indicator dye that perturbs the sample original pH. While the perturbation is modest in well-buffered seawater, applications of the method in environments with lower buffer capacity such as riverine, estuarine, sea-ice meltwater and lacustrine environments are increasingly common, and uncertainties related to larger potential dye perturbations need further evaluation. In this paper, we assess the effect of purified meta-Cresol Purple (mCP) dye addition on the sample pH and how to correct for this dye perturbation. We conducted numerical simulations by incorporating mCP speciation into the MATLAB CO2SYS program to examine the changes in water sample pH caused by the dye addition and to reveal the dye perturbation mechanisms. Then, laboratory experiments were carried out to verify the simulation results. The simulations suggest that the dye perturbation on sample pH is a result of total alkalinity (TA) contributions from the indicator dye and chemical equilibrium shifts that are related to both the water sample properties (pH, TA, and salinity) and the indicator dye solution properties (pH and solvent matrix). The laboratory experiments supported the simulation results; the same dye solution can lead to different dye perturbations in water samples with different pH, TA, and salinity values. The modeled adjustments agreed well with the empirically determined adjustments for salinities > 5, but it showed greater errors for lower salinities with disagreements as large as 0.005 pH units. Adjustments are minimized when the pH and salinity of the dye are matched to the sample. When the dye is used over a wide range of salinity, we suggest that it should be prepared in deionized water to minimize the dye perturbation effect on pH in the fresher sample waters with less well-constrained perturbation adjustments. We also suggest that the dye perturbation correction should be based on double dye addition experiments performed over a wide range of pH, TA, and salinity. Otherwise, multiple volume dye addition experiments are recommended for each sample to determine the dye perturbation adjustment. We further create a MATLAB function dyeperturbation.m that calculates the expected dye perturbation. This function can be used to validate empirically-derived adjustments or in lieu of empirical adjustments if dye addition experiments are unfeasible (e.g., for historical data). This study of dye perturbation evaluation and correction will improve the accuracy of the pH data, necessary for monitoring the long-term anthropogenic-driven changes in the seawater carbonate system.

First report of stalk bacterial soft rot of sugarcane caused by Dickeya zeae in China.

In late September 2019, seven stalks of about 1400 stalks of sugarcane cultivar Zhongzhe 1 exhibited soft rot symptoms in a trial plot in Beihai city, Guangxi province of China. Symptoms included scorched and collapsed leaves, maceration of stalks, and sour smelling exudates from the stalks (Supplementary Fig. S1). Severely diseased stalks had collapsed and were dead. Internal stalk fragments of 5 × 5 mm were collected at the junction of healthy and diseased tissue after surface-sterilization of stalks with 70% ethanol for one minute, and three times rinsing with sterile distilled water. Stalk fragments were placed on Luria-Bertani agar medium (1 % w/v tryptone, 0.5 % w/v yeast extract, 1 % w/v NaCl, 1 % w/v agar, pH7.0) and plates were put in an incubator at 30°C for 48h. Four types of bacterial colonies were obtained, and small and white colonies with irregular margins were the most dominant. A single colony of each type was diluted in sterile distilled water and aliquots of each suspension were streaked on fresh medium plates to obtain pure cultures. Ten eight-week-old stalks (11 th leaf stage) of sugarcane plants, which derived from cuttings of symptomless cultivar Zhongzhe 1, were inoculated by injection of 300 µl of bacterial suspension (3.5x108 CFU/ml) into the stalks. Another 10 stalks were injected with pure water and served as control. The inoculated plants were kept in a greenhouse at 25-37℃.Among the four types of bacteria, only strain BH9 induced symptoms that were identical to those of diseased canes observed in the field (Supplementary Fig. S1). Elongated water-soaked lesions were observed around the inoculation sites three days post inoculation. Five of the 10 BH9-inoculated plants had collapsed two days later. Water-soaked stalks had a sour smell similar to the filed diseased plants. Eight days post inoculation, all BH9-inoculated plants exhibited symptoms but control plants remained symptomless up to 30 days after inoculation. Uniform white colonies with irregular margins were isolated from the inoculated stalks that developed soft rot symptom, and these bacteria caused again stalk soft rot symptoms when inoculated to a new batch of 10 healthy plants. The 16S rRNA gene of strain BH9 was amplified by PCR with primer pair fD2/rP1 and the PCR amplicons from three independent colonies were sequenced. The sequences of the three amplicons were identical (Accession No. MT723897). BLAST alignments of the 16S rDNA sequence from BH9 strain with the GenBank database revealed that BH9 belonged to the genus Dickeya (98.5% identity between D. zeae BH9 and D. zeae EC1). Further PCR assays and sequencing of three genes, DNA polymerase III gamma subunit gene dnaX with primers dnaXf/dnaXr, DNA gyrase gene gyrB with primers gyrBf1/gyrBr1, and recombinase A gene recA with primers recAf/recAr, were performed to identify the species within the genus Dickeya (Zhang et al., 2014). BH9 sequences of these genes (Accession No. MT723898 to MT723900) had highest identity (97.5%, 97.6%, and 97.7%, respectively) with those from D. zeae EC1 (GenBank accession No. CP006929.1). To determine the evolutionary relationship of BH9 to other Dickeya species and strains, a phylogenetic analysis was performed using dnaX, gyrB, and recA sequences. As shown in Supplementary Fig. S2, BH9 clustered with D. zeae strains and formed a lineage distinguishable from other Dickeya species. Among the closest strains, D. zeae NCPPB3531 (Accession No. CM001980.1) was isolated from potato and D. zeae CSL RW192 (Accession No. CM001972.1) from river water (Pritchard et al., 2013). Consequently, strain BH9 was identified as D. zeae. This bacterial species has been reported to cause soft rot in rice (Pu et al., 2012), banana (Zhang et al., 2014), maize (Martinez-Cisneros et al., 2014), and clivia (Hu et al., 2018). To the best of our knowledge, this is the first report of a bacterial stalk rot caused by D. Zeae in sugarcane. In fact, low incidence of D. zeae-caused stalk soft rot was recently found in sugarcane fields in Fusui County, about 150 km north to Beihai. Given the potential threat of this disease to the local sugarcane industry, the mode of transmission, cultivar resistance, and measures to control the disease should be investigated.

Molecular characterization of carbendazim resistance of Fusarium species complex that causes sugarcane pokkah boeng disease.

BACKGROUND: Pokkah boeng is one of the most serious and devastating diseases of sugarcane and causes significant loss in cane yield and sugar content. Although carbendazim is widely used to prevent fungal diseases, the molecular basis of Fusarium species complex (FSC) resistance to carbendazim remains unknown. RESULTS: The EC50 (fungicide concentration that inhibits 50% of mycelial growth) values of carbendazim for 35 FSC isolates collected in cane growing regions of China were ranged from 0.5097 to 0.6941 µg mL- 1 of active ingredient (a.i.), in an average of 0.5957 µg a.i. mL- 1. Among carbendazim-induced mutant strains, SJ51M (F. verticillioides) had a CTG rather than CAG codon (Q134L) at position 134 of the FVER_09254 gene, whereas in the mutant strain HC30M (F. proliferatum) codon ACA at position 351 of the FPRO_07779 gene was replaced by ATA (T351I). Gene expression profiling analysis was performed for SJ51M and its corresponding wild type strain SJ51, with and without carbendazim treatment. The gene expression patterns in SJ51 and SJ51M changed greatly as evidenced by the detection of 850 differentially expressed genes (DEGs). Functional categorization indicated that genes associated with oxidation-reduction process, ATP binding, integral component of membrane, transmembrane transport and response to stress showed the largest expression changes between SJ51M and SJ51. The expression levels of many genes involved in fungicide resistance, such as detoxification enzymes, drug efflux transporters and response to stress, were up-regulated in SJ51M compared to SJ51 with and without carbendazim treatment. CONCLUSION: FSC was sensitive to carbendazim and had the potential for rapid development of carbendazim resistance. The transcriptome data provided insight into the molecular pathways involved in FSC carbendazim resistance.