Insights into Reactive Oxygen Species Production-Scavenging System Involved in Sugarcane Response to Xanthomonas albilineans Infection under Drought Stress.

Plants must adapt to the complex effects of several stressors brought on by global warming, which may result in interaction and superposition effects between diverse stressors. Few reports are available on how drought stress affects Xanthomonas albilineans (Xa) infection in sugarcane (Saccharum spp. hybrids). Drought and leaf scald resistance were identified on 16 sugarcane cultivars using Xa inoculation and soil drought treatments, respectively. Subsequently, four cultivars contrasting to drought and leaf scald resistance were used to explore the mechanisms of drought affecting Xa-sugarcane interaction. Drought stress significantly increased the occurrence of leaf scald and Xa populations in susceptible cultivars but had no obvious effect on resistant cultivars. The ROS bursting and scavenging system was significantly activated in sugarcane in the process of Xa infection, particularly in the resistant cultivars. Compared with Xa infection alone, defense response via the ROS generating and scavenging system was obviously weakened in sugarcane (especially in susceptible cultivars) under Xa infection plus drought stress. Collectively, ROS might play a crucial role involving sugarcane defense against combined effects of Xa infection and drought stress.

Identification of a sugarcane bacilliform virus promoter that is activated by drought stress in plants.

Sugarcane (Saccharum spp.) is an important sugar and biofuel crop in the world. It is frequently subjected to drought stress, thus causing considerable economic losses. Transgenic technology is an effective breeding approach to improve sugarcane tolerance to drought using drought-inducible promoter(s) to activate drought-resistance gene(s). In this study, six different promoters were cloned from sugarcane bacilliform virus (SCBV) genotypes exhibiting high genetic diversity. In ß-glucuronidase (GUS) assays, expression of one of these promoters (PSCBV-YZ2060) is similar to the one driven by the CaMV 35S promoter and >90% higher compared to the other cloned promoters and Ubi1. Three SCBV promoters (PSCBV-YZ2060, PSCBV-TX, and PSCBV-CHN2) function as drought-induced promoters in transgenic Arabidopsis plants. In Arabidopsis, GUS activity driven by promoter PSCBV-YZ2060 is also upregulated by abscisic acid (ABA) and is 2.2-5.5-fold higher when compared to the same activity of two plant native promoters (PScRD29A from sugarcane and PAtRD29A from Arabidopsis). Mutation analysis revealed that a putative promoter region 1 (PPR1) and two ABA response elements (ABREs) are required in promoter PSCBV-YZ2060 to confer drought stress response and ABA induction. Yeast one-hybrid and electrophoretic mobility shift assays uncovered that transcription factors ScbZIP72 from sugarcane and AREB1 from Arabidopsis bind with two ABREs of promoter PSCBV-YZ2060. After ABA treatment or drought stress, the expression levels of endogenous ScbZIP72 and heterologous GUS are significantly increased in PSCBV-YZ2060:GUS transgenic sugarcane plants. Consequently, promoter PSCBV-YZ2060 is a possible alternative promoter for genetic engineering of drought-resistant transgenic crops such as sugarcane.

Identification of ARF transcription factor gene family and its defense responses to bacterial infection and salicylic acid treatment in sugarcane.

Auxin response factor (ARF) is a critical regulator in the auxin signaling pathway, involved in a variety of plant biological processes. Here, gene members of 24 SpapARFs and 39 SpnpARFs were identified in two genomes of Saccharum spontaneum clones AP85-441 and Np-X, respectively. Phylogenetic analysis showed that all ARF genes were clustered into four clades, which is identical to those ARF genes in maize (Zea mays) and sorghum (Sorghum bicolor). The gene structure and domain composition of this ARF family are conserved to a large degree across plant species. The SpapARF and SpnpARF genes were unevenly distributed on chromosomes 1-8 and 1-10 in the two genomes of AP85-441 and Np-X, respectively. Segmental duplication events may also contribute to this gene family expansion in S. spontaneum. The post-transcriptional regulation of ARF genes likely involves sugarcane against various stressors through a miRNA-medicated pathway. Expression levels of six representative ShARF genes were analyzed by qRT-PCR assays on two sugarcane cultivars [LCP85-384 (resistant to leaf scald) and ROC20 (susceptible to leaf scald)] triggered by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections and salicylic acid (SA) treatment. ShARF04 functioned as a positive regulator under Xa and Aaa stress, whereas it was a negative regulator under SA treatment. ShARF07/17 genes played positive roles against both pathogenic bacteria and SA stresses. Additionally, ShARF22 was negatively modulated by Xa and Aaa stimuli in both cultivars, particularly LCP85-384. These findings imply that sugarcane ARFs exhibit functional redundancy and divergence against stressful conditions. This work lays the foundation for further research on ARF gene functions in sugarcane against diverse environmental stressors.

Genetic Divergence and Population Structure of Xanthomonas albilineans Strains Infecting Saccharum spp. Hybrid and Saccharum officinarum.

Leaf scald caused by Xanthomonas albilineans (Xa) is a major bacterial disease in sugarcane that represents a threat to the global sugar industry. Little is known about the population structure and genetic evolution of this pathogen. In this study, 39 Xa strains were collected from 6 provinces in China. Of these strains, 15 and 24 were isolated from Saccharum spp. hybrid and S. officinarum plants, respectively. Based on multilocus sequence analysis (MLSA), with five housekeeping genes, these strains were clustered into two distinct phylogenetic groups (I and II). Group I included 26 strains from 2 host plants, Saccharum spp. hybrid and S. officinarum collected from 6 provinces, while Group II consisted of 13 strains from S. officinarum plants in the Zhejiang province. Among the 39 Xa strains, nucleotide sequence identities from 5 housekeeping genes were: ABC (99.6-100%), gyrB (99.3-100%), rpoD (98.4-100%), atpD (97.0-100%), and glnA (97.6-100%). These strains were clustered into six groups (A-F), based on the rep-PCR fingerprinting, using primers for ERIC2, BOX A1R, and (GTG)5. UPGMA and PCoA analyses revealed that group A had the most strains (24), followed by group C with 11 strains, while there was 1 strain each in groups B and D-F. Neutral tests showed that the Xa population in S. officinarum had a trend toward population expansion. Selection pressure analysis showed purification selection on five concatenated housekeeping genes from all tested strains. Significant genetic differentiation and infrequent gene flow were found between two Xa populations hosted in Saccharum spp. hybrids and S. officinarum. Altogether, these results provide evidence of obvious genetic divergence and population structures among Xa strains from China.

Effects of borneol combined with astragaloside IV and Panax notoginseng saponins regulation of microglia polarization to promote neurogenesis after cerebral ischaemia.

OBJECTIVE: To study the effect of borneol combined with astragaloside IV and Panax notoginseng saponins (BAP) on promoting neurogenesis by regulating microglia polarization after cerebral ischaemia-reperfusion(CI/R) in rats. METHODS: A focal CI/R injury model was established. Evaluated the effects of BAP on ischaemic brain injury, on promoting neurogenesis, on inhibiting Inflammatory microenvironment and TLR4/MyD88/NFκB signalling pathway. A microglia oxygen-glucose deprivation reoxygenation (OGD/R) model was established that evaluated the effects of BAP on regulating the polarization of microglia and inflammatory microenvironment. RESULTS: BAP can inhibit the expression of TLR4, MyD88 and NFκB proteins, reduce IL-1ß and increase IL-10, reduce M1 type microglia and increase M2 microglia. The proliferation of neural stem cells increased, synaptic gap decreased, synaptic interface curvature increased, expression of SYN and PSD95 proteins increased, which improved the neurological dysfunction and reduced the volume of cerebellar infarction and nerve cell injury. CONCLUSION: BAP can reduce CI/R injury and promote neurogenesis, the effect is related to inhibition of the activation of TLR4/MyD88/NFκB, regulating the polarization of microglia from M1 type to M2 type and inhibition of inflammatory response.

Systematic and functional analysis of non-specific lipid transfer protein family genes in sugarcane under Xanthomonas albilineans infection and salicylic acid treatment.

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins that play a significant regulatory role in a wide range of physiological processes. To date, no genome-wide survey and expression analysis of this gene family in sugarcane has been performed. In this study we identified the nsLTP gene family in Saccharum spontaneum and carried out expression profiling of nsLTPs in two sugarcane cultivars (Saccharum spp.) that have different resistance to leaf scald caused by Xanthomonas albilineans (Xa) infection. The effect of stress related to exogenous salicylic acid (SA) treatment was also examined. At a genome-wide level, S. spontaneum AP85-441 had 71 SsnsLTP genes including 66 alleles. Tandem (9 gene pairs) and segmental (36 gene pairs) duplication events contributed to SsnsLTP gene family expansion. Five SsnsLTP proteins were predicted to interact with five other proteins. Expression of ShnsLTPI.8/10/Gb.1 genes was significantly upregulated in LCP85-384 (resistant cultivar), but downregulated in ROC20 (susceptible cultivar), suggesting that these genes play a positive regulatory role in response of sugarcane to Xa infection. Conversely, ShnsLTPGa.4/Ge.3 appears to act as a negative regulator in response Xa infection. The majority (16/17) of tested genes were positively induced in LCP85-384 72 h after SA treatment. In both cultivars, but particularly in LCP85-384, ShnsLTPIV.3/VIII.1 genes were upregulated at all time-points, suggesting that the two genes might act as positive regulators under SA stress. Meanwhile, both cultivars showed downregulated ShnsLTPGb.1 gene expression, indicating its potential negative role in SA treatment responses. Notably, the ShnsLTPGb.1 gene had contrasting effects, with positive regulation of gene expression in response to Xa infection and negative regulation induced by SA stress. Together, our results provide valuable information for elucidating the function of ShnsLTP family members under two stressors and identified novel gene sources for development of sugarcane that are tolerant of environmental stimuli.

Sugarcane responses to two strains of Xanthomonas albilineans differing in pathogenicity through a differential modulation of salicylic acid and reactive oxygen species.

Leaf scald caused by Xanthomonas albilineans is one of the major bacterial diseases of sugarcane that threaten the sugar industry worldwide. Pathogenic divergence among strains of X. albilineans and interactions with the sugarcane host remain largely unexplored. In this study, 40 strains of X. albilineans from China were distributed into three distinct evolutionary groups based on multilocus sequence analysis and simple sequence repeats loci markers. In pathogenicity assays, the 40 strains of X. albilineans from China were divided into three pathogenicity groups (low, medium, and high). Twenty-four hours post inoculation (hpi) of leaf scald susceptible variety GT58, leaf populations of X. albilineans strain XaCN51 (high pathogenicity group) determined by qPCR were 3-fold higher than those of strain XaCN24 (low pathogenicity group). Inoculated sugarcane plants modulated the reactive oxygen species (ROS) homoeostasis by enhancing respiratory burst oxidase homolog (ScRBOH) expression and superoxide dismutase (SOD) activity and by decreasing catalase (CAT) activity, especially after infection by X. albilineans XaCN51. Furthermore, at 24 hpi, plants infected with XaCN51 maintained a lower content of endogenous salicylic acid (SA) and a lower expression level of SA-mediated genes (ScNPR3, ScTGA4, ScPR1, and ScPR5) as compared to plants infected with XaCN24. Altogether, these data revealed that the ROS production-scavenging system and activation of the SA pathway were involved in the sugarcane defense response to an attack by X. albilineans.

Genetic Variability and Molecular Evolution of Maize Yellow Mosaic Virus Populations from Different Geographic Origins.

Maize yellow mosaic virus (MaYMV) hosted in various gramineous plants was assigned to the genus Polerovirus (family Luteoviridae) in 2018. However, little is known about its genetic diversity and population structure. In this study, 509 sugarcane leaf samples with mosaic symptoms were collected in 2017 to 2019 from eight sugarcane-growing provinces in China. Reverse-transcription PCR results revealed that four positive-sense RNA viruses were found to infect sugarcane, and the incidence of MaYMV among samples from Fujian, Sichuan, and Guangxi Provinces was 52.1, 9.8, and 2.5%, respectively. Based on 82 partial MaYMV sequences and 46 whole-genome sequences from different host plants, phylogenetic analysis revealed that MaYMV populations are very closely associated with their source geographical regions (China, Africa, and South America). Pairwise identity analysis showed significant variability in genome sequences among MaYMV isolates with genomic nucleotide identities of 91.1 to 99.9%. In addition to codon mutations, insertions or deletions also contributed to genetic variability in individual coding regions, especially in the readthrough protein (P3-P5 fusion protein). Low gene flow and significant genetic differentiation of MaYMV were observed among the three geographical populations, suggesting that environmental adaptation is an important evolutionary force that shapes the genetic structure of MaYMV. Genes in the MaYMV genome were subject to strong negative or purification selection during evolution, except for the movement protein (MP), which was under positive selection pressure. This finding suggests that the MP may play an important role in MaYMV evolution. Taken together, our findings provide basic information for the development of an integrated disease management strategy against MaYMV.

Genome-wide analysis of mitogen-activated protein (MAP) kinase gene family expression in response to biotic and abiotic stresses in sugarcane.

To systematically analyze mitogen-activated protein (MAP) kinase gene families and their expression profiles in sugarcane (Saccharum spp. hybrids; Sh) under diverse biotic and abiotic stresses, we identified 15 ShMAPKs, 6 ShMAPKKs and 16 ShMAPKKKs genes in the sugarcane cultivar R570 genome. These were also confirmed in one S. spontaneum genome and two transcriptome datasets of sugarcane trigged by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections. Phylogenetic analysis revealed that four subgroups were present in each ShMAPK and ShMAPKK family and three sub-families (RAF, MEKK and ZIK) presented in the ShMAPKKK family. Conserved protein motif and gene structure analyses supported the evolutionary relationships of the three families inferred from the phylogenetic analysis. All of the ShMAPK, ShMAPKK and ShMAPKKK genes identified in Saccharum spp. R570 were distributed on chromosomes 1-7 and 9-10. RNA-seq and qRT-PCR analyses indicated that ShMAPK07 and ShMAPKKK02 were defense-responsive genes in sugarcane challenged by both Aaa and Xa stimuli, while some genes were upregulated specifically by Aaa and Xa infection. Additionally, ShMAPK05 acted as a negative regulator under drought and salinity stress, but served as a positive regulator under salicylic acid (SA) treatment. ShMAPK07 plays a positive role under drought stress, but a negative role under SA treatment. ShMAPKKK01 was negatively modulated by both salinity stress and SA treatment, whereas ShMAPKKK06 was positively regulated by both of the two stress stimuli. Our results suggest that members of MAPK cascade gene families regulate adverse stress responses through multiple signal transduction pathways in sugarcane.

First Report of Pantoea stewartii subsp. stewartii Causing Bacterial Leaf Wilt of Sugarcane in China.

The pathogen of Pantoea stewartii subsp. stewartii (Pss) that is the causal agent of Stewart's bacterial wilt of corn also infects numerous experimental hosts of graminaceous plants (Pepper et al., 1967; Wang et al., 2012). However, little is known about this pathogen naturally infecting sugarcane. In 2017, we observed some sugarcane cultivars showing leaf blade bleaching at the disease initiation stage, which further resulted in development of blight and necrotic lesions (Figure 1-A and -B) in Zhanjiang, Guangdong province of China. To diagnose this putative disease, five symptomatic leaf samples were collected from different sugarcane cultivars. The Pss was found to infect these samples using the nested PCR with Pss-specific outer primers PS1/PS4 and inner primers Ps2r/Ps3r that targeted at the 16S rRNA gene of this pathogen (Wang et al., 2009). The expected 262-bp fragments from positive samples were amplified, cloned, and sequenced (GenBank accession no. MW015795-MW015799). BLASTN analysis revealed that these isolates had more than 99.5% nucleotide identify (222 bp out of 262 bp) with each other and with Pss strains (ATCC 8199 and DC283) as well as P. stewartii subsp. indologenes strains (SR2-12 and LMG 2632) after sequences were trimmed at the 5'- and 3'-terminal of inner primer sequences. In addition, these leaf samples were surface-sterilized with 75% alcohol followed by macerated and chopped in sterile water. Upon plating on solid NA medium at 28 °C for 24-36 h, the bacterial colonies exhibited yellow color with circular, convex, smooth and translucent edges (Figure 1-C). Straight rods and non-encapsulated cells were detected under transmission electron microscopy (Figure 1-D). Moreover, an identical colony termed as PSCN1 was isolated from sugarcane cultivar YZ08-1095 and was further confirmed by the PCR with a universal primer pair 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1387R (5'-GGGCGGWGTGTACAAGGC-3') that targeted at bacterial 16S rRNA gene (Marchesi et al., 1998). A 1362-bp DNA fragment sequence was obtained from PSCN1 strain and deposited on GenBank library (accession no. MW015767). Sequence analysis showed that PSCN1 shared 99.9-100% nucleotide identity (1315 bp out of 1362 bp) with the two reference strains of Pss (ATCC 8199 and DC283) after sequences were trimmed at the 5'- and 3'-terminal of primer sequences. According to Koch's postulates, pathogenicity test was carried out on YZ08-1095 plants with 3-5 fully developed leaf inoculated with the suspended cells (108 cells/ml) of PSCN1 strain by cutting the one-third of leaves before spraying with a suspension. Control plants were mock-inoculated with serial liquid nutrition agar medium. Two independent experiments were performed for pathogenicity assay and more than 28 plants of YZ08-1095 were used in each treatment. Plants were cultured in a growth chamber at 28 °C and 60% humidity under a 16 h light/8 h dark photoperiod. Leaves inoculated by the PSCN1 initially showed bleached, blight and wilting symptoms on leaf edges at seven days post-inoculation (dpi) (Figure 1-E and -F), which were similar to those symptoms observed in the fields. Control plants remained asymptomatic (Figure 1-G). The average incidence of diseased plant was 51.9% at 21 dpi. The bacteria were subsequently re-isolated from diseased leaves, and yielded colonies were completely identical to the PSCN1. Taken together, our data provides the valuable information for diagnosis and controlling this disease in sugarcane.

First report of Xanthomonas albilineans causing leaf scald on two chewing cane clones in Zhejiang province, China.

In recent years, outbreaks of leaf scald have been reported in two chewing cane clones "Guangdong Huangpi" and "Taoshang Guozhe" in Zhejiang province, China. From May to July 2019, we collected 11 and 13 leaf or stalk samples from symptomatic "Guangdong Huangpi" from four farms in Wenling and "Taoshang Guozhe" clones from three farms in Ruian, Zhejiang province, respectively. Leaves in young plants exhibited white pencil-line streaks (Supplement Fig. 1A & 1D) as well as partial or complete chlorosis of the leaf blade (Supplement Fig. 1B & 1E). Internal symptoms included an orange-red discoloration of the vascular bundles at the basal nodes of the stalk and discoloration extension into the internodes (Supplement Fig. 1C & 1F). Leaf and stalk tissues were used for bacterial isolation and purification on XAS medium, which is selective for Xanthomonas albilineans (Davis et al. 1994), using the streak plate method to obtain 24 isolates (Lin et al. 2018). Circular, convex, smooth, shiny and yellow colonies were isolated from all the samples. The pathogenicity of two isolates, XaCN30 from "Guangdong Huangpi" and XaCN43 from "Taoshang Guozhe", was confirmed with Koch's postulates according to the protocol reported by Lin et al. (2018). The incidences of diseased plants (56% and 63%) were observed in individual host clones at 28 d post-inoculation with isolates XaCN30 and XaCN43, respectively. Furthermore, all isolates were confirmed as X. albilineans via molecular methods. PCR amplification was conducted for all 24 isolates using the primer pairs XgyrB1F/XgyrB1R2 (Ntambo et al. 2019) and XAF1/XAR1 (Wang et al. 1999), which targeting the gyrB (encoding the b subunit of the DNA gyrase) and abc (encoding an ABC transporter) genes, and generating 904 bp and 608 bp amplicons, respectively. The PCR fragments were cloned into the pMD19-T vector (TaKaRa, Dalian, China). For each isolate, three single colonies of transformed Escherichia coli DH5α carrying targeted fragment were sequenced. These sequences were deposited into the GenBank with accession no. MT776053-MT776059 and MT776061-MT776077 for gyrB gene and MT776098-MT776104 and MT776106-MT776122 for abc gene. Based on the two concatenated DNA sequences of our 24 isolates, compared with 27 previously reported X. albilineans isolates obtained from the GenBank database, pairwise sequence identity analysis revealed that all 24 isolates from Zhejiang province had 99.4-100% identity with each other, 99.6-100% identity with 14 published domestic isolates, and 98.3-100% identity with 13 foreign isolates. Furthermore, phylogenetic analysis with MEGA 7.0 (Kumar et al. 2016) showed that the isolates from Zhejiang province clustered into two distinct groups (Supplement Fig. 2). One group consisted of 25 Chinese isolates (including all 11 isolates from Wenling) along with four isolates from the French West Indies (GPE PC73, GPE PC17, GPE PC86, and MTQ032), and one isolate from the USA (XaFL07-1), which were assigned to pulsed-field gel electrophoresis (PFGE) group B (Davis et al. 1997; Pieretti et al. 2012). A putative group was also proposed, which included all 13 isolates from Ruian, indicating that isolates from Ruian are distinct from the isolates isolated from other Chinese sugarcane-planting areas, including Wenling. We conclude that leaf scald disease in local clones of chewing cane are caused by X. albilineans in Zhejiang province in China, which will be helpful for leaf scald management in chewing cane, a cash crop.

Genome-wide identification and characterization of DCL, AGO and RDR gene families in Saccharum spontaneum.

RNA silencing is a conserved mechanism in eukaryotic organisms to regulate gene expression. Argonaute (AGO), Dicer-like (DCL) and RNA-dependent RNA polymerase (RDR) proteins are critical components of RNA silencing, but how these gene families' functions in sugarcane were largely unknown. Most stress-resistance genes in modern sugarcane cultivars (Saccharum spp.) were originated from wild species of Saccharum, for example S. spontaneum. Here, we used genome-wide analysis and a phylogenetic approach to identify four DCL, 21 AGO and 11 RDR genes in the S. spontaneum genome (termed SsDCL, SsAGO and SsRDR, respectively). Several genes, particularly some of the SsAGOs, appeared to have undergone tandem or segmental duplications events. RNA-sequencing data revealed that four SsAGO genes (SsAGO18c, SsAGO18b, SsAGO10e and SsAGO6b) and three SsRDR genes (SsRDR2b, SsRDR2d and SsRDR3) tended to have preferential expression in stem tissue, while SsRDR5 was preferentially expressed in leaves. qRT-PCR analysis showed that SsAGO10c, SsDCL2 and SsRDR6b expressions were strongly upregulated, whereas that of SsAGO18b, SsRDR1a, SsRDR2b/2d and SsRDR5 was significantly depressed in S. spontaneum plants exposed to PEG-induced dehydration stress or infected with Xanthomonas albilineans, causal agent of leaf scald disease of sugarcane, suggesting that these genes play important roles in responses of S. spontaneum to biotic and abiotic stresses.

Complete Genome Sequence Reveals Evolutionary and Comparative Genomic Features of Xanthomonas albilineans Causing Sugarcane Leaf Scald.

Leaf scald (caused by Xanthomonas albilineans) is an important bacterial disease affecting sugarcane in most sugarcane growing countries, including China. High genetic diversity exists among strains of X. albilineans from diverse geographic regions. To highlight the genomic features associated with X. albilineans from China, we sequenced the complete genome of a representative strain (Xa-FJ1) of this pathogen using the PacBio and Illumina platforms. The complete genome of strain Xa-FJ1 consists of a circular chromosome of 3,724,581 bp and a plasmid of 31,536 bp. Average nucleotide identity analysis revealed that Xa-FJ1 was closest to five strains from the French West Indies and the USA, particularly to the strain GPE PC73 from Guadeloupe. Comparative genomic analysis between Xa-FJ1 and GPE PC73 revealed prophage integration, homologous recombination, transposable elements, and a clustered regulatory interspaced short palindromic repeats (CRISPR) system that were linked with 16 insertions/deletions (InDels). Ten and 82 specific genes were found in Xa-FJ1 and GPE PC73, respectively, and some of these genes were subjected to phage-related proteins, zona occludens toxin, and DNA methyltransferases. Our findings highlight intra-species genetic variability of the leaf scald pathogen and provide additional genomic resources to investigate its fitness and virulence.

Identification of Differentially Expressed Proteins in Sugarcane in Response to Infection by Xanthomonas albilineans Using iTRAQ Quantitative Proteomics.

Sugarcane can suffer severe yield losses when affected by leaf scald, a disease caused by Xanthomonas albilineans. This bacterial pathogen colonizes the vascular system of sugarcane, which can result in reduced plant growth and plant death. In order to better understand the molecular mechanisms involved in the resistance of sugarcane to leaf scald, a comparative proteomic study was performed with two sugarcane cultivars inoculated with X. albilineans: one resistant (LCP 85-384) and one susceptible (ROC20) to leaf scald. The iTRAQ (isobaric tags for relative and absolute quantification) approach at 0 and 48 h post-inoculation (hpi) was used to identify and annotate differentially expressed proteins (DEPs). A total of 4295 proteins were associated with 1099 gene ontology (GO) terms by GO analysis. Among those, 285 were DEPs during X. albilineans infection in cultivars LCP 85-384 and ROC20. One hundred seventy-two DEPs were identified in resistant cultivar LCP 85-384, and 113 of these proteins were upregulated and 59 were downregulated. One hundred ninety-two DEPs were found in susceptible cultivar ROC20 and half of these (92) were upregulated, whereas the other half corresponded to downregulated proteins. The significantly upregulated DEPs in LCP 85-384 were involved in metabolic pathways, the biosynthesis of secondary metabolites, and the phenylpropanoid biosynthesis pathway. Additionally, the expression of seven candidate genes related to photosynthesis and glycolytic pathways, plant innate immune system, glycosylation process, plant cytochrome P450, and non-specific lipid transfer protein was verified based on transcription levels in sugarcane during infection by X. albilineans. Our findings shed new light on the differential expression of proteins in sugarcane cultivars in response to infection by X. albilineans. The identification of these genes provides important information for sugarcane variety improvement programs using molecular breeding strategies.

Global Gene Responses of Resistant and Susceptible Sugarcane Cultivars to Acidovorax avenae subsp. avenae Identified Using Comparative Transcriptome Analysis.

Red stripe disease in sugarcane caused by Acidovorax avenae subsp. avenae (Aaa) is related to serious global losses in yield. However, the underlying molecular mechanisms associated with responses of sugarcane plants to infection by this pathogen remain largely unknown. Here, we used Illumina RNA-sequencing (RNA-seq) to perform large-scale transcriptome sequencing of two sugarcane cultivars to contrast gene expression patterns of plants between Aaa and mock inoculations, and identify key genes and pathways involved in sugarcane defense responses to Aaa infection. At 0-72 hours post-inoculation (hpi) of the red stripe disease-resistant cultivar ROC22, a total of 18,689 genes were differentially expressed between Aaa-inoculated and mock-inoculated samples. Of these, 8498 and 10,196 genes were up- and downregulated, respectively. In MT11-610, which is susceptible to red stripe disease, 15,782 genes were differentially expressed between Aaa-inoculated and mock-inoculated samples and 8807 and 6984 genes were up- and downregulated, respectively. The genes that were differentially expressed following Aaa inoculation were mainly involved in photosynthesis and carbon metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, and plant-pathogen interaction pathways. Further, qRT-PCR and RNA-seq used for additional validation of 12 differentially expressed genes (DEGs) showed that eight genes in particular were highly expressed in ROC22. These eight genes participated in the biosynthesis of lignin and coumarin, as well as signal transduction by salicylic acid, jasmonic acid, ethylene, and mitogen-activated protein kinase (MAPK), suggesting that they play essential roles in sugarcane resistance to Aaa. Collectively, our results characterized the sugarcane transcriptome during early infection with Aaa, thereby providing insights into the molecular mechanisms responsible for bacterial tolerance.

Improved Primers for the Specific Detection of Leifsonia xyli subsp. xyli in Sugarcane Using a Conventional PCR Assay.

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.

Molecular cloning, characterization, and expression profiling analysis of Cry toxin receptor genes from sugarcane shoot borer Chilo infuscatellus (Snellen).

The sugarcane shoot borer Chilo infuscatellus (Snellen) is known for causing severe damage to sugarcane yield in China. Methods have been developed to control this pest, including Cry toxin pesticide and transgenic Bt plants. In order to investigate the molecular mechanism of the Cry toxin binding process and provide a basis for understanding the insect's resistance mechanism, we used a high throughput sequencing platform to perform a de novo transcriptome assembly across different larval developmental stages and analyzed Cry toxin receptors based on our assembled transcripts. We cloned twelve Cry toxin receptor genes including 1 cadherin (Cad), 7 aminopeptidase-Ns (APNs), 3 alkaline phosphatases (ALPs), and 1 ATP-binding cassette transporter subfamily C2 (ABCC2), and three of them with full length. The sublethal dosage of Cry1Ac toxin was applied to sugarcane shoot borer and identified some Cry toxin receptor genes that were significantly induced after 48 h of exposure. Furthermore, quantitative RT-PCR was conducted to detect the expression profiles of these genes. Our transcriptome sequence data provided a valuable molecular resource for further study and the identified Cry toxin receptor data gave insights for improved research into the mechanism of Bt resistance.

Presence of Diverse Sugarcane Bacilliform Viruses Infecting Sugarcane in China Revealed by Pairwise Sequence Comparisons and Phylogenetic Analysis.

Sugarcane bacilliform viruses (SCBV), which belong to the genus Badnavirus, family Caulimoviridae, are an important DNA virus complex that infects sugarcane. To explore the genetic diversity of the sugarcane-infecting badnavirus complex in China, we tested 392 sugarcane leaf samples collected from Fujian, Yunnan, and Hainan provinces for the occurrence of SCBV by polymerase chain reaction (PCR) assays using published primers SCBV-F and SCBV-R that target the reverse transcriptase/ribonuclease H (RT/RNase H) regions of the viral genome. A total of 111 PCR-amplified fragments (726 bp) from 63 SCBV-positive samples were cloned and sequenced. A neighbor-joining phylogenetic tree was constructed based on the SCBV sequences from this study and 34 published sequences representing 18 different phylogroups or genotypes (SCBV-A to -R). All SCBV-tested isolates could be classified into 20 SCBV phylogenetic groups from SCBV-A to -T. Of nine SCBV phylogroups reported in this study, two novel phylogroups, SCBV-S and SCBV-T, that share 90.0-93.2% sequence identity and show 0.07-0.11 genetic distance with each other in the RT/ RNase H region, are proposed. SCBV-S had 57.6-92.2% sequence identity and 0.09-0.66 genetic distance, while SCBV-T had 58.4-90.0% sequence identity and 0.11-0.63 genetic distance compared with the published SCBV phylogroups. Additionally, two other Badnavirus species, Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV), which originally clustered in phylogenetic groups SCBV-E and SCBV-F, respectively, are first reported in China. Our findings will help to understand the level of genetic heterogeneity present in the complex of Badnavirus species that infect sugarcane.

Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Badnavirus Species in Sugarcane.

Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.

Lower gastrointestinal tract bleeding caused by dieulafoy-like lesion synchronous meckel diverticulum: A rare case report.

Meckel diverticulum is an embryonic remnant of the Gastrointestinal duct which causes symptoms < 5% in the 2% population. Painless bleeding and abdominal pain are the most often reported symptoms. Dieulafoy lesion/dieulafoy-like lesion often cause upper gastrointestinal (GI) tract bleeding, but massive lower gastrointestinal bleeding is rare. We reported a 19-year-old male presented massive lower GI tract bleeding caused by Meckel diverticulum synchronous dieulafoy-like lesion.