Sugarcane Smut Resistance Research Articles

Field assessment and combining ability analysis for smut resistance in sugarcane during continuous ratoon planting in China

Field assessment and combining ability analysis for smut resistance in sugarcane during continuous ratoon planting in China

Ceramides involve sugarcane defense response to Sporisorium scitamineum

AbstractSugarcane smut, a widespread and destructive disease induced by Sporisorium scitamineum, has a significant effect on sugarcane cultivation. Sphingolipids, crucial components of membranes, play a role in plant resistance regulation. However, the specific function of sphingolipids in sugarcane smut resistance has not been understood. This study revealed that following the S. scitamineum infection, ceramide (Cer) and hydroxyceramide (hCer) levels specifically increased in the sugarcane variety ROC22, along with significant alterations in plant hormone levels. Analysis of lipid composition in S. scitamineum‐sensitive and ‐resistant sugarcane varieties indicated that the resistant variety YT93‐159 had lower Cer and hCer levels compared to the sensitive variety ROC22. Additionally, YT93‐159 displayed an increased level of jasmonoyl‐isoleucine (JA‐Ile) and upregulated the expression of genes linked to the pathway of jasmonic acid (JA). Through experiments using the sugarcane protoplast system, it was observed that exogenous ceramide could influence the expression of genes associated with the salicylic acid and JA pathways. This research was the initial examination into the lipidomics‐based molecular mechanisms underlying sugarcane's resistance to S. scitamineum, offering a theoretical foundation for enhancing sugarcane resistance through genetic engineering.

Determining morphological and biochemical indices to select for smut-resistant sugarcane varieties

ABSTRACT In Sri Lanka, smut in sugarcane (Saccharum spp. hybrid) is caused by Sporisorium scitamineum and results in significant crop yield and economic losses. Current field evaluation methods to identify smut resistance in sugarcane are time consuming and do not show an association between disease resistance and the resistance mechanisms. This research was conducted to identify morphological and biochemical characters as selection indices of smut-resistant varieties to strengthen the existing varietal selection program. Fifty accessions were selected from the sugarcane germplasm collection and tested for their resistance to S. scitamineum by the standard inoculation method. The disease incidence and Area Under Disease Progress Curve (AUDPC) were assessed in the first two crop cycles. Pearson correlation coefficients were used to identify relationships of AUDPC with evaluated nine morphological and biochemical characteristics. Principal component analysis (PCA) was used to identify the indices associated with smut resistance and select the superior sugarcane varieties. Significant negative correlations (P < 0.05) were detected between AUDPC and total phenols in the leaves, bud hardness, foliage inclination angle (FIA) and number of scale leaves in the buds. The tested nine morphological and biochemical indices formed four major clusters by PCA with a cumulative contribution rate of 66%. The findings revealed that total phenols in the leaf, bud hardness, FIA and the number of scale leaves in the bud are useful, simple and non-laborious indices to determine sugarcane smut resistance in varietal selection.

Assessment of the rhizosphere fungi and bacteria recruited by sugarcane during smut invasion.

Whip smut is one of the most serious and widely spread sugarcane diseases. Plant-associated microbes play various roles in conferring advantages to the host plant. Understanding the microbes associated with sugarcane roots will help develop strategies for the biocontrol of smut. Therefore, the present study explored microbe-mediated sugarcane response to smut invasion via 16S rRNA and ITS metabarcoding survey of the rhizosphere soils of resistant and susceptible sugarcane varieties. The bacterial and fungal diversity in the rhizosphere soils differed between the resistant and susceptible varieties. The bacterial genera Sphingomonas, Microcoleus_Es-Yyy1400, Marmoricola, Reyranella, Promicromonospora, Iamia, Phenylobacterium, Aridibacter, Actinophytocola, and Edaphobacter and one fungal genus Cyphellophora were found associated with smut resistance in sugarcane. Detailed analysis revealed that the majority of bacteria were beneficial, including the actinomycete Marmoricola and Iamia and Reyranella with denitrification activity. Analysis of bacterial network interaction showed that three major groups interacted during smut invasion. Meanwhile, seven of these genera appeared to interact and promote each other's growth. Finally, functional annotation based on the Functional Annotation of Prokaryotic Taxa (FAPROTAX) database predicted that the abundant bacteria are dominated by oxygenic photoautotrophy, photoautotrophy, and phototrophy functions, which may be related to smut resistance in sugarcane. The present study thus provides new insights into the dynamics of the sugarcane rhizosphere microbial community during smut invasion.

Genetic identification of SNP markers and candidate genes associated with sugarcane smut resistance using BSR-Seq.

Sugarcane smut caused by Sporisorium scitamineum is one of the most severe fungal diseases worldwide. In this study, a cross was made between a smut-resistant variety YT93-159 and a smut-susceptible variety ROC22, and 312 progenies were obtained. Two bulks of progenies were then constructed, one consisted of 27 highly smut resistant progenies and the other 24 smut susceptible progenies. Total RNAs of the progenies of each bulk, were pooled and subject to bulked segregant RNA-sequence analysis (BSR-Seq). A total of 164.44 Gb clean data containing 2,341,449 SNPs and 64,999 genes were obtained, 7,295 of which were differentially expressed genes (DEGs). These DEGs were mainly enriched in stress-related metabolic pathways, including carbon metabolism, phenylalanine metabolism, plant hormone signal transduction, glutathione metabolism, and plant-pathogen interactions. Besides, 45,946 high-quality, credible SNPs, a 1.27 Mb region at Saccharum spontaneum chromosome Chr5B (68,904,827 to 70,172,982), and 129 candidate genes were identified to be associated with smut resistance. Among them, twenty-four genes, either encoding key enzymes involved in signaling pathways or being transcription factors, were found to be very closely associated with stress resistance. RT-qPCR analysis demonstrated that they played a positive role in smut resistance. Finally, a potential molecular mechanism of sugarcane and S. scitamineum interaction is depicted that activations of MAPK cascade signaling, ROS signaling, Ca2+ signaling, and PAL metabolic pathway and initiation of the glyoxalase system jointly promote the resistance to S. scitamineum in sugarcane. This study provides potential SNP markers and candidate gene resources for smut resistance breeding in sugarcane.

Development of a SCAR marker based on SCoT polymorphisms for sugarcane smut resistance

Development of a SCAR marker based on SCoT polymorphisms for sugarcane smut resistance

WGCNA Identifies a Comprehensive and Dynamic Gene Co-Expression Network That Associates with Smut Resistance in Sugarcane.

Sugarcane smut is a major fungal disease caused by Sporisorium scitamineum, which seriously reduces the yield and quality of sugarcane. In this study, 36 transcriptome data were collected from two sugarcane genotypes, YT93-159 (resistant) and ROC22 (susceptible) upon S. scitamineum infection. Data analysis revealed 20,273 (12,659 up-regulated and 7614 down-regulated) and 11,897 (7806 up-regulated and 4091 down-regulated) differentially expressed genes (DEGs) in YT93-159 and ROC22, respectively. A co-expression network was then constructed by weighted gene co-expression network analysis (WGCNA), which identified 5010 DEGs in 15 co-expressed gene modules. Four of the 15 modules, namely, Skyblue, Salmon, Darkorange, and Grey60, were significantly associated with smut resistance. The GO and KEGG enrichment analyses indicated that the DEGs involving in these four modules could be enriched in stress-related metabolic pathways, such as MAPK and hormone signal transduction, plant-pathogen interaction, amino acid metabolism, glutathione metabolism, and flavonoid, and phenylpropanoid biosynthesis. In total, 38 hub genes, including six from the Skyblue module, four from the Salmon module, 12 from the Darkorange module, and 16 from the Grey60 module, were screened as candidate hub genes by calculating gene connectivity in the corresponding network. Only 30 hub genes were amplifiable with RT-qPCR, of which 27 were up-regulated upon S. scitamineum infection. The results were consistent with the trend of gene expression in RNA-Seq, suggesting their positive roles in smut resistance. Interestingly, the expression levels of AOX, Cyb5, and LAC were higher in ROC22 than in YT93-159, indicating these three genes may act as negative regulators in response to S. scitamineum infection. This study revealed the transcriptome dynamics in sugarcane challenged by S. scitamineum infection and provided gene targets for smut resistance breeding in sugarcane.

Quantitative Trait Loci Mapping and Development of KASP Marker Smut Screening Assay Using High-Density Genetic Map and Bulked Segregant RNA Sequencing in Sugarcane (Saccharum spp.).

Sugarcane is one of the most important industrial crops globally. It is the second largest source of bioethanol, and a major crop for biomass-derived electricity and sugar worldwide. Smut, caused by Sporisorium scitamineum, is a major sugarcane disease in many countries, and is managed by smut-resistant varieties. In China, smut remains the single largest constraint for sugarcane production, and consequently it impacts the value of sugarcane as an energy feedstock. Quantitative trait loci (QTLs) associated with smut resistance and linked diagnostic markers are valuable tools for smut resistance breeding. Here, we developed an F1 population (192 progeny) by crossing two sugarcane varieties with contrasting smut resistance and used for genome-wide single nucleotide polymorphism (SNP) discovery and mapping, using a high-throughput genotyping method called “specific locus amplified fragment sequencing (SLAF-seq) and bulked-segregant RNA sequencing (BSR-seq). SLAF-seq generated 148,500 polymorphic SNP markers. Using SNP and previously identified SSR markers, an integrated genetic map with an average 1.96 cM marker interval was produced. With this genetic map and smut resistance scores of the F1 individuals from four crop years, 21 major QTLs were mapped, with a phenotypic variance explanation (PVE) > 8.0%. Among them, 10 QTLs were stable (repeatable) with PVEs ranging from 8.0 to 81.7%. Further, four QTLs were detected based on BSR-seq analysis. aligning major QTLs with the genome of a sugarcane progenitor Saccharum spontaneum, six markers were found co-localized. Markers located in QTLs and functional annotation of BSR-seq-derived unigenes helped identify four disease resistance candidate genes located in major QTLs. 77 SNPs from major QTLs were then converted to Kompetitive Allele-Specific PCR (KASP) markers, of which five were highly significantly linked to smut resistance. The co-localized QTLs, candidate resistance genes, and KASP markers identified in this study provide practically useful tools for marker-assisted sugarcane smut resistance breeding.

Genome Characteristics Reveal the Biocontrol Potential of Actinobacteria Isolated From Sugarcane Rhizosphere.

To understand the beneficial interaction of sugarcane rhizosphere actinobacteria in promoting plant growth and managing plant diseases, this study investigated the potential role of sugarcane rhizospheric actinobacteria in promoting plant growth and antagonizing plant pathogens. We isolated 58 actinobacteria from the sugarcane rhizosphere, conducted plant growth-promoting (PGP) characteristics research, and tested the pathogenic fungi in vitro. Results showed that BTU6 (Streptomyces griseorubiginosus), the most representative strain, regulates plant defense enzyme activity and significantly enhances sugarcane smut resistance by regulating stress resistance-related enzyme (substances (POD, PAL, PPO, TP) in sugarcane) activity in sugarcane. The genomic evaluation indicated that BTU6 has the ability to biosynthesize chitinase, β-1,3-glucanase, and various secondary metabolites and plays an essential role in the growth of sugarcane plants under biotic stress. Potential mechanisms of the strain in improving the disease resistance of sugarcane plants and its potential in biodegrading exogenous chemicals were also revealed. This study showed the importance of sugarcane rhizosphere actinobacteria in microbial ecology and plant growth promotion.

Sugarcane Smut: Current Knowledge and the Way Forward for Management.

Whip smut of sugarcane is the most serious and widely spread disease of sugarcane and causes a significant reduction in cane quantity and quality. The severity of this disease often depends on the pathogen races, environmental conditions, cultivar genotype and the interaction among these three factors. Under optimum climatic conditions, this disease has the potential to cause total crop failure. Resistance screening is an ongoing process due to the variability among smut pathogen isolates. Multiple races and mutation ability of smut pathogen makes the breeding task more complex. A number of studies on various aspects of the disease epidemiology and management have been published. Due to many overlapping characteristics within the species complex, there is a dearth of information on early detection and strategies to control the smut pathogen. Furthermore, there is a need to coordinate these findings to expedite its research and control. In this paper, we summarize the disease etiology, especially disease impact on the qualitative and quantitative parameters of sugarcane. We also gathered research progress on molecular-based detection and available information on genetic variability in S. scitamineum. The research on the set of management options needed to effectively cope with the disease are reviewed herein. The present review is expected to be helpful for the further investigation on smut resistance in sugarcane.

English

Sugarcane smut caused by Sporisorium scitanmineumis the most severe sugarcane disease that causes major economic losses in sugarcane production in China, and disease resistance breeding is an important way of preventing and controlling this disease. In this study, BC3F1lines derived from the cross between YC 73-226 and YCE 06-111 were used to generate sugarcane smut-resistant and -susceptible gene pools using bulked segregant analysis (BSA). Eighty-nine random primers of start codon targeted (SCoT) polymorphisms were screened, whereas only primer SCoT44 could stably amplify the specific fragment (HE-Ss44) in the resistant pool. Then, several primer pairs of sequence characterized amplified regions (SCARs) were designed based on the sequence alignment of HE-Ss44 (920 bp), which was recovered after purification, and only one pair of SCAR primers (Ss44-F2/R2, forward: 5'-GGCGGGCACCGTCGAGTCCACAT-3'; reverse: 5'-CCGTCCGTCGG TCTCGTCCTTACG-3') could stably amplify a 400-bp specific band in resistant gene pool and its individuals. A validation test of SCAR marker Ss44-F2/R2 was performed using 34 sugarcane cultivars with known smut resistance, which revealed a selection accuracy of 82.35% between marker detection and known smut resistance. Moreover, Pearson’s correlation analysis also showed that the SCAR marker Ss44-F2/R2 was significantly correlated (r= 0.583, P= 0.0003 &lt; 0.01) with the smut resistance trait in sugarcane. In addition, the nucleotide sequence of HE-Ss44 linked with smut-resistancewas not aligned to the homologous sequence in GenBank (NCBI), and the accession number was MG740763. The SCAR marker Ss44-F2/R2 developed in this study can be used for the rapid detection of smut resistance in sugarcane and may be utilized as reference for the improvement of sugarcane smut resistance based on molecular marker-assisted selection.© 2021 Friends Science Publishers

Physiological Mechanisms of Improved Smut Resistance in Sugarcane Through Application of Silicon.

Sugarcane smut caused by Sporisorium scitamineum is a severe, global sugarcane disease with severe economic losses and is difficult to prevent. To explore more effective control techniques for smut, the effects and physiological mechanism of silicon (Si) on smut resistance in two smut-susceptible cultivars, ROC22 and Badila, were investigated. The results show that Si application significantly enhances smut resistance in ROC22 and Badila, and the incidence of sugarcane smut decreased by 11.57–22.58% (ROC22) and 27.75–46.67% (Badila). The incidence of smut is negatively correlated with the amount of Si applied and the Si content in sugarcane leaves, stems, and roots (highly significantly negatively correlated with stem Si content). Under S. scitamineum stress, the activities of pathogenesis-related enzymes, chitinase and β-1,3-glucanase, secondary metabolism-related enzymes such as polyphenoloxidase (PPO) and phenylalanine-ammonia-lyase (PAL), and the contents of secondary metabolites, total soluble phenol, and lignin in sugarcane leaves treated with Si were significantly higher than those without Si (CK). The results also demonstrated that the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2), the superoxide dismutase (SOD) activity of sugarcane leaves treated with Si increased in the seedling and tillering stages, and the peroxidase (POD) activity decreased in the seedling stage, which caused the accumulation of reactive oxygen species (ROS) that in turn triggered defense responses. Moreover, MDA and H2O2 levels decreased, and the activities of SOD and POD increased at the jointing stage, which was beneficial to the removal of excessive ROS. Collectively, these results suggest that Si modulates pathogenesis-related protein activity, secondary metabolism, and active oxygen metabolism of sugarcane that positively regulate resistance to smut. This study is the first to reveal the physiological mechanism of Si in improving smut resistance in sugarcane, and the results provide a theoretical basis for the development of Si fertilizers to control sugarcane smut.

Proteomic Analysis of the Resistance Mechanisms in Sugarcane during Sporisorium scitamineum Infection.

Smut disease is caused by Sporisorium scitamineum, an important sugarcane fungal pathogen causing an extensive loss in yield and sugar quality. The available literature suggests that there are two types of smut resistance mechanisms: external resistance by physical or chemical barriers and intrinsic internal resistance mechanisms operating at host–pathogen interaction at cellular and molecular levels. The nature of smut resistance mechanisms, however, remains largely unknown. The present study investigated the changes in proteome occurring in two sugarcane varieties with contrasting susceptibility to smut—F134 and NCo310—at whip development stage after S. scitamineum infection. Total proteins from pathogen inoculated and uninoculated (control) leaves were separated by two-dimensional gel electrophoresis (2D-PAGE). Protein identification was performed using BLASTp and tBLASTn against NCBI nonredundant protein databases and EST databases, respectively. A total of thirty proteins spots representing differentially expressed proteins (DEPs), 16 from F134 and 14 from NCo310, were identified and analyzed by MALDI-TOF/TOF MS. In F134, 4 DEPs were upregulated and nine were downregulated, while, nine were upregulated and three were downregulated in NCo310. The DEPs were associated with DNA binding, metabolic processes, defense, stress response, photorespiration, protein refolding, chloroplast, nucleus and plasma membrane. Finally, the expression of CAT, SOD, and PAL with recognized roles in S. scitamineum infection in both sugarcane verities were analyzed by real-time quantitative PCR (RT-qPCR) technique. Identification of genes critical for smut resistance in sugarcane will increase our knowledge of S. scitamineum-sugarcane interaction and help to develop molecular and conventional breeding strategies for variety improvement.

Identification of novel QTLs controlling sugarcane smut resistance and yield traits

Sugarcane yield depends upon various agro-morphological traits, viz., sugar recovery, stalk number, cane girth, cane height and smut resistance. Identification of Quantitative Traits Loci (QTLs) controlling these traits could greatly help sugarcane breeders in marker-assisted selection of sugarcane lines for various breeding programs. Structure and TASSEL software based integration of genotypic and phenotypic data of 103 sugarcane genotypes was resulted in the identification of eighty seven (87) highly associated alleles (p≤0.05), 34 alleles with smut resistance: 27 alleles with sugar recovery: 13 alleles with cane weight and 20 alleles with each of cane girth and height. The phenotypic variance (R2-values) explained by these linked alleles ranged 3.1-24.6% for smut resistance, 2.67- 22.5% for sugar recovery, 2.81-23.46% for cane height, 2.9-14.34% of cane weight and 1.75-12.8% for cane girth. The varying proportions of phenotypic variance explained by these linked alleles indicated that these traits were controlled by additive genetic effects of multiple genes. It also shows that these traits are the genuine quantitative traits. Moreover, the alleles depicting maximum degree of association for sugar recovery (51-131), cane girth (82-184), cane height (52-121), cane weight and smut resistance (51-145 & 51-146) could help in marker assisted selection of sugarcane lines for these traits.

Putative orthologs of Ustilago maydis effectors screened from the genome of sugarcane smut fungus - Sporisorium scitamineum

Smut caused by Sporisorium scitamineum is one of the most economically important diseases of sugarcane. In order to comprehend smut resistance in sugarcane, pathogenic determinants of this intriguing smut fungus need to be identified. However, the knowledge on molecular mechanisms behind its pathogenicity is inadequate and none of the predicted putative effectors of this smut fungus has been functionally characterised. Based on the established close genomic synteny between S. scitamineum and Ustilago maydis, in this preliminary study, we have identified putative orthologs of functionally characterised U. maydis effectors viz., Pep1, Cmu1, Tin2, Srt1, Stp1 and Pit1 in S. scitamineum genome, using blast - based searches. Further, in silico analysis revealed that the identified candidate S. scitamineum orthologs share higher sequence identity with Sporisorium reilianum than U. maydis sequences. Signal peptides, transmembrane and other conserved domains predicted in the identified putative S. scitamineum effector orthologs were consistent with the reported U. maydis effectors. In addition, most of the identified putative effector orthologs of S. scitamineum also shared considerable homology with the sequences of other closely-related smut fungi and codon sites with evidence of positive selection and episodic diversifying selection were detected in a few sequences. Temporal gene expression analysis revealed that transcript expression of the identified putative S. scitamineum effector orthologs increased significantly after 3 days post inoculation and was expressed in distinct patterns throughout the infection cycle. The results of this study also indicate that these candidate effectors could be conserved among other closely - related smut fungi.

Development of an RAPD-based SCAR marker for smut disease resistance in commercial sugarcane cultivars of Pakistan

A 300-bp RAPD-derived Sequence Characterized Amplified Region (SCAR) marker linked with resistance to sugarcane smut disease was developed. Bulked segregant analysis and RAPD were conducted using 480 random decamers in the initial screening of 12 sugarcane cultivars (two completely smut resistant, two completely smut susceptible, four moderately smut resistant, and four moderately smut susceptible). Eighty-four of these primers proved to be polymorphic while only one (B17) produced a reproducible polymorphic fragment, which appeared to co-segregate in repulsion with sugarcane smut resistance. A 300-bp B1SM-F/B1SM-R SCAR marker was designed based on the sequence alignment of the B17 products. Testing of this SCAR marker on these12 cultivars confirmed its specificity on two completely and four partially resistant cultivars. Further screening of this SCAR marker on seven additional sugarcane cultivars of Pakistan verified the earlier findings. Therefore, it was deduced that the B1SM-F/B1SM-R SCAR marker is linked with smut resistance trait in sugarcane. NCBI blast of the SCAR marker sequence showed no homology to any of the earlier identified sequences in GenBank. The newly discovered B1SM-F/B1SM-R SCAR marker for sugarcane smut resistance will facilitate sugarcane breeding programs since it can be used for marker-assisted selection in sugarcane.

Comparative proteomics reveals that central metabolism changes are associated with resistance against Sporisorium scitamineum in sugarcane.

BackgroundSugarcane smut, which is caused by Sporisorium scitamineum, has been threatening global sugarcane production. Breeding smut resistant sugarcane varieties has been proven to be the most effective method of controlling this particular disease. However, a lack of genome information of sugarcane has hindered the development of genome-assisted resistance breeding programs. Furthermore, the molecular basis of sugarcane response to S. scitamineum infection at the proteome level was incomplete and combining proteomic and transcriptional analysis has not yet been conducted.ResultsWe identified 273 and 341 differentially expressed proteins in sugarcane smut-resistant (Yacheng05-179) and susceptible (ROC22) genotypes at 48 h after inoculation with S. scitamineum by employing an isobaric tag for relative and absolute quantification (iTRAQ). The proteome quantitative data were then validated by multiple reaction monitoring (MRM). The integrative analysis showed that the correlations between the quantitative proteins and the corresponding genes that was obtained in our previous transcriptome study were poor, which were 0.1502 and 0.2466 in Yacheng05-179 and ROC22, respectively, thereby revealing a post-transcriptional event during Yacheng05-179-S. scitamineum incompatible interaction and ROC22-S. scitamineum compatible interaction. Most differentially expressed proteins were closely related to sugarcane smut resistance such as beta-1,3-glucanase, peroxidase, pathogenesis-related protein 1 (PR1), endo-1,4-beta-xylanase, heat shock protein, and lectin. Ethylene and gibberellic acid pathways, phenylpropanoid metabolism and PRs, such as PR1, PR2, PR5 and PR14, were more active in Yacheng05-179, which suggested of their possible roles in sugarcane smut resistance. However, calcium signaling, reactive oxygen species, nitric oxide, and abscisic acid pathways in Yacheng05-179 were repressed by S. scitamineum and might not be crucial for defense against this particular pathogen.ConclusionsThese results indicated complex resistance-related events in sugarcane-S. scitamineum interaction, and provided novel insights into the molecular mechanism underlying the response of sugarcane to S. scitamineum infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3146-8) contains supplementary material, which is available to authorized users.

Early Selection for Smut Resistance in Sugarcane Using Pathogen Proliferation and Changes in Physiological and Biochemical Indices.

Sugarcane smut disease, caused by Sporisorium scitamineum, significantly decreases yield and use of resistant cultivars is the most cost-effective measure for disease control. Current field testing methods for identification of smut resistance are time-consuming and hindered by environmental variability. Our goal was to develop an efficient and reliable resistance identification technique that is rapid, performed in a controlled environment, and stable. Nine sugarcane cultivars with different phenotypic resistance levels were selected. TaqMan quantitative real-time polymerase chain reaction analysis was performed to measure copy number changes of smut pathogen in sugarcane buds at 0–7 days after needle puncture inoculation. There was a positive correlation between time after inoculation and the amount of smut pathogen in the sugarcane bud. This reached a peak value on 7 days, and the copy number of S. scitamineum increased in the following order: YZ03-258, FN40, YZ01-1413, GT02-467, ROC22, YT96-86, YZ03-103, FN39, LC05-136. After smut pathogen inoculation, differences in the physiological and biochemical indices of the nine cultivars were observed. Peroxidase, ascorbate peroxidase, catalase, superoxide dismutase, β-1,3-glucanase, and malondialdehyde were grouped into three main components, and the cumulative contribution rate was 80.177%, revealing that these are useful physiological and biochemical indicators of smut resistance. Subordinate function analysis indicated that the levels of smut resistance of the nine genotypes were (high to low): YZ03-258, FN40, YZ01-1413, GT02-467, ROC22, YZ03-103, YT96-86, FN39, LC05-136, which is similar to the results from copy number determination of smut pathogens. The results suggest that after artificial needle inoculation, rapid identification of physiological resistance to sugarcane smut was achieved based on copy number increases in the sugarcane smut pathogen and the physiological and biochemical changes in sugarcane bud during the early phase of infection.

Molecular variation of Sporisorium scitamineum in Mainland China revealed by internal transcribed spacers

Sugarcane smut caused by the fungus Sporisorium scitamineum is a worldwide disease and also one of the most prevalent diseases in sugarcane production in mainland China. To study molecular variation in S. scitamineum, 23 S. scitamineum isolates from the 6 primary sugar-cane production areas in mainland, China (Guangxi, Yunnan, Guangdong, Hainan, Fujian, and Jiangxi Provinces), were assessed using internal transcribed spacer (ITS) methods. The results of ITS sequence analysis showed that the organisms can be defined at the genus level, including Ustilago and Sporisorium, and can also differentiate between closely related species. This method was not suitable for phylogenetic relationship analysis of different S. scitamineum isolates and could not provide support regarding their race ascription at the molecular level. The results of the present study will be useful for studies examining the molecular diversity of S. scitamineum and for establishing a genetic foundation for their pathogenicity differentiation and new race detection. In addition, our results can provide useful information for the pathogen selection principle in sugarcane smut resistance breeding and variety distribution.

ScChi, encoding an acidic class III chitinase of sugarcane, confers positive responses to biotic and abiotic stresses in sugarcane.

Chitinases (EC 3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank accession no. KF664180), a Class III chitinase gene, encoded a 31.37 kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Real-time quantitative PCR (RT-qPCR) results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with Sporisorium scitamineum. The ScChi also showed an obvious induction of transcription after treatment with SA (salicylic acid), H2O2, MeJA (methyl jasmonate), ABA (abscisic acid), NaCl, CuCl2, PEG (polyethylene glycol) and low temperature (4 °C). The expression levels of ScChi and six immunity associated marker genes were upregulated by the transient overexpression of ScChi. Besides, histochemical assay of Nicotiana benthamiana leaves overexpressing pCAMBIA 1301-ScChi exhibited deep DAB (3,3′-diaminobenzidinesolution) staining color and high conductivity, indicating the high level of H2O2 accumulation. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane.