Draft Genome Sequence Resource for the Orange Rust Pathogen of Sugarcane Puccinia kuehnii.

Puccinia kuehnii is an obligate biotrophic fungal pathogen that causes orange rust of sugarcane, which is prevalent in many countries around the globe. In the United States, orange rust was first detected in sugarcane in Florida in 2007 and poses a persistent and economically damaging threat to the sugarcane industry in this region. Here, we generated the first genome assemblies for two isolates of P. kuehnii (1040 and 2143) collected in Florida in 2017 from two sugarcane cultivars, CL85-1040 and CP89-2143, respectively. These two rust genome resources will be of immense value for future genomic studies, particularly further exploration of the predicted secretomes that may help define key pathogenicity determinants for this economically important pathogen.

Comparison of Progress of Brown Rust and Orange Rust and Conditions Conducive for Severe Epidemic Development During the Sugarcane Crop Season in Florida.

Brown rust (caused by Puccinia melanocephala) and orange rust (caused by P. kuehnii) are two major diseases of sugarcane in Florida. To better understand the epidemiology of these two rusts, disease severity and weather variables were monitored for two seasons in cultivars CL90-4725 (susceptible to brown rust and resistant to orange rust) and CL85-1040 (susceptible to orange rust and resistant to brown rust). Brown rust was most severe during mid-May to mid-July, whereas orange rust severity peaked during two periods: mid-May to early August and then November to December. Overall, disease severity was higher for orange rust than for brown rust. Maximum disease severity was correlated with the number of hours at night with an average temperature of 20 to 22.2°C for brown rust one season and orange rust both seasons. Slightly higher correlation was obtained when relative humidity above 90% was included in the number of hours at night with an average temperature of 20 to 22.2°C for brown rust but not orange rust, suggesting that leaf wetness is not a limiting factor for either disease in Florida. Epidemics of brown rust began at lower night temperatures (16.7 to 22.2°C) in one season, but epidemics of orange rust lasted longer under higher temperatures. The correlation of rust severity on recently emerged leaves with conducive temperatures recorded in 10-, 20-, or 30-day windows starting 7 days before disease assessment suggested that earlier inoculum production is needed to create severe epidemics that result in yield loss.

Occurrence of a novel mastrevirus in sugarcane germplasm collections in Florida, Guadeloupe and Réunion.

BACKGROUND: In Africa and Asia, sugarcane is the host of at least seven different virus species in the genus Mastrevirus of the family Geminiviridae. However, with the exception of Sugarcane white streak virus in Barbados, no other sugarcane-infecting mastrevirus has been reported in the New World. Conservation and exchange of sugarcane germplasm using stalk cuttings facilitates the spread of sugarcane-infecting viruses. METHODS: A virion-associated nucleic acids (VANA)-based metagenomics approach was used to detect mastrevirus sequences in 717 sugarcane samples from Florida (USA), Guadeloupe (French West Indies), and Réunion (Mascarene Islands). Contig assembly was performed using CAP3 and sequence searches using BLASTn and BLASTx. Mastrevirus full genomes were enriched from total DNA by rolling circle amplification, cloned and sequenced. Nucleotide and amino acid sequence identities were determined using SDT v1.2. Phylogenetic analyses were conducted using MEGA6 and PHYML3. RESULTS: We identified a new sugarcane-infecting mastrevirus in six plants sampled from germplasm collections in Florida and Guadeloupe. Full genome sequences were determined and analyzed for three virus isolates from Florida, and three from Guadeloupe. These six genomes share >88% genome-wide pairwise identity with one another and between 89 and 97% identity with a recently identified mastrevirus (KR150789) from a sugarcane plant sampled in China. Sequences similar to these were also identified in sugarcane plants in Réunion. CONCLUSIONS: As these virus isolates share <64% genome-wide identity with all other known mastreviruses, we propose classifying them within a new mastrevirus species named Sugarcane striate virus. This is the first report of sugarcane striate virus (SCStV) in the Western Hemisphere, a virus that most likely originated in Asia. The distribution, vector, and impact of SCStV on sugarcane production remains to be determined.

Morphological and Physiological Responses of Sugarcane to Leifsonia xyli subsp. xyli Infection.

Ratoon stunt, caused by the bacterium Leifsonia xyli subsp. xyli, is one of the major sugarcane diseases worldwide. The objectives of this study were to determine the variation in morphology and DNA sequence of L. xyli subsp. xyli strains isolated in China, to compare the changes that occurred in vascular ultrastructure and levels of endogenous hormone abscisic acid (ABA), auxins (indoleacetic acid [IAA]), and gibberellic acids (GA3) in sugarcane stalks. Experiments were also conducted with two sugarcane varieties, 'ROC22' and 'Badila', in the greenhouse to understand the cytological and physiological mechanisms of L. xyli subsp. xyli-induced growth stunting. There were three treatments in the experiments: (i) healthy plants (L. xyli subsp. xyli-free plants), (ii) infected plants (L. xyli subsp. xyli-infected seedcanes treated with hot water, and (iii) infected plants (healthy seedcanes dipped in L. xyli subsp. xyli cell culture). The results showed that sequence coverage of a locally isolated strain, LxxGXBZ01, was 99.99%, and the average nucleotide identity between LxxGXBZ01 and the other well-characterized Brazilian isolate LxxCTCB07 was 93.61%. LxxGXBZ01 occurred in different sizes and shapes in xylem vessels of infected plants. In comparison with healthy stalks, the secondary walls of the vessel element in L. xyli subsp. xyli-infected stalks were degraded with uneven wall thickness, deformities, sticky substances, and electron-dense substances accumulated inside the cells. Compared with the healthy and hot-water treatments, the contents of IAA and GA3 were significantly lower, while that of ABA was significantly higher in the L. xyli subsp. xyli-infected stalks. The information obtained in this study will expand our understanding of ratoon stunt etiology and cytological and physiological bases of the disease manifestation.

Orange Rust Effects on Leaf Photosynthesis and Related Characters of Sugarcane.

Orange rust of sugarcane (Saccharum spp. hybrids), caused by Puccinia kuehnii, is a relatively new disease in the Western Hemisphere that substantially reduces yields in susceptible sugarcane genotypes. The objective of this study was to determine the physiological mechanisms of orange rust-induced reductions in sugarcane growth and yield by quantifying effects of the disease on leaf SPAD index (an indication of leaf chlorophyll content), net photosynthetic rate, dark respiration, maximum quantum yield of CO2 assimilation, carbon fixation efficiency, and the relationships between these leaf photosynthetic components and rust disease ratings. Plants growing in pots were inoculated with the orange rust pathogen using a leaf whorl inoculation method. A disease rating was assigned using a scale from 0 to 4 with intervals of 0.5. At disease ratings ≥2, the rust-infected leaf portion of inoculated plants showed significant reductions in SPAD index, maximum quantum yield, carbon fixation efficiency, stomatal conductance, leaf transpiration rate, and net photosynthetic rate; but the rusted portion of the infected leaves had increased intercellular CO2 concentration and leaf dark respiration rate. Although leaf SPAD index, photosynthetic rate, stomatal conductance, and transpiration rate at the rust-infected portion decreased linearly with increased rust rating, the effect of orange rust on photosynthetic rate was much greater than that on stomatal conductance and transpiration. Unlike earlier reports on other crops, reduction in leaf photosynthesis by orange rust under low light was greater than that under high light conditions. These results help improve the understanding of orange rust etiology and physiological bases of sugarcane yield loss caused by orange rust.

Leaf Whorl Inoculation Method for Screening Sugarcane Rust Resistance.

Brown rust, caused by Puccinia melanocephala, and orange rust, caused by P. kuehnii, are agronomically important diseases of sugarcane in Florida. Cultivar resistance is the best means of controlling these diseases. Natural infection has been the primary means of assessing resistance in sugarcane cultivars against rusts; unfortunately, natural infection is not always efficient in identifying resistant cultivars due to variable environmental conditions. Therefore, a more reliable screening method is needed to effectively select resistant genotypes. An inoculation technique was evaluated for identification of brown and orange rust resistance in sugarcane cultivars. Inoculations were performed in the field by placing a 0.5-ml urediniospore suspension in the leaf whorl of three individual sugarcane stalks per plant using a pipette. Symptoms developed on leaves of all the susceptible cultivars after 4 weeks, and appeared as a band of pustules. Plants were rated for their reaction to rust 4 weeks after inoculation. The optimum concentrations of inoculum for expression of brown and orange rust symptoms were determined. The most severe brown rust and orange rust symptoms were observed using inoculum containing 105 and 104 urediniospores/ml, respectively. Clones in several stages of the Canal Point breeding program were screened for their rust reaction by leaf whorl inoculation. The technique enabled rapid screening of a large number of cultivars in field plantings using a small amount of inoculum and limited man hours.

Isolation of nucleotide binding site-leucine rich repeat and kinase resistance gene analogues from sugarcane (Saccharum spp.).

BACKGROUND: Resistance gene analogues (RGAs) have been isolated from many crops and offer potential in breeding for disease resistance through marker-assisted selection, either as closely linked or as perfect markers. Many R-gene sequences contain kinase domains, and indeed kinase genes have been reported as being proximal to R-genes, making kinase analogues an additionally promising target. The first step towards utilizing RGAs as markers for disease resistance is isolation and characterization of the sequences. RESULTS: Sugarcane clone US01-1158 was identified as resistant to yellow leaf caused by the sugarcane yellow leaf virus (SCYLV) and moderately resistant to rust caused by Puccinia melanocephala Sydow & Sydow. Degenerate primers that had previously proved useful for isolating RGAs and kinase analogues in wheat and soybean were used to amplify DNA from sugarcane (Saccharum spp.) clone US-01-1158. Sequences generated from 1512 positive clones were assembled into 134 contigs of between two and 105 sequences. Comparison of the contig consensuses with the NCBI sequence database using BLASTx showed that 20 had sequence homology to nuclear binding site and leucine rich repeat (NBS-LRR) RGAs, and eight to kinase genes. Alignment of the deduced amino acid sequences with similar sequences from the NCBI database allowed the identification of several conserved domains. The alignment and resulting phenetic tree showed that many of the sequences had greater similarity to sequences from other species than to one another. CONCLUSION: The use of degenerate primers is a useful method for isolating novel sugarcane RGA and kinase gene analogues. Further studies are needed to evaluate the role of these genes in disease resistance.

Natural Allelic Variations in Highly Polyploidy Saccharum Complex.

Sugarcane (Saccharum spp.) is an important sugar and biofuel crop with high polyploid and complex genomes. The Saccharum complex, comprised of Saccharum genus and a few related genera, are important genetic resources for sugarcane breeding. A large amount of natural variation exists within the Saccharum complex. Though understanding their allelic variation has been challenging, it is critical to dissect allelic structure and to identify the alleles controlling important traits in sugarcane. To characterize natural variations in Saccharum complex, a target enrichment sequencing approach was used to assay 12 representative germplasm accessions. In total, 55,946 highly efficient probes were designed based on the sorghum genome and sugarcane unigene set targeting a total of 6 Mb of the sugarcane genome. A pipeline specifically tailored for polyploid sequence variants and genotype calling was established. BWA-mem and sorghum genome approved to be an acceptable aligner and reference for sugarcane target enrichment sequence analysis, respectively. Genetic variations including 1,166,066 non-redundant SNPs, 150,421 InDels, 919 gene copy number variations, and 1,257 gene presence/absence variations were detected. SNPs from three different callers (Samtools, Freebayes, and GATK) were compared and the validation rates were nearly 90%. Based on the SNP loci of each accession and their ploidy levels, 999,258 single dosage SNPs were identified and most loci were estimated as largely homozygotes. An average of 34,397 haplotype blocks for each accession was inferred. The highest divergence time among the Saccharum spp. was estimated as 1.2 million years ago (MYA). Saccharum spp. diverged from Erianthus and Sorghum approximately 5 and 6 MYA, respectively. The target enrichment sequencing approach provided an effective way to discover and catalog natural allelic variation in highly polyploid or heterozygous genomes.

Species-specific detection and identification of fusarium species complex, the causal agent of sugarcane pokkah boeng in China.

BACKGROUND: Pokkah boeng disease caused by the Fusarium species complex results in significant yield losses in sugarcane. Thus, the rapid and accurate detection and identification of the pathogen is urgently required to manage and prevent the spreading of sugarcane pokkah boeng. METHODS: A total of 101 isolates were recovered from the pokkah boeng samples collected from five major sugarcane production areas in China throughout 2012 and 2013. The causal pathogen was identified by morphological observation, pathogenicity test, and phylogenetic analysis based on the fungus-conserved rDNA-ITS. Species-specific TaqMan real-time PCR and conventional PCR methods were developed for rapid and accurate detection of the causal agent of sugarcane pokkah boeng. The specificity and sensitivity of PCR assay were also evaluated on a total of 84 isolates of Fusarium from China and several isolates from other fungal pathogens of Sporisorium scitamineum and Phoma sp. and sugarcane endophyte of Acremonium sp. RESULT: Two Fusarium species (F. verticillioides and F. proliferatum) that caused sugarcane pokahh boeng were identified by morphological observation, pathogenicity test, and phylogenetic analysis. Species-specific TaqMan PCR and conventional PCR were designed and optimized to target their rDNA-ITS regions. The sensitivity of the TaqMan PCR was approximately 10 pg of fungal DNA input, which was 1,000-fold over conventional PCR, and successfully detected pokkah boeng in the field-grown sugarcane. CONCLUSIONS/SIGNIFICANCE: This study was the first to identify two species, F. verticillioides and F. proliferatum, that were causal pathogens of sugarcane pokkah boeng in China. It also described the development of a species-specific PCR assay to detect and confirm these pathogens in sugarcane plants from mainland China. This method will be very useful for a broad range of research endeavors as well as the regulatory response and management of sugarcane pokkah boeng.

Promoting utilization of Saccharum spp. genetic resources through genetic diversity analysis and core collection construction.

Sugarcane (Saccharum spp.) and other members of Saccharum spp. are attractive biofuel feedstocks. One of the two World Collections of Sugarcane and Related Grasses (WCSRG) is in Miami, FL. This WCSRG has 1002 accessions, presumably with valuable alleles for biomass, other important agronomic traits, and stress resistance. However, the WCSRG has not been fully exploited by breeders due to its lack of characterization and unmanageable population. In order to optimize the use of this genetic resource, we aim to 1) genotypically evaluate all the 1002 accessions to understand its genetic diversity and population structure and 2) form a core collection, which captures most of the genetic diversity in the WCSRG. We screened 36 microsatellite markers on 1002 genotypes and recorded 209 alleles. Genetic diversity of the WCSRG ranged from 0 to 0.5 with an average of 0.304. The population structure analysis and principal coordinate analysis revealed three clusters with all S. spontaneum in one cluster, S. officinarum and S. hybrids in the second cluster and mostly non-Saccharum spp. in the third cluster. A core collection of 300 accessions was identified which captured the maximum genetic diversity of the entire WCSRG which can be further exploited for sugarcane and energy cane breeding. Sugarcane and energy cane breeders can effectively utilize this core collection for cultivar improvement. Further, the core collection can provide resources for forming an association panel to evaluate the traits of agronomic and commercial importance.