Analysis of the resistance mechanisms in sugarcane during Sporisorium scitamineum infection using RNA-seq and microscopy.

Smut caused by biotrophic fungus Sporisorium scitamineum is a major disease of cultivated sugarcane that can cause considerable yield losses. It has been suggested in literature that there are at least two types of resistance mechanisms in sugarcane plants: an external resistance, due to chemical or physical barriers in the sugarcane bud, and an internal resistance governed by the interaction of plant and fungus within the plant tissue. Detailed molecular studies interrogating these two different resistance mechanisms in sugarcane are scarce. Here, we use light microscopy and global expression profiling with RNA-seq to investigate these mechanisms in sugarcane cultivar CP74-2005, a cultivar that possibly possesses both internal and external defence mechanisms. A total of 861 differentially expressed genes (DEGs) were identified in a comparison between infected and non-infected buds at 48 hours post-inoculation (hpi), with 457 (53%) genes successfully annotated using BLAST2GO software. This includes genes involved in the phenylpropanoid pathway, cell wall biosynthesis, plant hormone signal transduction and disease resistance genes. Finally, the expression of 13 DEGs with putative roles in S. scitamineum resistance were confirmed by quantitative real-time reverse transcription PCR (qRT-PCR) analysis, and the results were consistent with the RNA-seq data. These results highlight that the early sugarcane response to S. scitamineum infection is complex and many of the disease response genes are attenuated in sugarcane cultivar CP74-2005, while others, like genes involved in the phenylpropanoid pathway, are induced. This may point to the role of the different disease resistance mechanisms that operate in cultivars such as CP74-2005, whereby the early response is dominated by external mechanisms and then as the infection progresses, the internal mechanisms are switched on. Identification of genes underlying resistance in sugarcane will increase our knowledge of the sugarcane-S. scitamineum interaction and facilitate the introgression of new resistance genes into commercial sugarcane cultivars.

Confirmation that the Novel Cercozoa Phytocercomonas venanatans Is the Cause of the Disease Chlorotic Streak in Sugarcane.

A cercomonad, named Phytocercomonas venanatans, is confirmed as the cause of the sugarcane disease chlorotic streak. This was achieved by establishing aseptic liquid cultures of the pathogen isolated from internal pieces of sugarcane stalk tissue. Actively motile cultures of the pathogen were inoculated into sugarcane roots, stalks, and leaf whorls. Infected plants subsequently developed the characteristic symptoms of chlorotic streak. Infection was confirmed by PCR screening of plant tissues and by reisolation of the pathogen into aseptic culture followed by PCR and microscopic confirmation. P. venanatans is the first reported pathogenic cercomonad able to systemically infect higher plants and the first plant pathogenic cercozoan able to be successfully grown in axenic culture on common microbiological media.

Phytocercomonas venanatans, a New Species of Cercozoa Associated with Chlorotic Streak of Sugarcane.

Chlorotic streak is a global disease of commercial sugarcane (Saccharum spp. hybrids). The disease is transmitted by wet soil, water, as well as in diseased planting material. Although first recognized almost 90 years ago and despite significant research effort, the identity of the causal agent has been elusive. Metagenomic high throughput sequencing (HTS) facilitated the discovery of novel protistan ribosomal and nuclear genes in chlorotic streak-infected sugarcane. These sequences suggest a possible causal agent belonging to the order Cercomonadida (Rhizaria, phylum Cercozoa). An organism with morphological features similar to cercomonads (=Cercomonadida) was isolated into pure axenic culture from internal stalk tissues of infected sugarcane. The isolated organism contained DNA sequences identical to those identified in infected plants by HTS. The DNA sequences and the morphology of the organism did not match any known species. Here we present a new genus and species, Phytocercomonas venanatans, which is associated with chlorotic streak of sugarcane. Amplicon sequencing also supports that P. venanatans is associated with this disease. This is the first reported member from Cercomonadida showing a probable pathogenic association with higher plants.

New Method of Controlling Sugarcane Smut using Flutriafol Fungicide.

Sugarcane smut, caused by Sporisorium scitamineum, is one of the most devastating diseases of sugarcane worldwide. Triazole fungicides such as propiconazole and triadimefon have been routinely used as dip treatments to protect seed-cane (stalk cuttings) from infection by sugarcane smut fungus. However, dip treatments can be applied only to mother stock (nursery) planting materials because of the logistics of dipping large quantities of seed-cane and it is difficult to dispose of the large volumes of waste fungicide solution in an environmentally safe manner. There was a need for a new fungicide that can be applied using more practical methods. The efficacy of flutriafol fungicide to control smut was evaluated in inoculated and infected stalks of a highly susceptible cultivar using various application methods. In a 2007 experiment, flutriafol fungicide was equally effective or better in controlling sugarcane smut as a dip than were two fungicides, propiconazole and triadimefon, registered in Australia. In 2009, two experiments determined the effectiveness of flutriafol when applied by mixing with fertilizer or spraying on the seed-cane at planting. All fungicide treatments significantly suppressed smut in one experiment but, in the second experiment, flutriafol-fertilizer mix treatments were ineffective. In 2010, two experiments verified the efficacy and effective rates of flutriafol against sugarcane smut when applied through existing spray equipment designed to spray fungicide on cuttings as they drop into the planting furrow on a commercial planter. Area under the disease progress curve values of smut incidence were reduced significantly to 47 and 56% with low (125 g a.i. ha-1) and high (375 g a.i. ha-1) application rates, respectively, in both experimental sites compared with the inoculated control plots. Sugar yield increased by 46 to 65% in one experiment and 157 to 203% in the second experiment compared with the inoculated control. This research formulated a more practical method of application of flutriafol and suggests greater applications of this fungicide for the management of sugarcane smut.

Pathogenic Variation in Spore Populations of Sporisorium scitamineum, Causal Agent of Sugarcane Smut in Australia.

An incursion of sugarcane smut in Queensland was identified in 2006 in Bundaberg, Ingham, and Mackay. In 2008, two cultivars, 'Q205' and 'Q170', were highly susceptible in Bundaberg but remained disease free on a heavily infested farm in Mackay. A glasshouse experiment was established to determine whether the differences in disease expression were due to the sources of the two cultivars sourced from Bundaberg and Mackay in 2009. These were inoculated with the Mackay population of Sporisorium scitamineum spores. These cultivars had the same levels of disease, indicating that the variation in the clonal source of the cultivars was not responsible for the observed differences. A second glasshouse experiment in 2013 confirmed that highly susceptible Q205 and Q170 were resistant to the Mackay population of S. scitamineum but remained susceptible to the Bundaberg population. In 2010, S. scitamineum populations of smut fungi from Mackay and Bundaberg were compared in a field trial and the Mackay population had significantly less disease than the Bundaberg population on four cultivars (Q170, Q205, 'Q174', and 'Q138') but had significantly more disease in 'Q188'. These results confirmed the field observations and suggested that there was a differential response among the cultivars for the smut fungi collected from Mackay compared with Bundaberg and are genetically different. This finding suggests that a mixture of spores should be used to inoculate sugarcane clones for resistance screening.

Variation in acquisition of Fiji disease virus by Perkinsiella saccharicida (Hemiptera: Delphacidae).

Fiji leaf gall, caused the Fiji disease virus (genus Fijivirus, family Reoviridae, FDV), is a serious disease of sugarcane, Saccharum officinarum L., in Australia and several other Asia-Pacific countries. In Australia FDV is transmitted only by the planthopper Perkinsiella saccharicida Kirkaldy (Hemiptera: Delphacidae), in a propagative manner. Successful transmission of FDV by single planthoppers confined to individual virus free plants is highly variable, even under controlled conditions. The research reported here addresses two possible sources of this variation: (1) gender, wing form, and life stage of the planthopper; and (2) genotype of the source plant. The acquisition of FDV by macropterous males, macropterous females, brachypterous females, and nymphs of P. saccharicida from infected plants was investigated using reverse transcription-polymerase chain reaction to diagnose FDV infection in the vector. The proportion of individuals infected with FDV was not statistically related to life stage, gender, or adult wing form of the vector. The acquisition of FDV by P. saccharicida from four cultivars of sugarcane was compared to assess the influence of plant genotype on acquisition. Those planthopper populations reared on diseased 'NCo310' plants had twice as many infected planthoppers as those reared on 'Q110', 'WD1', and 'WD2'. Therefore, variation in FDV acquisition in this system is not the result of variation in the gender, wing form and life stage of the P. saccharicida vectors. The cultivar used as the source plant to rear vector populations does affect the proportion of infected planthoppers in a population.

Genetic variability of genome segments 3 and 9 of Fiji disease virus field isolates.

Fiji leaf gall is an important disease of sugarcane in Australia and other Asia-Pacific countries. The causative agent is the reovirus Fiji disease virus (FDV). Previous reports indicate that there is variation in pathology between virus isolates. To investigate the amount of genetic variation found in FDV, 25 field isolates from Australia, Papua New Guinea and Malaysia were analysed by partial sequencing of genome segments S3 and S9. There was up to 15% divergence in the nucleotide sequence among the 25 isolates. A similar amount of divergence and pattern of relationships was found for each of the two genomic segments for most of the field isolates, although reassortment of genome segments seems likely for at least one of the Papua New Guinean isolates. The finding of a high level of variation in FDV isolated in different regions has implications for quarantine and disease management.