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Rust fungi (Pucciniales) are a diverse group of plant pathogens in natural and agricultural systems. They pose ongoing threats to the diversity of native flora and cause annual crop yield losses. Agricultural rusts are predominantly managed with fungicides and breeding for resistance, but new control strategies are needed on non-agricultural plants and in fragile ecosystems. RNA interference (RNAi) induced by exogenous double-stranded RNA (dsRNA) has promise as a sustainable approach for managing plant-pathogenic fungi, including rust fungi. We investigated the mechanisms and impact of exogenous dsRNA on rust fungi through in vitro and whole-plant assays using two species as models, Austropuccinia psidii (the cause of myrtle rust) and Coleosporium plumeriae (the cause of frangipani rust). In vitro, dsRNA either associates externally or is internalized by urediniospores during the early stages of germination. The impact of dsRNA on rust infection architecture was examined on artificial leaf surfaces. dsRNA targeting predicted essential genes significantly reduced germination and inhibited development of infection structures, namely appressoria and penetration pegs. Exogenous dsRNA sprayed onto 1-year-old trees significantly reduced myrtle rust symptoms. Furthermore, we used comparative genomics to assess the wide-scale amenability of dsRNA to control rust fungi. We sequenced genomes of six species of rust fungi, including three new families (Araucariomyceaceae, Phragmidiaceae, and Skierkaceae) and identified key genes of the RNAi pathway across 15 species in eight families of Pucciniales. Together, these findings indicate that dsRNA targeting essential genes has potential for broad-use management of rust fungi across natural and agricultural systems.

Following the detection of potato mop-top virus (PMTV) in New Zealand in 2018, three near-complete PMTV genomes (AS22, AS99, AS144) were assembled from soil samples taken from potato fields in Canterbury. Phylogenetic analysis revealed that these genomes form a distinct lineage, with limited genetic diversity, within the PMTV species. This analysis supports the hypothesis that these genomes share a common origin, possibly resulting from a single (or limited) incursion of PMTV into New Zealand. A single nucleotide polymorphism was identified in the region where a key diagnostic primer binds. The mismatch of the diagnostic primer has implications for the effectiveness of the Mumford diagnostic protocol currently recommended for use in New Zealand; we recommend that the alternative Pandey assay, for which no primer mismatch was detected, be validated and optimized for use on the viral genomes present in New Zealand.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. ‘CandidatusLiberibacter solanacearum’ is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid—a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the effects of genome reduction and genetic drift on the function ofCa. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyses the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate thatCLsoDHDPS is expressed inCa. L. solanacearum and expression is increased ∼2-fold in the insect host compared toin planta.CLsoDHDPS has increased aggregation propensity, implying mutations have destabilised the enzyme but are compensated for through elevated chaperone expression and a stabilised oligomeric state.CLsoDHDPS uses a ternary-complex kinetic mechanism, which is unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure ofCLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study reveals the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary association. We suggest that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.

Resistance to the pandemic strain of Austropuccinia psidii was identified in New Zealand provenance Leptospermum scoparium, Kunzea robusta, and K. linearis plants. Only 1 Metrosideros excelsa-resistant plant was found (of the 570 tested) and no resistant plants of either Lophomyrtus bullata or L. obcordata were found. Three types of resistance were identified in Leptospermum scoparium. The first two, a putative immune response and a hypersensitive response, are leaf resistance mechanisms found in other myrtaceous species while on the lateral and main stems a putative immune stem resistance was also observed. Both leaf and stem infection were found on K. robusta and K. linearis plants as well as branch tip dieback that developed on almost 50% of the plants. L. scoparium, K. robusta, and K. linearis are the first myrtaceous species where consistent infection of stems has been observed in artificial inoculation trials. This new finding and the first observation of significant branch tip dieback of plants of the two Kunzea spp. resulted in the development of two new myrtle rust disease severity assessment scales. Significant seed family and provenance effects were found in L. scoparium, K. robusta, and K. linearis: some families produced significantly more plants with leaf, stem, and (in Kunzea spp.) branch tip dieback resistance, and provenances provided different percentages of resistant families and plants. The distribution of the disease symptoms on plants from the same seed family, and between plants from different seed families, suggested that the leaf, stem, and branch tip dieback resistances were the result of independent disease resistance mechanisms.

Among the many responsibilities of the worldwide scientific community are advancing the knowledge base that underpins each scientific discipline, addressing the pressing scientific issues of the day (e.g., emerging infectious diseases, food security, and climate change), and perhaps most importantly, educating and training subsequent generations of scientists. Yet, around the globe, advances in scientific and communications technology, proliferation and mining of data, and increasing financial constraints of university systems have led to fundamental changes in our institutions of higher learning. Increasing emphasis on interdisciplinary and multidisciplinary approaches to problem solving in agriculture add to the complexity of providing robust preparation for the plant pathologists of the future. Thus, as the U.N. recognizes the year 2020 as the International Year of Plant Health, it is fair to ask if current approaches to graduate education in plant pathology are adequate to meet current and anticipated challenges and if the outcomes can be improved.

A new dsRNA virus from the oomycete

‘Candidatus Liberibacter solanacearum’ is an unculturable α-proteobacterium that is the causal agent of zebra chip disease of potato—a major problem in potato-growing areas, because it affects growth and yield. Developing effective treatments for ‘Ca. L. solanacearum’ has been hampered by the difficulty in functionally characterizing the proteins of this organism, largely because they are not easily expressed and purified in standard expression systems. ‘Ca. L. solanacearum’ has a reduced genome and its proteins are predicted to be prone to instability and aggregation. Among intracellular-dwelling bacteria, chaperone proteins are conserved and overexpressed to buffer against problems in protein folding. We mimicked this approach for expressing and purifying ‘Ca. L. solanacearum’ proteins in Escherichia coli by coexpressing them with chaperones. Neither of the representative ‘Ca. L. solanacearum’ enzymes, dihydrodipicolinate synthase (key in lysine biosynthesis) and pyruvate kinase (involved in glycolysis), were overexpressed in standard E. coli expression plasmids or strains. However, soluble dihydrodipicolinate synthase was successfully coexpressed with GroEL/GroES, while soluble pyruvate kinase was successfully coexpressed with either GroEL/GroES, dnaK/dnaJ/grpE, or a trigger factor. Both enzymes, believed to be key proteins for the organism, were purified by a combination of affinity chromatography and size-exclusion chromatography. Additionally, both ‘Ca. L. solanacearum’ enzymes are active and have the canonical tetrameric oligomeric structure in solution, consistent with other bacterial orthologs. This is the first study to successfully isolate and functionally characterize proteins from ‘Ca. L. solanacearum’. Thus, we provide a general strategy for characterizing its proteins, enabling new research and drug discovery programs to study and manage the pathogenicity of the organism.

Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade. Austropuccinia psidii has a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies into A. psidii pathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in TEs and a very low overall GC content of 33.8%. Compared to other Pucciniales, the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how TEs shaped the genome evolution of A. psidii and provide a greatly needed resource for strategic approaches to combat disease spread.

Resistance to the pandemic strain of

Austropuccinia psidii, originating in South America, is a globally invasive fungal plant pathogen that causes rust disease on Myrtaceae. Several biotypes are recognized, with the most widely distributed pandemic biotype spreading throughout the Asia-Pacific and Oceania regions over the last decade.Austropuccinia psidiihas a broad host range with more than 480 myrtaceous species. Since first detected in Australia in 2010, the pathogen has caused the near extinction of at least three species and negatively affected commercial production of several Myrtaceae. To enable molecular and evolutionary studies intoA. psidiipathogenicity, we assembled a highly contiguous genome for the pandemic biotype. With an estimated haploid genome size of just over 1 Gb (gigabases), it is the largest assembled fungal genome to date. The genome has undergone massive expansion via distinct transposable element (TE) bursts. Over 90% of the genome is covered by TEs predominantly belonging to the Gypsy superfamily. These TE bursts have likely been followed by deamination events of methylated cytosines to silence the repetitive elements. This in turn led to the depletion of CpG sites in transposable elements and a very low overall GC content of 33.8%. The overall gene content is highly conserved, when compared to other closely related Pucciniales, yet the intergenic distances are increased by an order of magnitude indicating a general insertion of TEs between genes. Overall, we show how transposable elements shaped the genome evolution ofA. psidiiand provide a greatly needed resource for strategic approaches to combat disease spread.

‘Candidatus Liberibacter solanacearum’ is an unculturable α-proteobacterium that is the putative causal agent of Zebra Chip (ZC) disease of potato. This disease is a major problem in potato growing areas in the United States and New Zealand, as it affects growth and yield of the crop which has resulted in millions of dollars of loss to the potato industries. ZC disease is characterised by browning and necrotic flecking of vascular and internal tissue, which when fried present as dark stripes and streaks within the chip rendering them commercially unacceptable. The potato-infecting clades of this bacterium are vectored by Bactericera cockerelli, the tomato potato psyllid. Vertical transmission via seed potatoes is another mechanism that can spread the disease. Current disease management strategies target the psyllid: as the pathogen is transmitted relativity quickly, these strategies are limited in control of the disease. Thus, new management strategies that target the bacterial pathogen are required. A number of high quality bacterial genomes are now available and comparative genomics has identified a number of potential targets. This bacterium has a relatively small, AT-rich genome that contains all the components of a type I secretion system, ABC transporters, as well as ten bifunctional protein genes that encode proteins with two different enzymatic domains. Two of the bifunctional genes encode proteins similar to those described as pathogenicity or virulence determinants in other organisms. The relevance of these bifunctional genes to pathogenicity and virulence of this species is discussed in relation to maintaining these domains in a relatively small, AT-rich genome and their putative pathogenicity/virulence roles.

Data from 190 plant pathogen eradication programs in the Global Eradication and Response Database (GERDA) were reviewed to identify characteristics that contributed to successful programs in 45 countries between 1912 and 2013. The most successful treatment (94%) was tissue culture, often in combination with thermotherapy to eradicate viral or bacterial pathogens from plants held in in germplasm collections. Whilst 6% of these programs had no reported outcome, there were no recorded failures of this strategy. Host removal and/or destruction was successful in 55% of the programs and was used against all the pathogen groups. The analysis was limited by the high percentage of unknown outcome results across the pathogen groups. A quarter (49 of 190) of the records contained no indication of the eradication treatment: in 43% of these cases an unknown treatment resulted in successful eradication. There were no obvious correlations between the characteristics of a pathogen (viral/viroid, bacterial/phytoplasma, fungal/oomycete or nematode) and the outcome of the eradication program. For many species there is only one record, or the taxa records were dominated by a few genera that do not represent the biological diversity of the pathogen group. No economic or other analysis was possible due to the large number of unknown result/ongoing programs and the lack of common data. Despite these limitations, GERDA is an important record of the outcomes of worldwide plant pathogen eradication programs since the second decade of the twentieth century. However, care should be exercised when extrapolating from these records to formulating responses to new taxa as pathogens emerge and/or adapt to new plant hosts as the biology of plant pathogens is extremely variable and this diversity is not represented by the records in the database.

Here, we report the draft genome sequence of “ Candidatus Liberibacter europaeus” ASNZ1, assembled from broom psyllids ( Arytainilla spartiophila ) from New Zealand. The assembly comprises 15 contigs, with a total length of 1.33 Mb and a G+C content of 33.5%.

The environmental and economic impacts of exotic fungal species on natural and plantation forests have been historically catastrophic. Recorded surveillance and control actions are challenging because they are costly, time-consuming, and hazardous in remote areas. Prolonged periods of testing and observation of site-based tests have limitations in verifying the rapid proliferation of exotic pathogens and deterioration rates in hosts. Recent remote sensing approaches have offered fast, broad-scale, and affordable surveys as well as additional indicators that can complement on-ground tests. This paper proposes a framework that consolidates site-based insights and remote sensing capabilities to detect and segment deteriorations by fungal pathogens in natural and plantation forests. This approach is illustrated with an experimentation case of myrtle rust (Austropuccinia psidii) on paperbark tea trees (Melaleuca quinquenervia) in New South Wales (NSW), Australia. The method integrates unmanned aerial vehicles (UAVs), hyperspectral image sensors, and data processing algorithms using machine learning. Imagery is acquired using a Headwall Nano-Hyperspec ® camera, orthorectified in Headwall SpectralView ® , and processed in Python programming language using eXtreme Gradient Boosting (XGBoost), Geospatial Data Abstraction Library (GDAL), and Scikit-learn third-party libraries. In total, 11,385 samples were extracted and labelled into five classes: two classes for deterioration status and three classes for background objects. Insights reveal individual detection rates of 95% for healthy trees, 97% for deteriorated trees, and a global multiclass detection rate of 97%. The methodology is versatile to be applied to additional datasets taken with different image sensors, and the processing of large datasets with freeware tools.

The pseudoscorpions, Nesochernes gracilis and Chelifer cancroides, appear to have potential for control of varroa mites (Varroa destructor), but have not been observed to predate on varroa in honey...

Tomato potato psyllid (TPP) Bactericera cockerelli (Scaron;ulc) (Hemiptera Triozidae) was first recorded in the North Island of New Zealand in 2006 Three years later the insect was found in the Oamaru area (South Island) Over the years there has been a rise in TPP numbers trapped in potato crops in Canterbury (South Island) Recently increased prevalence and severity of foliar and tuber symptoms related to plant infection with Candidatus Liberibacter solanacearum (CLso) vectored by TPP have been observed in trials and in commercial potato crops Moreover in the 201314 season the resulting zebra chip disease was observed for the first time in tubers at a processing plant in Canterbury It is concluded that Canterbury has a landscape where hosts are available yearround and a climate that does not seem to hinder TPP development The aim of this paper is to present a stocktake of TPP in Canterbury

Haplotypes A and B of 'Candidatus Liberibacter solanacearum' (CLso) are associated with diseases of solanaceous plants, especially Zebra chip disease of potato, and haplotypes C, D and E are associated with symptoms on apiaceous plants. To date, one complete genome of haplotype B and two high quality draft genomes of haplotype A have been obtained for these unculturable bacteria using metagenomics from the psyllid vector Bactericera cockerelli. Here, we present the first genomic sequences obtained for the carrot-associated CLso. These two genomic sequences of haplotype C, FIN114 (1.24 Mbp) and FIN111 (1.20 Mbp), were obtained from carrot psyllids (Trioza apicalis) harboring CLso. Genomic comparisons between the haplotypes A, B and C revealed that the genome organization differs between these haplotypes, due to large inversions and other recombinations. Comparison of protein-coding genes indicated that the core genome of CLso consists of 885 ortholog groups, with the pan-genome consisting of 1327 ortholog groups. Twenty-seven ortholog groups are unique to CLso haplotype C, whilst 11 ortholog groups shared by the haplotypes A and B, are not found in the haplotype C. Some of these ortholog groups that are not part of the core genome may encode functions related to interactions with the different host plant and psyllid species.

International audience; A novel candidate species of the liberibacter genus, Candidatus Liberibacter brunswickensis' (CLbr), was identified in the Australian eggplant psyllid, Acizzia solanicola. This is the first discovery of a species belonging to the liberibacter genus in Australia and the first report of a liberibacter species in the psyllid genus Acizzia. This new candidate liberibacter species has not been associated with plant disease, unlike other psyllid-vectored species in the genus including Candidatus Liberibacter asiaticus' (CLas), Candidatus Liberibacter africanus' (CLaf) and Ca. Liberibacter solanacearum' (CLso). This study describes novel generic liberibacter genus primers, used to screen Australian psyllids for the presence of microflora that may confound diagnosis of exotic pathogens. CLbr forms a unique clade in the liberibacter genus based on phylogenetic analysis of the 16S ribosomal ribonucleic acid (rRNA) region and multilocus sequence analysis (MLSA) of seven highly conserved genes, dnaG, gyrB, mutS, nusG, rplA, rpoB and tufB. The MLSA mapping approach described in this article was able to discriminate between two Ca. Liberibacter' species within a metagenomic data set and represents a novel approach to detecting and differentiating unculturable species of liberibacter. Further, CLbr can confound the Li et al. (2006) quantitative PCR (qPCR) diagnostic tests for CLas and CLaf.

‘Candidatus Liberibacter solanacearum’ contains two solanaceous crop-infecting haplotypes, A and B. Two haplotype A draft genomes were assembled and compared with ZC1 (haplotype B), revealing inversion and relocation genomic rearrangements, numerous single-nucleotide polymorphisms, and differences in phage-related regions. Differences in prophage location and sequence were seen both within and between haplotype comparisons. OrthoMCL and BLAST analyses identified 46 putative coding sequences present in haplotype A that were not present in haplotype B. Thirty-eight of these loci were not found in sequences from other Liberibacter spp. Quantitative polymerase chain reaction (qPCR) assays designed to amplify sequences from 15 of these loci were screened against a panel of ‘Ca. L. solanacearum’-positive samples to investigate genetic diversity. Seven of the assays demonstrated within-haplotype diversity; five failed to amplify loci in at least one haplotype A sample while three assays produced amplicons from some haplotype B samples. Eight of the loci assays showed consistent A–B differentiation. Differences in genome arrangements, prophage, and qPCR results suggesting locus diversity within the haplotypes provide more evidence for genetic complexity in this emerging bacterial species.

Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with alpha-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia.

The genetic diversity of sugarcane yellow leaf virus (SCYLV) was analyzed with 43 virus isolates from Réunion Island and 17 isolates from world-wide locations. We attempted to amplify by reverse-transcription polymerase chain reaction (RT-PCR), clone, and sequence four different fragments covering 72% of the genome of these virus isolates. The number of amplified isolates and useful sequence information varied according to each fragment, whereas an amplicon was obtained with diagnostic primers for 59 out of 60 isolates (98%). Phylogenetic analyses of the sequences determined here and additional sequences of 11 other SCYLV isolates available from GenBank showed that SCYLV isolates were distributed in different phylogenetic groups or belonged to single genotypes. The majority of isolates from Réunion Island were grouped in phylogenetic clusters that did not contain any isolates from other origins. The complete six ORFs (5612 bp) of five SCYLV isolates (two from Réunion Island, one from Brazil, one from China, and one from Peru) were amplified, cloned, and sequenced. The existence of at least three distinct genotypes of SCYLV was shown by phylogenetic analysis of the sequences of these isolates and additional published sequences of three SCYLV isolates (GenBank accessions). The biological significance of these genotypes and of the origin of the distinct lineage of SCYLV in Réunion Island remains to be determined.

The complete nucleotide sequences of Fiji disease virus (FDV) genome segments S2, S4 and S7 were determined. This now completes the sequencing of all ten dsRNA genome segments of the Fijivirus type member, FDV, which comprises a total of 29339 nt. FDV S2, S4 and S7 comprised 3820, 3568 and 2194 nt, respectively. S2 and S4 each contained a single open reading frame (ORF), which encoded putative proteins of 137 and 133 kDa, respectively, while S7 contained two ORFs, which encoded putative proteins of 42 and 37 kDa. The putative amino acid sequences of FDV S2 and S4 showed most similarity to the gene products of Rice black-streaked dwarf virus (RBSDV) S2 and RBSDV S3, respectively. The putative amino acid sequences of FDV S7 ORF I and II showed most similarity to Maize rough dwarf virus (MRDV) S6 ORF I and RBSDV S7 ORF II, respectively. Phylogenetic analyses showed that FDV was most closely related to the group 2 fijiviruses.

Marker-assisted selection for traits that are difficult to screen for, such as resistance to many sugarcane diseases, has the potential to facilitate the development of improved cultivars in sugarcane. Pachymetra root rot (PRR) and brown rust resistance ratings were obtained over two years for 192 I1 progeny (progeny produced by two heterozygous, non-inbred parental lines) of a sugarcane (Saccharum spp. hybrid) cross between two elite sugarcane clones, Q117 and 74C42. Approximately 1000 single-dose markers, including microsatellite (SSR), amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers, were scored across the population and maps containing approximately 400 markers were constructed for each parent. At p ≤ 0.01, two genomic regions, one from the female Q117 map and a different region from the 74C42 male map, plus an unlinked bi-parental simplex marker (single-dose marker present in both parents) were identified as associated with PRR over both years of data collection. These regions explained between 6 and 16% of the phenotypic variation. An additional region was identified in the female map as associated with PRR at p ≤ 0.01 in one year and p ≤ 0.05 in the second year. This region explained between 4 and 8% of the phenotypic variation. For brown rust, two genomic regions, one from the female map and one from the male map, plus an unlinked marker from both maps, were identified as associated with brown rust resistance at p ≤ 0.01 over two years of phenotypic data. Each region explained between 7 and 18% of the phenotypic variation. Several additional regions were identified in both maps as associated with brown rust at p ≤ 0.01 in one year and p ≤ 0.05 in the second year. These regions also explained between 5 and 11% of the phenotypic variation. To validate these markers and determine whether they would be useful in alternative germplasm, markers from each genomic region associated with PRR or brown rust were screened across a set of 154 elite sugarcane clones; PRR and brown rust ratings were available for 131 and 72 of the clones, respectively. For PRR, three of the 6 markers tested remained significantly associated (p ≤ 0.01) with resistance ratings in the elite clone set. For brown rust, only one of the seven markers tested remained significantly associated (p ≤ 0.01) with resistance in the elite clone set, with one other marker associated at p ≤ 0.05. These results suggest that these markers could be broadly effective in selecting for PRR and/or brown rust resistance in sugarcane breeding programs.

Commercial sugarcane, belonging to the genus Saccharum (Poaceae), is an important industrial crop accounting for nearly 70% of sugar produced worldwide. Compared to other major crops, efforts to improve sugarcane are limited and relatively recent, with the first introduction of interspecific hybrids about 80 yr ago. Progress in traditional breeding of sugarcane, a highly polyploid and frequently aneuploid plant, is impeded by its narrow gene pool, complex genome, poor fertility, and the long breeding/selection cycle. These constraints, however, make sugarcane a good candidate for molecular breeding. In the past decade considerable progress has been made in understanding and manipulating the sugarcane genome using various biotechnological and cell biological approaches. Notable among them are the creation of transgenic plants with improved agronomic or other important traits, advances in genomics and molecular markers, and progress in understanding the molecular aspects of sucrose transport and accumulation. More recently, substantial effort has been directed towards developing sugarcane as a biofactory for high-value products. While these achievements are commendable, a greater understanding of the sugarcane genome, and cell and whole plant physiology, will accelerate the implementation of commercially significant biotechnology outcomes. We anticipate that the rapid advancements in molecular biology and emerging biotechnology innovations would play a significant role in the future sugarcane crop improvement programs and offer many new opportunities to develop it as a new-generation industrial crop.

Populations of the planthopper vector Perkinsiella saccharicida on sugarcane cultivars resistant (cvs Q110 and Q87), moderately resistant (cvs Q90 and Q124) and susceptible (evs NCo310 and Q 102) to Fiji disease with known field resistance scores were monitored on the plant (2000-2001) and ratoon (2001-2002) crops. In both crops, the vector population remained very low, reaching its peak in the autumn. The vector population was significantly higher on cultivars susceptible to Fiji disease than on cultivars moderately resistant and resistant to Fiji disease. The number of R saccharicida adults, nymphs and oviposition sites per plant increased with the increase in the Fiji disease susceptibility. The results suggest that under low vector density, cultivar preference by the planthopper vector mediates Fiji disease resistance in sugarcane. To obtain resistance ratings in the glasshouse that reflect field resistance, glasshouse-screening trials should be conducted under both low and high vector densities, and the cultivar preference of the planthopper vector recorded along with Fiji disease incidence.

The genome of an Australian isolate of Sugarcane bacilliform virus (SCBV-IM) was cloned, sequenced and analysed. The genome consisted of 7687 nucleotides and contained three open reading frames which were similar in size and organisation to those of other badnaviruses. SCBV-IM was found to be most similar to the SCBV-Morocco isolate with amino acid sequence similarity of 91.4 %, 83.8 % and 85.3 % in the ORF I, II and III coding regions, respectively. Phylogenetic analysis of the SCBV-IM ORF III deduced amino acid sequence showed that SCBV isolates were more closely related to each other than to other badnaviruses. Amplification of SCBV sequences from three different sugarcane varieties revealed considerable variability in the viral populations, both within single infected plants as well as between infected plants, suggesting that the SCBV isolates sequenced to date may not be representative of the range of virus variability.

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

Sugarcane mosaic potyvirus (SCMV) is an important pathogen of sugarcane (Saccharum L. interspecific hybrids) and can cause significant yield losses in susceptible cultivars. Pathogen-derived resistance using the viral coat protein (CP) gene has been successfully demonstrated in other host/virus combinations and is being evaluated in sugarcane. Sugarcane plants were cotransformed with the CP gene of sugarcane mosaic virus (SCMV) and the neomycin phosphotransferaseII (nptII) (selectable marker) gene by microprojectile bombardment. These genes were constructed with either the Emu (an artificial promoter) or the ubiquitin (Ubi) promoter. Over 80 transgenic lines were assessed for nptII activity by dot-blot and ELISA. There was considerable variability between lines in npt expression, with the most active Ubi line producing npt enzyme to a level equivalent to 0.03% of total soluble protein. Initial results show no correlation between the number of copies of the transgene and the level of npt expression. Plants containing the CP transgene have been selected, propagated and challenged with SCMV in glasshouse trials. Ten lines were resistant to challenge inoculation with the virus and these lines are being further characterised.

We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.

Current management options to minimize the impact of canegrubs (larvae of melolonthine scarabs) on Australian sugarcane are heavily dependent on the use of synthetic insecticides. In developing a successful integrated pest management (IPM) programme for canegrubs, control options must be broadened. Plant resistance offers the potential for an easily deliverable and environmentally acceptable management option. Previous anecdotal reports suggest that Australian cultivars vary in their resistance to canegrubs. Many sugarcane clones (Saccharum spp. complex hybrids) have been screened and our studies have shown that there is variability in resistance through the reduction of the amount of tops, roots and stubble and in their effects on canegrub development and survival. Proteinase inhibitors, lectins and avidin have been identified as having activity against Australian canegrubs and are being introduced into the sugarcane genome. Toxins from Bacillus thuringiensis have shown little potential and are not being used further. We present a rationale for using resistant plants as a control option and for the incorporation of plant resistance into the breeding programme. Projected research will screen more sugarcane clones for resistance, study the nature of inheritance, identify chemical mechanisms involved in antibiotic effects, screen further antimetabolites for possible incorporation into the genome, prove the potential resistance of genetically engineered plants and incorporate plant resistance into an IPM programme for canegrubs.

A high titre (1:10,000) antiserum was raised in a rabbit against the coat protein of sugarcane mosaic potyvirus (SCMV), by injecting a preparation of recombinant coat protein purified from a fusion protein expressed in E. coli. The fusion protein consisted of the MalE maltose binding protein (MBP) and the viral coat protein separated by the protease factor Xa cleavage site. The fusion protein was encoded by the plasmid pMAL-cCPM, which was constructed by cloning a modified coat protein gene to the 3' end of the MBP/factor Xa coding region. The coat protein gene was modified by site-directed mutagenesis so that the ATG start codon in the original construct was replaced by the codon AGC, deleting the NcoI restriction site (C/CATGG) and creating a unique Eco47III site (AGC/GCT). Endonuclease restriction with Eco47III resulted in a DNA fragment with GCT as the first three nucleotides. This triplet encodes alanine, which is the proposed N-terminal amino acid residue of the mature native coat protein. This modified coat protein coding region was ligated directly behind the nucleotide code for the amino acid recognition sequence for factor Xa. Expression was induced with IPTG and the recombinant fusion protein was extracted from the bacterial lysate by amylose resin column affinity chromatography and the two domains separated by factor Xa proteolysis. The coat protein was then purified from the maltose binding protein by ion exchange chromatography in buffer containing 6 M urea. A highly purified sample which contained 150 micrograms of both full-length and truncated coat proteins, was recovered from a litre of bacterial broth. The antiserum reacted with native coat protein in SCMV-infected sugarcane, and with recombinant coat proteins expressed in E. coli and sugarcane protoplasts with little or no cross-reaction with sugarcane proteins.

Ascochitine, a phytotoxic metabolite, was purified from cultures of Phoma clematidina and identified by 1 H and 13 C nuclear magnetic resonance spectroscopy and electron impact and chemical ionization mass spectroscopy. This toxin-induced electrolyte leakage from leaf discs of Clematis cultivars that were susceptible to fungal infection, while there was no significant electrolyte leakage from leaf discs of the cultivar most resistant to fungal infection. The level of ascochitine production in vitro by P. clematidina isolates was related to isolate virulence. The fungal isolates could be characterized into two groups: (1) high virulence, high ascochitine production, and (2) low virulence, low ascochitine production. Ascochitine was isolated from P. clematidina -infected leaf discs, indicating the toxin is produced in vivo . Leaf tissues exposed to ascochitine solutions showed black flecking in proportion to the concentration of ascochitine. Scanning electron and light microscopy of infected leaves indicated that the fungal hyphae were well behind the necrotic zone in leaf spots, while transmission electron and light microscopy suggested that mitochondria and chloroplasts were the major organelles affected by ascochitine, although extensive cellular damage was evident. These observations suggest that ascochitine may be involved in the pathogenesis of P. clematidina against Clematis , by killing plant cells prior to hyphae ramification through the necrotic tissue.

Biotinylated Fiji disease fijivirus specific cDNA probes detected the presence of the virus in total nucleic acid extracts from infected sugarcane plants. Hybridised biotinylated probes were detected with streptavidin-alkaline phosphatase conjugate and the light generating substrate AMPPD. Samples were either blotted manually, or by alkaline capillary transfer using 100 mM NaOH. Transfer of nucleic acids to charge modified nylon with sodium hydroxide was superior to denaturation with glyoxal or formamide and salt-citrate buffer transfer as the bands were clearly resolved and no degradation of the FDV dsRNA was observed. Transfer of either total nucleic acid extracts or purified dsRNA in manifold blots generated false positive signals with the non-radio-active chemiluminescent detection systems tested. Manual or northern blots had a limit of detection for purified target double-stranded RNA of approximately 10 pg and 0.5 pg respectively. Manual blots were tested for practical application to screen germplasma for FDV infection. The virus was detected in leaf samples from FDV-infected plants, in some instances prior to development of the characteristic gall symptom.

A 450-bp region from one species of the segmented dsRNA genome of Fiji disease virus (FDV) was amplified from total nucleic acid extracts of diseased plants by reverse transcription with MMLV, followed by amplification with Taq DNA polymerase (RT-PCR). Other FDV-specific regions (c 150 bp and c 270 bp) were also amplified from the dsRNA template. FDV cDNA was only synthesised when the viral dsRNA template was boiled and quenched with FDV-specific or random hexamer primers. The reverse transcriptase/DNA polymerase enzyme rTth appeared to yield only the 150 bp fragment from the dsRNA template under the conditions used. The level of sensitivity of RT-PCR for purified FDV dsRNA was 100 ag, approximately 10(4)-fold more sensitive than detection with biotinylated DNA probe.

Four promoters derived from sugarcane bacilliform virus (SCBV) were compared and characterised. Three were obtained by PCR amplification of purified virion DNA extracted from three sugarcane cultivars. The fourth promoter was obtained by subcloning from an almost genome-length clone of SCBV. All promoters were able to drive stable expression of beta-glucuronidase in sugarcane. The PCR-derived promoter sequences shared more DNA homology with banana streak virus than to the subcloned SCBV. The subcloned promoter was the strongest expressing and was able to drive reporter gene expression in vitro and in the leaves, meristems and roots of glasshouse-grown sugarcane. Expression levels were at least equal to or higher than those measured for the maize polyubiquitin promoter.

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

The complete sequences of Fiji disease virus (FDV) genome segments 5 (S5), S6, S8 and S10 were obtained and comprised 3150 nt, 2831 nt, 1959 nt and 1819 nt, respectively. Each segment contained a single ORF which encoded putative proteins of 115 kDa, 97 kDa, 69 kDa and 63.0 kDa, respectively. The putative amino acid sequences encoded by S5 and S6 contained putative leucine zipper motifs while FDV S5 and S8 each contained an ATP-GTP-binding motif. At the amino acid level, FDV S5, S6, S8 and S10 showed most similarity to the corresponding segments of Rice black-streaked dwarf virus. Based on sequence similarities, it is predicted that FDV S8 encodes a minor core protein, while FDV S10 encodes an outer capsid protein. The evolutionary relationships of FDV to other reoviruses are discussed.

Fiji disease fijivirus (FDV) genomic segments 1 (S1) and 3 (S3) were completely sequenced. FDV S1 comprised 4,532 nt and was predicted to encode a 170.6 kDa protein. FDV S3 comprised 3,623 nt and was predicted to encode a 135.5 kDa protein. The terminal sequences of S1 and S3 were 5' AAGUUUUU......CAGCUAGCGUC 3' and 5' AAGUUUUU......CAGCAGAUGUC 3', respectively, and located immediately adjacent to these sequences were 12 bp imperfect inverted repeats. The predicted translation product of FDV S1 showed highest similarity to Rice black-streaked dwarf virus (RBSDV) S1 and is thought to encode the viral RNA-dependent RNA polymerase (RdRp). The predicted translation product of FDV S3 was found to be most similar to RBSDV S4 which is thought to encode the 'B-spike' protein. The FDV sequence contained an ATP/GTP binding motif and a leucine zipper motif, but these motifs were not found in the RBSDV sequence. Phylogenetic analysis based on the amino acid sequences of the RdRp of FDV S1 and other reoviruses revealed that the fijiviruses form a cluster close to the oryzaviruses. The RdRp sequences were grouped into genera that were consistent with the current reovirus classification scheme that is based on physico-chemical and biological properties.

Sugarcane bacilliform virus (SCBV) DNA molecules larger than the complete genome length of 7.6 kbp were detected in infected plants and in virions. We have confirmed that these high molecular weight nucleic acids were open circular DNA and viral in origin. Due to their open circular conformation, accurate size determination of the DNA molecules was not possible using conventional electrophoresis. Using field inversion gel electrophoresis (FIGE), however, the DNA appeared to increase in genome size increments, with sizes ranging from 1 to 4 genomes (31 kbp) detected. The DNA was packaged into virions, which may explain the observation of purified virions with lengths corresponding to one, two or three times the modal length of 130 nm. The DNA products were possibly concatamers formed during replication as a result of a terminal overlap on the sense strand, and were shown to be overlapped individual genome-length molecules and not covalently-bonded continuous DNA strands. Southern analysis indicated that SCBV sequences are not integrated into the sugarcane genome and that the high molecular weight DNA observed in the sugarcane accessions analysed represents SCBV concatamers.

The 5895 nucleotide long single-stranded RNA genome of Sugarcane yellow leaf virus Florida isolate (SCYLV-F) includes six major ORFs. All but the first of these are homologous to genes of known function encoded by viruses of the three newly defined genera in the Luteoviridae (‘luteovirids’), i.e. poleroviruses, luccccteoviruses and the enamoviruses. SCYLV-F ORFs 1 and 2 are most closely related to their polerovirus counterparts, whereas SCYLV-F ORFs 3 and 4 are most closely related to counterparts in luteovirus genomes, and SCYLV-F ORF5 is most closely related to the read-through protein gene of the only known enamovirus. These differences in affinity result from inter-species recombination. Two recombination sites in the genome of SCYLV-F map to the same genomic locations as previously described recombinations involving other luteovirids. A fourth type of luteovirid, Soybean dwarf virus, has already been described. Our analyses indicate that SCYLV-F represents a distinct fifth type.

This is the first report of sequence from Fiji disease fijivirus (FDV), the type member of the genus Fijivirus of the family Reoviridae. FDV genome segment (S9) comprised 1843 nt and contained two non-overlapping ORFs, separated by a 57 nt intergenic region. S9 ORF 1 comprised 1008 nt and encoded a 335-amino-acid polypeptide (predicted molecular mass 38.6 kDa), while ORF 2 comprised 627 nt and encoded a 208-amino-acid polypeptide (predicted molecular mass 23.8 kDa). The 5' and 3' non-coding regions were 49 and 102 nt, respectively. The S9 terminal sequences were 5' AAGUUUUU------UGUC 3', and located immediately adjacent to these sequences were 12 bp imperfect inverted repeats. The entire S9 ORF 1 and the hydrophilic regions of S9 ORF 2 were each expressed as a fusion protein with the maltose-binding protein in Escherichia coli. Antibodies produced against the ORF 1 fusion protein reacted strongly with a protein of approximately 39 kDa present in both crude extracts of FDV-infected sugarcane and partially purified FDV preparations. In contrast, antibodies raised against the modified ORF 2 fusion protein did not react with any proteins in the same samples. Further, polyclonal antibodies produced against partially purified FDV reacted with the ORF 1, but not the modified ORF 2, fusion protein. These results indicate that FDV S9 ORF 1 encodes a major structural protein, while ORF 2 probably encodes a non-structural protein.

We have sequenced the coat protein (CP) coding region of 11 field isolates of SCMV from Australia, USA and South Africa. The differences between the nucleotide sequences of the isolates was 0.2 to 4.1% and the encoded amino acid sequences differed by 0.0 to 3.5%. Phylogenetic analysis of the CP coding sequences of the SCMV isolates and the related potyviruses SCMV-MDB, JGMV, SrMV, MDMV-A and PVY showed that the SCMV isolates formed a tightly clustered group, with SCMV-MDB forming a separate branch. This indicated that (i) the SCMV isolates are of one strain (SCMV-A) and not geographically distinct species and (ii) SCMV-MDB is clearly distinct, and may represent another potyvirus species.