The IX Pathology and VI Molecular Biology Workshop of the International Society of Sugar Cane Technologists (ISSCT) was jointly organised. The workshop was hosted by the Colombian Sugarcane Research Centre (CENICAÑA) from 23-27 June 2008 at the Radisson Royal Hotel, in Cali, Colombia. It was organized by Drs Jorge I Victoria and Jershon López-Gerena. The Workshop was well attended by 44 participants, with 39 of them coming from overseas - Argentina (4), Australia (4), Brazil (3), Ecuador (1), France (3), Guadeloupe (2), Guatemala (2), India (2), Mauritius (2), Nicaragua (2), Panama (1), South Africa (2), USA (9), Venezuela (1) and Zimbabwe (1)- and 5 from Colombia.

The theme of the Workshop was ‘Advances and Challenges in Sugarcane Biotechnology and Plant Pathology’. The first three days were devoted to oral presentations and poster session, while during the last two days site visits were organized.  

There were 11 sessions at the workshop and a copy of the programme is given in annex 1. Altogether 37 oral presentations and 10 posters were presented.

The Workshop started with a plenary conference on ‘Biofuel production in Colombia and the role of research organizations’ presented by the General Director of CENICAÑA, Dr Alvaro Amaya Estévez. Two additional plenary conferences were given on day 2 and day 3 by Dr Erik Mirkov of Texas A & M Unversity, USA - ‘ Will transgenics have a role in the improvement of commercial sugar cane varieties?’ and by Dr Joe Tohme, Leader of agro biodiversity and biotechnology at CIAT, Colombia – Nutritional genomic to fight micronutrient deficiencies.  

Session 1 (Moderator: Asha Dookun-Saumtally)

Three papers were presented within Session 1. Topics covered were diverse, in that they reflected aspects of three key innovations in sugarcane agriculture, viz. bioethanol production, regulatory requirements ahead of commercialisation of genetically-modified sugarcane and biocontrol of pests. 

1. Isolation, selection and molecular characterization of wild yeasts found in ethanol production at Providencia Sugarmill. Buzón K, Parrado D, Quevedo B, Victoria J I and Avellaneda M C. Variety Program, Colombian Sugarcane Research Center-CENICAÑA, Colombia.

Ethanol production within the Colombian sugar industry is a rapidly expanding initiative, which is being driven primarily by price regulation and tax incentives from government. Although production is based on technology imported from India, efforts are being made on several fronts to adapt the technology to suit local conditions. The work of M. Carolina Avellaneda focused on the isolation of wild yeast strains that have the potential to out-perform commercial yeasts. A number of yeasts (18 isolates) were identified in samples taken after the heat exchanger, propagation reactor and fermentation reactor at the Providencia Mill in the Cauca Valley. Biochemical and molecular characterisation of the isolates revealed that ten of the 18 isolates were Saccharomyces and were capable of fermentative respiration to produce ethanol. All of the ethanol-producing isolates could tolerate ethanol concentrations of 12% (v/v) and were at least as efficient as commercial strains. Evidence presented by Ms Avellaneda indicates that the wild yeasts were endogenous within cane entering the mill, rather than factory contaminants. The protocol developed appears to be an effective means to isolate and characterise wild yeasts and, as such, has the potential to deliver yeast strains that are highly efficient for ethanol production. 

2. Fertile pollen detected in commercial sugarcane in northern Argentina. Lozzia M E, Cristóbal M E, Serino G, de Ullívarri R F and Tassara H J. Chacra Experimental Agricola Santa Rosa. Argentina.

As in many countries, the Argentine regulatory framework governing the commercial release of genetically modified (GM) crops encompasses both food safety and environmental issues. In terms of the latter, concern exists over the potential of GM crops becoming weeds or crossing with wild species that have the potential to become weeds. As Saccharum spotaneum exists as naturalised populations in Argentina, consideration of the potential for transgene spread from GM Saccharum spp hybrid crops is essential. The low temperatures (10 – 12 ^oC) experienced in Argentina in the period prior to anther opening (June and July) were believed sufficient to suppress male fertility. However, in his ISSCT Workshop presentation, Germán Serino revealed that, during June to August 2006, fertile sugarcane pollen was detectable in the northern regions of the Argentinean industry (between S 23^o 16’ and S 24^o 50’), although prevalence decreased southwards, such that no fertile pollen was detected between S 26^o 30’ and S 27^o 55’. The unexpectedly high pollen fertility encountered in the Northern regions was ascribed to unusually warm winter conditions. It is important to note that final interpretation of these observations and the impact that they may have on the assessment of the risk of transgene spread in Argentina will require assessment of pollen longevity, distance of dispersal and, ultimately, seed viability. 

3. Molecular and morphological characterization of the species of the Chrisopidae genus found in the Cauca Valley (Colombia). Cadena P, Ángel F, Gomez L A and González R. Variety Program. Colombian Sugarcane Research Center-CENICAÑA, Colombia.

The Colombian sugar industry has a long and proud history of successful biocontrol of sugarcane pests and the insect vectors of pathogens. In his ISSCT Workshop presentation, Luis Gomez outlined Cenicaña’s strategy for reducing the negative impact of the yellow sugarcane aphid (Sipha flava), which reportedly reduces sugar yield (ton sucrose/ha) by up to 52%. Currently, the effects of this pest are minimised by insecticide application, host plant resistance and natural predation. Attempts at biocontrol have been made through the introduction of commercially-reared North American lacewings (Chrysoperla spp.) but release results were erratic and difficult to interpret.  Consequently, Cenicaña researchers are searching for local lacewings that may serve as more effective biocontrol agents of the aphid. Development of molecular technologies for the analysis of the lacewing phylogeny was outlined during this presentation. Generally, the rapid and simple molecular tests could discriminate amongst morphotypes, which were supported by traditional classifications based on the morphology of inner genitalia. In all, eleven chrysopid morphotypes have been revealed; knowledge which will serve as the foundation for the development of a biocontrol mechanism against the yellow sugarcane aphid. 

Session 2 (Moderator: Erik Mirkov)

Within this session, five presentations were given ranging from the molecular characterization of sugarcane borer species, genotyping of sugarcane varieties, mapping QTL for sucrose content and the understanding of sucrose accumulation in sugarcane.

4. Molecular and morphological characterization of some sugarcane borer species. Cadena P, Gomez L A, Ángel F and Victoria J I. Variety Program–Colombian Sugarcane Research Centre, CENICAÑA., Colombia.

A molecular and morphological diversity study based on the sequence of the mitochondrial cytochrome oxidase gene, COII, from 108 borer specimens collected along the Cauca Valley was carried out in the year 2005 to determine possible new races of the three borer species in the region: Diatraea saccharalis, D. indigenella, and Blastobasis gramineae that may explain recent borer population increases in the region. The studies indicated that D. saccharalis was the main species behind this outbreak, and that there were no new races that explained the recent borer outbreak, according to genetic differentiation indexes based on PCR-RFLP and sequence of a COII gene fragment. Changes in climate or agricultural practices may explain the outbreak. A survey in the year 2006 indicates that the borer population has decreased after control practices were further implemented.  

5. Microsatellites as a tool for assessing genetic variability and protection of CC varieties. Espinosa K, Ángel F, Victoria J I and López-Gerena J. Variety Program Colombian Sugarcane Research Centre – CENICAÑA., Colombia.

A group of 131 varieties used as parents in Cenicaña’s breeding program and 5 S. officinarum clones were fingerprinted with microsatellites. 35 microsatellite primer pairs yielded 177 bands that were analyzed by Jaccard distances and UPGMA clustering. For germplasm protection, DNA from 1350 varieties from the breeding program were extracted and are stored in separate locations. SSR primers CV29, CV37, and CV38 (provided by Walter Maccheroni, Canavialis) are able to identify 95% of the varieties. Fingerprints allowed in assessing the pedigree of CC85-92, a cultivar adopted in 70% of the sugarcane area in Colombia that was originated from a polycross between Co775 and varieties ICA75-4, CP52-78, and CP55-30, assigning CP52-78 as the male parent for this highly adopted cultivar.  

6. Developing a sugarcane molecular identity database for use in breeding.  Pan Y B, Scheffler B E, and Richard E P, JR. USDA-ARS, Houma,LA, USA.

A molecular identity database has been assembled for 655 clones from the breeding programs at Houma, Louisiana (Ho), Canal Point, Florida (CP), LSU Ag Center, Louisiana (L), and the quarantine at Houma (Q), using 21 SSR markers with High Throughput (HT) DNA genotyping technology. A total of 144 robust, peaks are scored to build the database. The intended use of this database includes identification of mislabeled parental clones, as molecular descriptors for newly released varieties, determine genetic relatedness of elite clones, assessing the quality of crosses, and identifying F1 hybrids from crosses with wild germplasm.  

7. Understanding the biology of sucrose accumulation: Along the path to increasing sucrose content through transgenesis. Watt D, Huckett B and Botha F. South African Sugarcane Research Institute (SASRI), South Africa.

Progress was reported on SASRI’s work to identify the key control steps in sucrose accumulation. The objective of this work is to increase the sucrose content from 30% of fresh mass in the culm, which would account for only 65% of the predicted storage capacity of the sugarcane plant. Early work focused on changes in gene expression, biochemistry, and physiology occurring along internode maturation in one or a few varieties with different sucrose accumulation capacities. Gene expression studies moved to focus on key metabolic pathways identified by subtractive DNA libraries and boutique arrays. A small set of candidates was identified, and some of them studied on transgenic plants grown in the field. Currently, physiological evidence points to source-sink communication as crucial to sucrose metabolism, where feedback inhibition between sucrose concentration in the culm and leaf photosynthesis limits further accumulation of sucrose. New candidate genes will be tested via simple and stacked transgenic approaches to attempt unlocking the key to sucrose accumulation.  

8. Mapping QTL controlling sugar content and biomass components in sugarcane. López-Gerena J, Campillo N and Victoria J I. Variety Program. Colombian Sugarcane Research Center-CENICAÑA, Colombia.

QTL for sugar content and biomass accumulation were identified in a population of 300 individuals from a cross between the high sugar content cultivar MZC 74-275 and ICA 69-11. After preliminary mapping of QTL with SSR and AFLP markers, a single marker analysis with 65 SSR marker data points for 10 sugar and 4 biomass component traits in a semidry field experiment significantly identified 84 QTL. A new field experiment in both hilly and humid zone is being carried out to validate the position and effect of the QTL and to assess for environmental effects in QTL expression. QTL in this studies are candidates for MAS in the sugarcane breeding program at CENICAÑA. 

Session 3 (Moderator: Mike P Grisham)

Within Session 3 of the Workshop, the topics presented for discussion ranged from the issue of chimaeric transgenic plants production when biolistics are used in concert with direct somatic embryogenesis, mapping of QTLs for yellow spot resistance and the development of markers for smut resistance through the use of a novel combination of marker technologies. 

9. Early identification and rescue of chimaeric plants obtained from transformation protocols without a callus selection stage. Zimmermann J, Spedaletti Y and Serino G. Chacra Experimental Agricola Santa Rosa. Colonia Santa Rosa, Argentina.

Many institutes involved in GM sugarcane research are seeking ways to simplify and speed-up the transformation process, as well as to reduce somaclonal variation that sometimes results from tissue culture. Germán Serino reported that their efforts to reduce time in culture, viz. through imposition of a one-to-two week post-bombardment callus induction period where selection was restricted to the regeneration phase, resulted in plants with chimaeric phenotypes. As the gene-of-interest delivered a herbicide-resistant phenotype, the detection of such chimaeras was simple. The Argentinean group propose that insufficient selective growth of transgenic cells in the absence of a callus selection phase yields heterogenic masses of both transformed cells that develop into chimaeric embryos that which regenerate under non-stringent geneticin selection protocols. That group aims to further refine minimal culture and selection requirements, but which will reduce or eliminate chimaeras. 

10. Mapping QTLs for yellow spot resistance in sugar cane. Aljanabi S M, Parmessur Y, Dhayan S, Saumtally S and Dookun-Saumtally A.. Mauritius Sugar Industry Research Institute, Réduit, Mauritius.

In 1977 and 1978, 3 100 ha and 4 200 ha, respectively, of the Mauritian sugarcane crop were affected by yellow spot disease. In subsequent years, the disease was brought under control through the planting of resistant varieties. However, the disease recurred in 2003 at a severity similar to that experienced in the 1970s, which emphasised the importance of development of molecular markers of resistance or susceptibility for use in breeding initiatives. Consequently, the Mauritian group at MSIRI, under the leadership of Asha Dookun-Saumtally, have deployed a variety of marker technologies (including AFLPs and SSRs) in a quest to detect a QTL for the disease. The foundation of the study was a bi-parental cross between a resistant (M 134/75) and a susceptible (R 570) parent, which generated a progeny (227 individuals) with a segregation pattern for yellow spot infection suggestive of monogenic dominant inheritance (3:1). Of note is that the AFLP and SSR markers uncovered 666 polymorphisms that were present in the resistant but absent in susceptible parent. A genetic map of M 134/75 was constructed using 764 single-dose polymorphisms assigned to 102 linkage groups, each of which containing at least two markers in coupling. QTL analysis by means of QTLCartographer v1.17d and MAPMAKER/QTL v1.1 identified a single major QTL located on Linkage Group 87, flanked by an AFLP and SSR marker. A further AFLP marker has been placed at 1.9 cM from the major QTL. The genomic region identified by the MSIRI group explains 43.3% of phenotypic variation of the trait and is constant across environments and growing season. This work clearly demonstrates the huge potential of QTL analysis as an adjunct to sugarcane breeding. 

11. Developing a high throughput marker system for sugarcane smut resistance based on Motif Directed Profiling (MDP) and Targeted Region Amplified Polymorphism (TRAP) methodologies. Rutherford R S and Sweby D L. South African Sugarcane Research Institute. Mount Edgecombe. KwaZulu-Natal, South Africa. 

Session 4 (Moderator: Yong-Bao Pan)
Three papers were presented in this session and dealt with sugarcane transgenics and proteomics. 

12. Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. Molinari H B C^a, Bespalhok Filho J C^b, Marur C J^a, Daros E^b, Freitas de Campos M F^a, Rodrigues Portela de Carvalho J F^a, Pereira L F P^c and Vieira L G^a. ^a Plant Biotechnology Laboratory, Agricultural Research Institute of Parana (IAPAR) CP 481, CEP 86001-970 Londrina PR, Brazil, ^bDepartment of Agronomy and Plant  Protection, Federal University of Parana (UFPR), CEP 80035-050 Curtiba PR, Brazil, ^c Plant Biotechnology Laboratory (IAPAR), Embrapa Café, CP 481, CEP 86001-970, Londrina PR, Brazil.

This paper addressed stress inducible production of proline in transgenic sugarcane. The authors express the heterologous P5CS gene (for proline biosynthesis) from the drought resistant bean, Vigna angustifolia, under the control of the synthetic stress-induced AIPC promoter. A series of transgenic events with 1 to 4 insertions were generated and upon drought stress, the proline content was on average 2.5 times higher than in the controls, and that this lead to good drought tolerance. The authors further determined that the mode of action was not osmotic adjustment. Instead, they found that the mechanism was protection of the photosynthetic apparatus from damage due to reactive oxygen species.  

13. Proteomics analysis of sugarcane reaction to infection by Xanthomonas albilineans. Garces F, Hoy J and Chen Z Y. Louisiana State University, Baton Rouge, Louisiana, USA.

This paper presented the results of a proteomics approach to study sugarcane’s reaction to infection by Xanthomonas albilineans. The authors used a differential protein expression analysis using one resistant ( Ho 95-988) and one susceptible (CP 89-846) sugar cane variety. Their analysis was based on 2-dimensional electrophoresis. They compared protein profiles from infected and non-infected plants, and found proteins that were differentially up and down-regulated in the susceptible and resistant varieties. The proteins were recovered from the gels and subjected to peptide sequencing using LC-MS/MS.  

14. Comparison of Agrobacterium-mediated and biolistic methods for transformation of sugarcane. Joyce P, Kuwahata M, O’Connell A., Geijskes J, Turner N and Lakshmanan P. BSES Limited; Indooroopilly. Australia.

This paper presented a comparison of Agrobacterium-mediated and biolistic methods for transformation of sugarcane. The authors first test a variety of parameters in transient assays to optimize Agrobacterium-mediated transformation of embryogenic callus of the variety Q117. They used Agro strains LBA4404 and AGL1, and a pCambia vector with GUS as the reporter gene under the control of the CaMV 35S promoter. They found that vacuum infiltration worked well for transient expression. Further optimization was done with AGL1 and they found that dilution the Agro cultures to an O.D. of 0.4 was best as was a 4 day co-cultivation period on EM3 medium. . However they were not able to regenerate any transgenic plants, so they remade constructs using the maize Ubi 1 promoter rather than the CaMV35S promoter and generated over 200 plants. Most of the plants had only 1 or 2 insertion events. They also generated a large number of transgenic events using linear DNA with only minimal sequences, and found that blunt end DNA was best.  In a comparison between Agro and biolistic methods for expression levels, the biolistic approach lead to much higher expression levels, and that there was not a correlation with copy number.  

Session 5 (Moderator: Jack C. Comstock)

Three presentations were presented in this session two on leaf scald and its causal agent, Xanthomonas albilineans, and one on the causal agents of mosaic in Argentina.  

15. Leaf surface colonization and stalk infection by Xanthomonas albilinean of sugarcane varieties grown under high rainfall conditions.  Daugrois J H¹, Oriol P¹ and Rott P².  ¹ CIRAD Département BIOS, Station de Roujol, Petit-Bourg, Guadeloupe, F. W. I. and ² UMR CIRAD-INRA-Montpellier SupAgro Biologie et Génétique des Interactions Plante-Parasite, Montpellier, France.

Leaf surface colonization and stalk infection by Xanthomonas albilineans of sugarcane was determined in varieties grown under high rainfall conditions. The X. albilineans colonization of sugarcane leaf surface after aerial spread of the pathogen in Guadeloupe, varied according to the host cultivar. The stalks of two (out of 8) sugarcane varieties that exhibited the highest leaf surface populations were also the most infected by X. albilineans. Additionally, severity of leaf necrotic symptoms was correlated with intensity of leaf colonization. The authors concluded that resistance of sugarcane to leaf scald appears to be controlled by several mechanisms, from leaf surface colonization to stalk colonization. The discussion that followed this communication focussed on i/ the few reports of aerial spread of X. albilineans (up to date, aerial spread of X. albilineans was only reported in Florida/USA, Guadeloupe and Mauritius), ii/ possible recovery from stalk infection from one crop cycle to another, and iii/ importance of temperature on leaf scald symptom progress and severity (symptoms seem to be more severe when sugarcane is grown at high temperatures).  

16. Toward alleles tagging involved in Xanthomonas albilineans leaf infection resistance.  Hoarau J Y¹, Joubert C¹, D’Hont A¹, Roques D² and Daugrois J H^{2  1} UMR CIRAD-INRA-Montpellier, France and ² CIRAD Département BIOS, Station de Roujol, Petit-Bourg, Guadeloupe.

Preliminary results regarding tagging alleles involved in X. albilineans leaf infection resistance were reported by J.Y. Hoarau and collaborators. The objective of this study was to tag resistant alleles in the sugarcane genome that control aerial leaf infection by X. albilineans in Guadeloupe. The authors used a large unstructured population of 198 sugarcane clones that was recorded for severity of leaf symptoms, and 700 polymorphic AFLP markers. A set of six markers explained 31% of the total phenotypic variation in necrotic leaf symptom intensity. This first insight into X. albilineans leaf colonization resistance needs to be continued with additional markers to further tag sugarcane alleles involved in this resistance. During the discussion that followed the communication, the authors indicated that additional sugarcane clones will be tested for leaf resistance and screened using additional AFLP markers (maybe with microsatellite markers and DARTs). Additionally, it was mentioned that these markers may be used in the future to eliminate clones susceptible to leaf scald in breeding programs, although the number of markers to do this can not be determined at this stage. 

17. A survey of the causal agent of sugarcane mosaic in Argentina and neighboring regions.  Gómez M¹, Rago A M², and Serino G¹.  Chacra Experimental Agricola Santa Rosa. Salta, Argentina and INTA- EEA Famaillá. Tucumán, Argentina.

The first large-scale survey of the causal agent of sugarcane mosaic in Argentina and neighbouring regions was made. This survey was undertaken to determine the mosaic virus population present in Argentina and to improve control of mosaic with transgenic plants. A total of 522 plants showing mosaic symptoms plants were analyzed by RT-PCR, and leaves were sampled from 111 sites and 106 sugarcane varieties. The majority (95%) of samples from Argentina were infected by Sugarcane mosaic virus (SCMV). Sorghum mosaic virus (SrMV) was found in only 1.5% of the samples. SCMV was present in the 35 samples from Bolivia and Uruguay. SCMV and SrMV were both detected in symptomatic plants from Paraguay, but only few samples originated from this country. Some samples (4.4%) from Argentina tested negative for SCMV and SrMV and the virus present in these samples remains to be determined. Future work will be focussed on sequencing of the DNA fragments obtained by RT-PCR and diversity studies of the virus sequences. Conserved viral regions will be used to design gene constructs for sugarcane transformation. Discussion that followed this presentation was focussed on the putative identity of the virus present in the undetermined samples: Sugarcane streak mosaic virus (SCSMV), another Potyvirus or a new and undescribed virus are possibilities. 

Session 6. (Moderator: Philippe Rott)

Five papers were presented in this session: one on mosaic causing viruses in the United State, two on phytoplasmas and two on yellow leaf viruses. 

18. Virus strains causing mosaic in Louisiana and Florida sugarcane.  Grisham M P¹, Li R² and Comstock J C³.  ¹ USDA, ARS, SRRC, Houma, Louisiana, USA, ² USDA, ARS, BARC, Bellsville, MD, USA and ³ USDA-ARS Sugarcane Field Station, Canal Point, Florida, USA..

M Grisham described the virus strains causing mosaic in Louisiana and Florida. Previously, the various strains of Sugarcane mosaic virus (SCMV), H, I and M were so significantly different that they had been reclassified as Sorghum mosaic virus (SrMV).  Recent surveys in Louisiana showed that strains present were: SrMV-I, 66%; SrMV-H, 14% and SrMV-M 6%. In 7% of samples, RFLP analysis indicated that the strain was different from H, I and M. Furthermore, an unknown strain was present in 10% of samples, suggesting the presence of another virus causing mosaic in Louisiana. In Florida samples, SCMV-E was observed in symptomatic samples and no SCMV or SrMV symptomatic plants were observed. No SCMV was found in Louisiana and no SrMV in Florida.  No Sugarcane streak mosaic virus was detected in samples from both States. The results of this study showed that there has been a shift in the strain profile in Louisiana since 1990-1995, when SrMV-H was most abundant (90%), followed by SrMV-I (10%) and SrMV-M (3-5%). The difference in the strain distribution of Florida and Louisiana could not be explained. It was felt that there is a need to investigate the genetic diversity of SCMV internationally. 

19. Molecular characterization of sugarcane grassy shoot phytoplasma infecting sugarcane in India and its phylogenetic relations to closely related phytoplasmas.   Rao G P, Singh A K and Chand P. Sugarcane Research Station, Kunraghat Gorakhpur UP. India.  MLK PG College, Balrampur, UP. India.

Using nucleotide sequence analysis of 16SrRNA gene and 16S/23S rDNA established that sugarcane grassy shoot (SCGS) phytoplasma is closely related to that of sugarcane white leaf (SCWL). The two sugarcane phytoplasmas share a 97.5 to 98.8% homology with respect to their 16S rDNA sequences. SCGS phytoplasma also belongs to the rice yellow dwarf phytoplasma group. In contrast, sorghum grassy shoot phytoplasma is more distantly related to SCGS. Nested PCR is required to reliably detect SCGS. Resistance to the disease is present among cultivated varieties.   

20. Molecular investigation and phylogenetic analysis of sugarcane leaf yellows phytoplasma (16SrII ribosomal group) in India.  Gaur R K¹, Rao G P² and Marcone C³.  ¹ Department of Biotechnology, Faculty of Arts, Science and Commerce, Mody Institute of Technology and Sciences, Sikar-332311, Rajasthan, India, ² Sugarcane Research Station, Kunraghat, Gorakhpur-273008, Uttar Pradesh, India, ³ Dipartimento di Sciennze Farmaceutiche, Universita degli Studi di Salerno, 1-84084, Fisciano (Salerno), Italy.

The occurrence of Sugarcane yellows phytoplasma (SCYLP) associated with yellow leaf syndrome (YLS) exhibiting symptoms of sugarcane leaf yellows and yellowing of midribs was reported in India. A phytoplasma characteristic ~0.840 kb rDNA PCR product were amplified from DNAs of all infected sugarcane leaf samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs P1/P7 and fU3/rU5. RFLP analysis of PCR products with Hae III and Hha I endonuclease generated fragment profiles that were identical for all the samples.  The 16S rRNA sequence of the Indian SCYLP isolate (EU170474) showed the closest identity (99%) with that of SCYLP isolate in Cuba identified in Macroptilium lathyroides (AY725233) and other grasses like Cynodon dactylon (AB052871), Conyza canadensis (AY 725231) and Sorghum halpense (AY 725232), which belong to 16SrXII (Stolbur group). This was the first report of 16SrXII group phytoplasma affecting sugarcane in India. 

21. Sugarcane yellow leaf virus in Mauritius.  Joomun N and Dookun-Saumtally A.. Mauritius Sugar Industry Research Institute, Réduit, Mauritius.

A Dookun-Saumtally reported on a more sensitive detection of Sugarcane yellow leaf virus (SCYLV) using real-time fluorescent Taqman ® RT-PCR assay.  It was found to be 100x more sensitive than conventional RT-PCR. This method allows the simultaneous detection of the virus and an internal sugarcane control. The O-met internal positive control increases the reliability of the test by eliminating false negatives. Also, genetic diversity studies using a two-step RT-PCR showed the presence of REU, predominantly, as well as CUB and BRA-PER in the Mauritian germplasm collection.  Mixed infection of REU with either CUB or BRA-PER genotype was also present. 

22. Variation of sugar cane yellow leaf disease status in the French West Indies. Daugrois J H¹, Edon-Jock C¹, Fernandez E², Girard J C², and Rott P². ¹ CIRAD Département BIOS, Petit-Bourg, Guadeloupe, F. W. I. and ² UMR CIRAD-INRA_Montpellier SupAgro Biologie et Génétique des Interactions Plante-Parasite, Montpellier, France.

The incidence of SCYLV was compared in the two geographically close islands of Guadeloupe and Martinique. Infection is lower in Guadeloupe, although the vector population and cultivated varieties are the same. In Guadeloupe, REU genotype predominates over BRA-PER and REU.  In Martinique, BRA-PER is more important and sometimes with mixed infection of REU. The differences observed between the two islands suggest the occurrence of local effects and interactions. 

Session 7. (Moderator: Jeff Hoy)

Three papers were presented on sugarcane yellow leaf virus on the following topics: incidence, yield effect and control and improved detection. 

23. Incidence of sugarcane yellow leaf virus in commercial varieties in Mauritius. Khoodoo M H R, Behary-Panray N, Joomun J, Dookun-Saumtally A, Ganeshan S and Saumtally S. Mauritius Sugar Industry Research Institute. Réduit, Mauritius.

An island-wide survey for the presence of sugarcane yellow leaf virus in 22 commercial cultivars revealed that 58.8 % of the over 3000 samples were infected with the virus.  The incidence of infection ranged from zero for M 1176/77 to 100 % for R 579.  Although severe symptoms were observed in varieties M 695/69, M 52/78, M387/85, M1186/86, M 1400/86, M 2004/88, M 703/89, M 2593/92, R 570, R 573, and R 575 the presence of the virus did not correlate to the presence of the virus.  The presence of the aphid vector, Melanaphis sacchariwas low and the spread of the virus was thought to be primarily by seed cane. 

24. Yield effect and control of yellow leaf disease under under tropical conditions in Ecuador.  Garces F, Mendoza J, Valladares C, Fiallos F and Burbano C.  Centro de Investigación de la cana de azúcar del Ecuador (CINCAE), Guayaquil. Ecuador.

During 2001 to 2005, the incidence of sugarcane yellow leaf virus increased dramatically in Ecuador.  Yield trials using disease-free free plants obtained via meristem culture had 20 % more cane production and 36 % more TSH than infected plants.  Plants treated with systemic acquired resistance products had increased yields compared to untreated plants.  The best method of control was a combination of using virus-free plants derived from meristem culture along with systemic acquired resistance products. 

25. Improved detection of sugarcane yellow leaf virus based on genetic diversity studies of sugarcane yellow leaf virus.  Girard J C¹, Fernandez E², Royer M², Daugrois J H¹, Edon-Jock C and Rott P¹.  ¹ UMR CIRAD-INRA-Montpellier SupAgro Biologie et Génétique des Interactions Plante-Parasite, Montpellier, France, ² CIRAD Département BIOS, Station de Roujol, Petit-Bourg, Guadeloupe, F. W. I.

Sugarcane yellow leaf virus is difficult to recognize because of the lack of and in some cases non-specific symptoms.  Prior to the recognition of the disease and diagnostic techniques were developed the virus probably was spread worldwide without any knowledge.  Because there are four different genotypes of the virus, diagnosis is not equally as sensitive with all genotypes. CUB (for the Cuba) isolate was the most difficult with the available primers.  New primers were developed that allowed detection of all isolates.  The distribution of CUB isolates is being investigated using the newly developed primers. 

Session 8.  (Moderator: Germán Serino)

Three papers on sugarcane smut were presented by the Australians on the following topics:  molecular detection of smut spores, smut biology and breeding for resistance. 

26. Molecular detection of sugarcane smut spores on spore trap tapes in Australia. Braithwaite K¹, Bade G², Croft B³, and Magarey R⁴. ¹ BSES Limited. Indoorpoopilly, Australia, ² BSES Limited, Bundaberg, Australia, ³ BSES Limited, Woodford, Australia, ⁴ BSES Limited, Tully, Australia.

With the introduction of sugarcane smut in Australia, Burkard spores traps were used to detect sugarcane smut spores in areas prior to the visual detection of the disease.  Initially smut spores were identified by visual inspection of the collection tapes.  Because dirt particles and other spores visual confirmation was slow and difficult.  A DNA extraction and PCR based assay method was developed that allowed easy confirmation of the presence of smut spores.  In several sugarcane growing areas smut was identified on the tape samples prior to the actual visual observation of smut whips in the same areas.  The conformation of the aerial spread of smut into area where smut had not been observed helped to influence growers to shift to smut resistant varieties.    

27. Some aspects of the biology of the sugarcane smut fungus (Ustilago scitaminea) in Australia. Bhuiyan S A, Croft B J and Cox M C.  BSES Limited, Australia.

Sugarcane smut spores are a major method of spread of smut long distances. However, the survival of smut spores is influenced by temperature and moisture. Spores germinate under temperatures ranging from 12 to 36 ° C. Under moist conditions smut spores survived only for short time periods (2-to-3 months) in the soil, sugarcane trash and in the laboratory. However, survival of smut spore in under dry condition is greatly extended. Smut spores will survive for up to 6 months on machinery and on cotton clothing and these mechanisms could in theory spread the pathogen.   

28. Breeding smut-resistant sugarcane varieties in Australia. Croft B J, Cox M C and Bhuiyan S A.. BSES Limited, Australia.

Prior to the introduction of sugarcane smut into Australia over 1700 sugarcane clones had been screened for their reaction to the pathogen, Ustilago scitaminea. Because of the lack of any previous screening against smut 69 % of the clones were susceptible.  After the introduction of smut in Eastern Australia massive smut screenings were undertaken. The proportion of smut-resistant crosses increased from 0.4 % in 2000 to 52 % in 2007. This plus inoculating seedlings at the time of transplanting to the field and in the first clonal stage will rapidly shift resistance in the BSES-CSIRO Variety Improvement Program.   

Session 9. (Moderator: G. P. Rao)

Three papers were presented; one on techniques of detecting disease variability, one on Ramu Stunt and one on transient gene expression in sugarcane using Agrobacterium tumefaciens. 

29. Techniques for determining disease variability and yield loss in commercial sugarcane. Grisham M P, Johnson R M, Viator R P and Zimba P V. USDA, ARS, SRRC, Sugarcane Research Laboratory, Houma, Louisiana, USA.

The usefulness of tools applied in precision agriculture to plant pathology were explained. The influence of environmental conditions and cultural practices on the incidence of brown rust was investigated. Infection was positively correlated with soil properties, particularly the levels of phosphorus and sulphur. It was deduced that excess fertilizer applications could bring about a higher rust incidence and thereby negatively affecting sucrose and cane yields. Remote sensing using a fibre optic spectrometer was utilised to determine leaf infection by SCMV or SrMV. Analysis of mild and severe SCMV leaf reflectance measurements were correctly classified in 75 and 68% of the cases, respectively. Leaves infected by SCYLV were correctly identified in 77% of the time. 

30. A preliminary diagnostic test for Ramu stunt disease of sugarcane. Braithwaite K¹, Croft B² and Magarey R³. ¹ BSES Limited. Indoorpoopilly, Australia, BSES, ² BSES Limited, Woodford, Australia and ³ Limited, Tully, Australia.

Evidence was presented suggesting that the causal agent of Ramu stunt disease is a virus. Viral preparations from infected leaves showed a 36 kDA protein to be consistently detected and isometric viral particles can be observed from the infected cv Ragnar. Sequences of RNA with homology to viral RNA-dependant RNA polymerase have been cloned. A test that can detect a 1 kb RT-PCR product in leaf RNA extracts and the insect vector has been developed for the disease. 

31. Transient gene expression in sugarcane using Agrobacterium tumefaciens. Bonilla M L, Ángel F and Victoria J I. Variety Program. Colombian Sugarcane Research Center-CENICAÑA..

Ms M.L. Bonilla of CENICANA, Columbia discussed the transient gene expression in sugarcane using Agrobacterium tumefaciens. She discussed the advantages and significance of Agrobacterium mediated transformation over particle bombardment and electroporation. This was discussed with several examples of different combination of vectors and gene constructs for successful transformation in sugarcane.  

Session 10.  (Moderator: Jershon López-Gerena)
Five papers were presented on sugarcane rusts. 

32. Is an alternative to host plant resistance needed for rust control? Can fungicides provide it? Hoy J. Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, Louisiana, USA..

The epidemiology of brown rust in Louisiana was discussed with major emphasis on yield losses, resistance and fungicidal control. The magnitude of demonstrated losses strongly suggested for exploring the application of fungicides. Strobilurin fungicides were found more effective than triazole fungicides in reducing rust severity and yield losses. However, combinations of these two fungicides types were more superior. Two applications of fungicides can provide better economic benefits against rust incidence and infection severity. However, research is further required for the time and mode of application of fungicides against rust infection for maximise economic benefit, and researches are in progress. Till today, the host resistance is the most desirable control method of brown rust in Louisiana. But adaptability of the pathogen and problems in regular replacement of commercial sugarcane varieties in sugarcane are the major hurdles. The necessacity for opportunistic fungicidal control option for management of the new introduction of orange rust into Florida and possibility of coming introduction into Louisiana and Texas was also suggested. 

33. Characterization of the Bru1 (brown rust resistance) locus; distribution in sugarcane cultivars. Royaert S¹, Le Cunff L², Costet L¹, Raboin J M¹, Hoarau J Y³, Telismart H¹, Hervouet C², Garsmeur O², Nibouche S¹ and D’Hont A². ¹CIRAD, UMR PVBMT, Pôle de Protection des Plantes, Station de Ligne Paradis, Chemin de l’IRAT, Saint-Pierre, Réunion, France, ²CIRAD, UMR DAP, Avenue Agropolis, Montpellier Cedex 5, France, ³CIRAD, Station de Roujol, Petit-Bourg, Guadeloupe, France.

A major dominant gene Bru1, conferring brown rust resistance was identified in cultivar R570 which was shown to confer resistance to eight isolates from Brazil, Colombia, Zimbabwe, USA (Florida), Reunion and Guadeloupe. The target haplotype map encompasses 15 markers that cosegregate with Bru1 and markers, and the physical map encompasses 16BAC clones was discussed. The markers surrounding Bru1 in R570 were surveyed in nearly 400 international sugarcane cultivars were also phenotyped for rust resistance in Reunion and Guadeloupe islands and found that Bru1 was present in most of the resistant cultivars. Thus this represents an alternative source of resistance to the rust pathogen. The PCR marker in perfect linkage disequilibrium with Bru1 can also be used as diagnostic for the presence of Bru1 in experimental sugarcane cultivars. 

34. Sugarcane Rust Inoculations. Comstock J C, Sood S G and Glynn N C. USDA-ARS Sugarcane Field Station, Canal Point, Florida.

Sugarcane brown and orange rust field inoculation technique was described where the plants were inoculated by placing 0.1 ml spore suspension of rust urediospores in the whorl of 3-month-old plants.  The optimum spore concentration was 10⁵ spores per ml^-1and the whorl inoculated screens gave results comparable to historical ratings.  A rating based on pustule type was used with emphasis on pustule sporulation to classify susceptible reactions.  The technique is reliable and uses a relatively small amount of inoculum allowing a large number of clones to be evaluated. 

35. Genetic variability o f brown rust of sugarcane (Puccinia melanocephala H. and P. Sydow) in Colombia. Cardona L M, Ángel F, López-Gerena J, Angel S J C and Victoria J I. Colombian Sugarcane research Centre- CENICAÑA, Colombia.

Brown rust severity has increased on resistant varieties (CC 85-92, CC 84-75, CC 93-3895, CC 92-2804 and CC 94-5827) during the last two years.  No molecular variation in the rust pathogen was determined using ITS 1F/ ITS4 primers and restriction enzymes (ALU I and Hinf I).  More genomic regions in the rust pathogen are being evaluated.  The reason for the increased severity of rust symptoms is unknown. 

36. Field evaluation of brown rust (Puccinia melanocephala H. and P. Sydow) in different sugarcane varieties in the Cauca Valley. Angel S J C, Guzmán R M L, Cardona. L M and Victoria J I. Colombian Sugarcane research Centre- CENICAÑA, Colombia.

Data on the variation of brown rust severity was presented in different areas of the Cauca Valley. Based on microscopic evaluation only the brown rust pathogen, Puccinia melanocephala, was present. Based on the differences in rust symptom expression a new race may be present. 

Session 11. (Moderator: Salem Saumtally)
A single paper on orange rust in Guatemala was presented prior to a discussion on the disease. 

37. Orange rust disease in Guatemala. Ovalle W, Orozco H, Quemí J and Melgar M.  Centro Guatemalteco de Investigación y Capacitación de la cana de azúcar- CENGICAÑA, Guatemala.

The present of sugarcane orange rust was detected in September 2007. The course of action in response to the new disease was reported. Surveys for orange rust determined the disease widespread on CP 72-2086 in Guatemala with low severity of symptoms. The orange rust reaction of the promising cultivars was being determined.  CP 72-2086 is being withdrawn from commercial production with no new plantings of it. Resistant cultivars, CP 88-1165, CP 73-1547, CP 88-1508, CG 96-135 and CG 98-10, are being expanded. Rust screening of clones in the variety development program has been initiated. 

Orange Rust Discussion: Moderator: Jorge I. Victoria

A lively discussion on orange rust occurred. As of the Workshop the orange rust was confirmed in Florida (USA), Costa Rica, Guatemala, Nicaragua and Panama. Jack Comstock (USA), Werner Ovalle (Guatemala), Jaime Vega (Nicaragua), and Abby Guerra (Panama) presented information of orange rust in their countries. Photographs were presented showing the difference between the brown and orange rust symptoms, the pathogens, Puccinia melanocephala and P. kuehnii and the epidemiology of the two diseases. Orange rust tolerates warmer temperatures and its infection occurs during the warmer months and warmer locations where brown rust normally does not occur. Disease reactions of the commercial cultivars in the various countries were also presented. 

Visits

During the last two days of the Workshop, visits were organized to:
(i)                   CENICANA laboratories, glasshouses, nurseries
(ii)                 Trapiche La Palestina Panela mill
(iii)                Incuaca sugar mill
(iv)                Providencia Sugar mill
(v)                  Nurseries, commercial cane fields, Colombian open quarantine station, and transgenic fields
(vi)                Visit to a cane museum

A summary of the main outcome of the visits is given below.