Volume 6 Special Issue 2 2012
Sugarcane Pathology

ISBN 978-4-903313-97-9

Guest Editors

A. Ramesh Sundar, Rasappa Viswanathan

Sugarcane Breeding Institute, Coimbatore, India

CONTENTS AND ABSTRACTS

Asha Dookun-Saumtally, Salem Saumtally, Nawshad Joomun (Mauritius) Biotechnological Applications to Sugarcane Pathogens (pp 1-11)

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ABSTRACT

Invited Review: Sugarcane (Saccharum spp) is an important cash crop accounting for nearly two thirds of the world sugar production. Much effort has and is still being devoted to breeding resistant varieties, as the crop is vulnerable to a number of diseases that affect production. These constraints have triggered the adoption of biotechnological tools to counteract the impact of diseases on sugarcane. The increasing use of biotechnology is enabling early detection of pathogens of fungal, viral, bacterial and phytoplasma origin, allowing preventive measures to be adopted much earlier, before the appearance of disease symptoms as well as enabling the increased use of clean seed for establishment of nurseries and for the exchange of germplasm. The strategy of developing transgenic sugarcane varieties for resistance to several diseases is now well established and several transgenic lines are being evaluated in the field in a number of countries, although no commercial transgenic cane is grown thus far. Recent advances in sugarcane genomics as well as the genomics of sugarcane pathogens will soon provide information on disease resistance genes enabling new measures to be adopted in the breeding for disease resistance.

Sandy Snyman, Tania van Antwerpen, Sumita Ramgareeb, Sharon McFarlane (South Africa) Sugarcane Pathogens: Detection and Elimination through in Vitro Culture (pp 12-18)

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Invited Mini-Review: Commercial sugarcane cultivation, which involves: (a) multiple ratoons in the field for several years, (b) propagation by means of vegetative stem cuttings, (c) large areas under sugarcane monoculture, (d) production in tropical and subtropical climates, can exacerbate pathogen infestation and the spread of diseases. This paper reviews important diseases in sugarcane, methodology for detection of pathogens and highlights several interventions for pathogen elimination such as hot water treatment of stem sections and in vitro culture using meristematic cells as the initiation material. These have ensured a multi-faceted approach for disease management and enabled the production of material without pathogens such as Leifsonia xyli subsp. xyli, Xanthomonas albilineans, sugarcane mosaic virusand sugarcane yellow leaf virus. Additionally, international germplasm exchange may benefit by incorporating aspects of in vitro culture at various stage of the process.

Govind P. Rao, Smriti Mall (India), Carmine Marcone (Italy) Recent Biotechnological Approaches in Diagnosis and Management of Sugarcane Phytoplasma Diseases (pp 19-29)

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Invited Review: Phytoplasmas have been reported to be associated with two major sugarcane diseases viz., grassy shoot disease (GSD) and white leaf disease (WLD). Sugarcane yellow leaf syndrome is a new syndrome of sugarcane caused by a luteovirus and a phytoplasma. Sugarcane green grassy shoot (SCGGS) and ramu stunt are also associated with sugarcane from Thailand and Papua New Guinea, respectively. GSD and WLD are causing significant economic losses to sugarcane yield and sugar recovery in Asian countries. Both these phytoplasmas have been spreading very rapidly to newer locations with the help of infected seed material and leafhopper vectors. Hence it would be important to diagnose and manage these phytoplasmas at an early stage of sugarcane growth to avoid further spread and significant losses caused by them. Because of unreliable and unspecific symptoms, the identification and characterization of the associated phytoplasma at an early stage of plant growth is problematic and unreliable. The introduction of molecular genetic methods into plant mycoplasmology about 15 years ago greatly improved the diagnosis of phytoplasma infections in plant and insect hosts. PCR offers several advantages over other methods including versatility, relative simplicity, specificity and high sensitivity, which can be increased by a two-step PCR (nested PCR). It has also become possible to differentiate, characterize and classify the phytoplasmas on a phylogenetic basis, using mainly sequence analysis of ribosomal DNA (rDNA). The biotechnological and molecular assays developed in recent years have been utilized at a large scale to characterize these pathogens at an early stage of growth and for screening planting seed materials for possible elimination and management of theses phytoplasma at an early stage of their growth and propagation. We summarize research being conducted on characterization and management of these phytoplasma by novel biotechnological and molecular approaches.

Rasappa Viswanathan, Muthusamy Poongothai, Palaniyandi Malathi, Amalraj R. Sundar (India) Sugarcane Wilt: New Insights into the Pathogen Identity, Variability and Pathogenicity (pp 30-39)

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Invited Review: Wilt of sugarcane, a fungal disease is known to cause significant damage to sugarcane production and productivity in India and other countries for the past one century. Although sugarcane wilt is known in India for a long time, research work on this important disease is totally lacking. The causal organism was found to vary with time and investigator and could not reproduce the disease under artificial conditions in the field. We have made detailed disease surveys in 13 major sugarcane growing states in the country and 263 Fusarium isolates were isolated. We have established the variation in Fusarium isolates associated with sugarcane wilt, based on cultural, morphological, pathogenic and molecular characterization of 117 isolates. Critical observations in the conventional techniques combined with molecular biological approaches clearly established that Fusarium sacchari as the causal agent of the disease. Other Fusarium sp isolated from wilt infected sugarcane stalks were found to be either secondary invaders or non-pathogenic in nature. We have developed an artificial simulation technique to induce wilt in sugarcane under field conditions and efficient management strategy through biocontrol was also developed.

Rasappa Viswanathan (India) Molecular Basis of Red Rot Resistance in Sugarcane (pp 40-50)

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Invited Review: Red rot of sugarcane caused by Colletotrichum falcatum Went is one of the devastating diseases of sugarcane causing significant loss to sugarcane production in India and other Asian countries. Complex polyploidy and lack of information on inheritance to red rot in sugarcane make breeding for red rot resistance more difficult. Hence researchers have studied the mechanism of red rot resistance in sugarcane in detail. Initial studies based on biochemical tools identified oxidative enzymes and red rot pigments in disease resistance. Further studies revealed the role of pathogenesis-related (PR) proteins and 3-deoxyanthocyanidin phytoalexins especially luteolinidin and apigeninidin in red rot resistance. Recent studies using semiquantitative RT-PCR after pathogen inoculation from sugarcane cultivars varying in red rot resistance, revealed differential accumulation of transcripts of the flavanoid biosynthetic pathway like coumarate-4-hydroxylase, chalcone synthase, chalcone reductase, flavanoid 3′-5′ hydroxylase and flavanoid glycosyl transferase and this transcript analysis, further confirmed the role of sugarcane phytoalexins in red rot resistance. Similarly the role of PR- proteins like chitinase and b-1,3-glucanase was established at the transcript level. Detailed molecular studies using differential display (DD)-RT-PCR identified expression of more number of differentially expressed transcripts during the host-pathogen interaction. Full length sequences of many potential transcripts were identified and are being characterized. Also to identify specific proteins involved in host resistance, proteomic approach has been attempted by optimizing sample preparation from stalk tissues, 2-D electrophoresis (2-DE), down stream processing of identified spots and bioinformatics. Several resistance associated proteins spots were identified and they are being analyzed critically. Overall, application of molecular techniques was found to be useful in identifying transcripts/proteins involved in host defence. Further studies are in progress to validate their specific involvement in red rot resistance.

Durairaj Mohanraj, Purushothaman Padmanaban, Rasappa Viswanathan (India) Screening for Red Rot Resistance in Sugarcane (pp 51-62)

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Invited Review: Red rot of sugarcane caused by Colletotrichum falcatum Went is a serious fungal disease affecting sugarcane stalks, the economical part of sugarcane in different countries. In India, the disease has been a menace for sugarcane cultivation and many elite varieties were removed from cultivation due to their susceptibility to the disease. The commercial varieties are recommended for cultivation in India if they possess red rot resistance. Hence screening for red rot resistance became an integral part of varietal development in the country. Plug method of pathogen inoculation is the most common method used for red rot screening in sugarcane and here, severity score is being assigned based on the red rot symptoms inside the canes. The key parameters of the lesion such as lesion width, nodal transgression and nature and severity of white spots determine the disease severity. Since a bore-hole injury is caused on the stalk to inoculate the pathogen, there is an apprehension that natural barrier is breached to introduce the pathogen in this method. So many other methods were developed to facilitate the pathogen to enter the cane tissues in a natural way. However the disease reaction in these methods is influenced by environmental factors which lead to disease escapes under field conditions. Recently a controlled condition testing was developed to screen large number of progenies using cane tops in a short time. Overall, the plug method is used to assess resistance potential of a genotype in a foolproof way in sugarcane varieties proposed for commercial cultivation and the controlled condition testing is followed initially to screen large population.

Amalraj Ramesh Sundar, Nagarathinam Selvaraj, Muthumeena Muthiah, Ashwin Ramadass, Palaniyandi Malathi, Rasappa Viswanathan (India) Induced Resistance – A Potential Supplementary Strategy for the Management of Red Rot in Sugarcane (pp 63-72)

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Invited Review: Induced resistance (IR) is the phenomenon by which a plant exhibits an increased level of resistance without changes of its basic genetic constitution. IR utilizes the plant’s own defense mechanisms to restrict pathogen development. Resistance can be induced locally on the pre-treated parts of the plant as well as systemically on untreated parts and is then called systemic acquired resistance (SAR). Several chemicals such as salicyclic acid, acibenzolar-S-methyl, iso-nicotinic acid, jasmonic acid, etc. are known to induce resistance in certain plants, but only a few of them fulfil the criteria for being real plant activating compounds. The introduction of novel synthetic signal molecules is thought to be the first step for the integration of the principle of IR in a modern environmentally friendly plant protection concept. Plant diseases account for a considerable proportion of crop loss worldwide. In sugarcane, red rot disease caused by the fungus Colletotrichum falcatum is one of the important pathogens causing significant loss to the growers and the sugar industry. Many of the commercially superior varieties are out of cultivation because of either high levels of red rot susceptibility or breakdown of resistance, which is attributed to the emergence of new virulent pathotypes. In addition to breeding for durable red rot resistance, other viable options need to be explored for successful management of the disease. In this background, exploitation of the inherent SAR potential of highly successful commercial varieties, otherwise susceptible to red rot is a viable option to manage the disease. With the advent of tools of biotechnology, it has now become possible to understand better the molecular basis of IR and to regulate defense genes for augmenting disease resistance in crop plants. This review attempts to comprehensively present all the available information on inducing disease resistance in sugarcane, with a special focus on the red rot disease.

Porntip Wongkaew (Thailand) Sugarcane White Leaf Disease Characterization, Diagnosis Development, and Control Strategies (pp 73-84)

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Invited Mini-Review: Sugarcane white leaf (SCWL) is the most destructive disease of sugarcane in Thailand where major cane and sugar production of the world are situated. The severity degree and symptom variance are depended on soil fertility, temperature, cane sette quality, cultural practice and the phloem-colonized phytoplasma amount. The leafhopper Matsumuratettix hiriglyphicus is the first known vector that transmits SCWL phytoplasma. Recently at least other six leafhoppers have been found containing SCWL phytoplasma DNA. However, only Yamatotettix flavovitatus has been closely investigated and found its transmissibility but with lesser extent. Outbreak of SCWL disease is mainly activated by transportation and propagation of endemic cane setts. This SCWL phytoplasma is closely related to Thailand sugarcane green grassy shoot (SCGGS) and India sugarcane grassy shoot (SCGS) with 96-98% similarity in their 16S-23S rDNA sequences. Some diversity among SCWL phytoplasma isolates from different sources and growing locations has also been indicated within the range of 89-98% similarity by the 16S-23S rDNA sequence analyses. In addition to existed conventional diagnosis methods, the electrochemical DNA sensors have been proposed as a new tool for reliable routine practice. At present, control of the SCWL disease is difficult due to their systemic nature and the lack of desirable resistant variety. Application of insecticide is ineffective either. The disease control measures thus are mostly emphasized on the other strategies such as physical and chemical treatment of the cane setts, disease free plant production via tissue cultures, sanitation and crop rotations, and regulatory quarantine. Cooperative assistance among growers and involving association is strongly needed to accomplish effective control.

María Blanch, María-Estrella Legaz, Carlos Vicente (Spain) Structure and Biosynthesis of a Xanthan-like Polysaccharide Produced by Xanthomonas albilineans (pp 85-90)

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Invited Mini-Review: Leaf scald is a vascular disease of sugarcane plants caused by Xanthomonas albilineans. Scalded leaves show white-yellowish streaks alternating with green zones in parallel to the main veins. The white-yellowish streaks show both phloem and xylem completely occluded by a gum and the overall mesophyll appears to be full of this bacterial secretion. The gum in conducting tissues has been purified from juices obtained from scalded stalks and it was identified as a xanthan-like polysaccharide composed by repeated tetrameric units containing two rests of fructose, one of mannose and one of glucuronic acid. Hydrolysis of xanthan with selective mannosidases and b-1,4-glucanases reveals that the macromolecule consists of a linear, b-1,4-backbone of b-glucose units to which mannose in a-1,3 bonds is linked. Since xanthans contain glucuronic acid, the ability of Xanthomonas to produce an active UDP glucose dehydrogenase is often seen as a virulence factor. X. albilineans produces a UDP-glucose dehydrogenase growing on sucrose. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid by using molecular oxygen and NADPH. The value of pI of the purified enzyme is 8.98 and its molecular mass has been estimated as about 14 kDa. The enzyme is inhibited by UDP-glucose concentrations higher than 1.3 mM X. albilineans cultured axenically does not secrete xanthan to liquid media but it is produced by bacteria growing in sugarcane tissues. This host-dependence can be explained on the basis of the action of bacterial proteases upon the dehydrogenase. Glycoproteins from sugarcane, the natural host of the bacterium, produced as a response to the bacterial invasion assure the production of the active enzyme by inhibiting bacterial proteases.

Jean Heinrich Daugrois, Rosiane Boisne-Noc (Guadeloupe F.W.I.), Patrice Champoiseau (Guadeloupe F.W.I./USA), Philippe Rott (France) The Revisited Infection Cycle of Xanthomonas albilineans, the Causal Agent of Leaf Scald of Sugarcane (pp 91-97)

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Invited Mini-Review: Leaf scald, a sugarcane disease caused by Xanthomonas albilineans, has been largely studied since its discovery in 1911. Numerous data were obtained and reported, from isolation, transmission and diagnosis of the pathogen to the complete sequence of its genome. Although X. albilineans was thought to be exclusively transmitted by infected cuttings and contaminated cutting implements for more than 80 years, the existence of an epiphytic life of this pathogen was reported in the late 1990s. We highlight herein the role of the epiphytic populationsof X. albilineans in plant and field contamination and the impact of environmental conditions, especially rainfall, on these populations of the pathogen. Data obtained from experimental research in Guadeloupe showed the capacity of X. albilineans, when rainfall is sufficient, to spread by aerial means within the sugarcane canopy in a field or between fields. This new epidemiological trait helps X. albilineans in contaminating rapidly healthy sugarcane plants and, in certain geographical locations, it can be much more important than disease spread by cutting implements. These new epidemiological data of sugarcane leaf scald lead us to propose a revisited infection cycle for X. albilineans.

María Francisca Perera, María Paula Filippone, Aldo Sergio Noguera, María Inés Cuenya, Atilio Pedro Castagnaro (Argentina) An Overview of the Sugarcane Mosaic Disease in South America (pp 98-107)

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Invited Review: Sugarcane mosaic, one of the most important viral diseases of sugarcane, is widely distributed in the world and its economic significance varies among regions. Economic losses depend on varietal susceptibility, virus strains, its interaction with other diseases, vector population and environmental conditions. Although not a major problem in some countries, sugarcane mosaic has caused substantial yield losses in other countries (Argentina, Brazil) due to severe outbreaks. Numerous strains of Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) are commonly associated with mosaic symptoms. Both viruses are members of the SCMV subgroup in the genus Potyvirus of the family Potyviridae and their genetic variability could be effectively assessed only through DNA sequence comparisons. The greater genetic variability of viruses associated with sugarcane mosaic needs to be taken into consideration in breeding and biotechnology programmes for resistance to mosaic. The most effective way to control sugarcane mosaic has been through the use of resistant cultivars, which requires a complete understanding of the genetic diversity of the pathogens as well as their interaction with cultivars; resistance breakdown can occur when new strains or viruses appear. However, the production of healthy and genetically pure seed cane could be an available tool to reduce the pathogenic load in sugarcane-growing areas. This could be achieved through hydro-heat-treatment followed by apical meristem in vitro culture and micropropagation. It is also relevant to implement extreme quarantine measures to prevent the entry of new pathogens or variants of the established ones through germplasm exchange.

Marcos Cesar Gonçalves, Luciana Rossini Pinto, Silvana Creste Souza, Marcos Guimarães A. Landell (Brazil) Virus Diseases of Sugarcane. A Constant Challenge to Sugarcane Breeding in Brazil (pp 108-116)

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Invited Review: Sugarcane mosaic virus (SCMV) and Sugarcane yellow leaf virus (SCYLV) are the two main viruses infecting sugarcane in Brazil. The authors analyze the emergence and current status of these pathogens and what has being done to minimize losses and prevent new outbreaks in sugarcane. It was shown that SCMV is the only causal agent of mosaic in sugarcane in the country. Most sugarcane cultivars are believed to be tolerant or intermediate resistant to mosaic, although new cases have been reported in the field. One recently characterized SCMV isolate comprises a novel severe strain capable of infecting cultivars previously considered to be resistant. SCYLV, the causal agent of sugarcane yellow leaf, is widely distributed in Brazil and other sugarcane producing countries causing significant yield losses. The virus became widespread in the field and in parental clones used in sugarcane breeding programmes. Sensitive and reliable detection methods for SCYLV were developed and have been routinely applied for diagnosis while screening for resistance, virus elimination for germplasm exchange, and production of virus-free seed cane. Screening for resistance to sugarcane mosaic and sugarcane yellow leaf is considered a primary and essential step in sugarcane breeding programmes in Brazil.

Trinh Xuan Hoat, Dang Vu Thi Thanh (Viet Nam), Matthew Dickinson (UK), Ngo Gia Bon, Mai Van Quan, Vu Duy Hien, Nguyen Duc Thanh, Le Thanh Thuy, Ngo Vinh Vien (Viet Nam) Disease Problems of Sugarcane in Vietnam, with Special Reference to Phytoplasma (pp 117-123)

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Invited Mini-Review: Seventeen diseases of sugarcane have been found in Vietnam including 13 fungal, 2 bacterial and 2 phytoplasmal diseases. Among them, the most important diseases are smut, pineapple, red rot, sugarcane white leaf and sugarcane grassy shoot diseases. We applied nested PCR using universal primers for detection and characterization of phytoplasmas from infected tissues. PCR products of the expected size (1200bp) were obtained from the 16S rRNA of the phytoplasma. The RFLP profiles indicated that all the samples were infected by the same pathogen. Nucleotide sequence analysis of the 16S rRNA genes revealed that the phytoplasma causing sugarcane grassy shoot disease in Vietnam is very similar to the phytoplasmas causing sugarcane grassy shoot disease in India sharing a sequence similarity of 99%. Phylogenetic trees showed that SCGS and SCWL strains collected from Vietnam and other reported SCGSs belong to the 16SrXI subgroup RYD (Rice Yellow Dwarf) 16S rRNA Group.

Lilik Koesmihartono Putra, Tri Asmira Damayanti (Indonesia) Major Diseases Affecting Sugarcane Production in Indonesia (pp 124-129)

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Invited Mini-Review: Although a large number of sugarcane pathogens have been recorded in Indonesia, the current major sugarcane diseases affecting sugar production in Indonesia are ratoon stunting disease, smut, mosaic and leaf scorch. A new type of mosaic disease caused by Sugarcane streak mosaic virus is now widely distributed in most commercial sugarcane areas in Java (Indonesia). An integrated management strategy is used for controlling the disease and minimizing their impact on sugar production.

Heather McCafferty, Yun J. Zhu (USA) Transgenic Approach to Improve the Resistance of Sugarcane to Sugarcane Yellow Leaf Virus (SCYLV) (pp 130-133)

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Invited Mini-Review: Genetic transformation of sugarcane using various methods has been reported. In this review, an overview of the sugarcane yellow leaf virus (SCYLV) will be presented. The technical approaches used to create transgenic sugarcane through transformation, selection and regeneration will be reviewed. The genetic engineering methods for sugarcane include Agrobacterium-mediated transformation, electroporation and particle bombardment. In particular, work in Hawaii and Florida with constructs conferring resistance to sugarcane yellow leaf virus will be highlighted. Finally, thoughts about the future of sugarcane transformation will be discussed.

Ana Carolina Wanderley-Nogueira, Ederson Akio Kido, Nina da Mota Soares-Cavalcanti, Luis Carlos Belarmino, João Pacífico Bezerra-Neto, William Lee Burnquist, Sabrina Moutinho Chabregas, José Ivo Baldani, Ana Maria Benko-Iseppon (Brazil) Insight on Pathogen Defense Mechanisms in the Sugarcane Transcriptome (pp 134-148)

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Original Research Paper: Recognition of pathogen and activation of defense mechanisms is a common feature known from all multicellular organisms. Among higher plants, systemic acquired resistance (SAR) is known to activate pathogenesis-related (PR) genes after recognition of the pathogen mediated by a resistance (R) gene. Both gene classes (R and PR) represent the main mechanism against biotic and sometimes also abiotic stresses. Therefore, the identification of SAR metabolism components is an important concern regarding plant breeding. Sugarcane (Saccharum officinarum) is a major tropical and subtropical crop, grown in more than 120 countries, being especially important due to its value for the production of bioethanol, constituting a renewable energy source, besides the sugar production. The present work brings an overview on sugarcane resistance and pathogenesis-related genes, regarding their structure, abundance and role in the plant-pathogen metabolic pathway and also regarding their distribution, as compared with rice. For this purpose a collection of 282,818 expressed sequences tags have been evaluated to identify R and PR genes as well as important factors identified in the classic plant-pathogen metabolic KEGG pathway using rice full length cDNA as seed-sequences. The identified sugarcane genes have been also used to screen four SuperSAGE libraries with 8,787,313 tags, allowing the identification of the main activated and repressed genes under abiotic stress (drought/salinity) conditions. The 1,460 identified genes have been plotted on a rice virtual karyotype inferring about their distribution, considering a putative synteny as a measure to infer about their relative position within rice and sugarcane chromosomes. The results revealed interesting insights on the variability and complexity of defense genes in sugarcane.

Velusamy Jayakumar, Seiya Tsushima, Palaniyandi Malathi, Rasappa Viswanathan (India) Elution, Biological Activity and Characterization of Phytotoxin Produced by SugarcaneRed RotPathogen Colletotrichum falcatum (pp 149-163)

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Original Research Paper: An attempt was made to assess the chemical nature of the toxic compound(s) present in the culture broth of Colletotrichum falcatum by selection of only biologically active fractions at every stage of toxin elution and characterization. In liquid culture, C. falcatum produced toxic metabolites that are soluble in water, methanol and diethyl ether. The column eluted semi-purified form of secondary metabolites produced severe necrotic lesions on sugarcane leaves at a concentration of 5000 mg/l and the toxin was found to be host specific. Production of necrotic lesion size in sugarcane leaves increased with increase in concentration and time of toxin inoculation, i.e. severe necrotic lesions were noticed after 72 h at 10000 mg/l and after 96 h at 5000 mg/l. The biologically active column eluted fractions viz., RP1, RP2, RP3 and RP8 were analyzed with HPLC and GC-MS for structural characterization. The analysis of biologically active fractions identified seven toxic compounds, viz., C₁₂H₃₀O₄, C₁₆H₄₀O₅, C₂₁H₅₀O₇, C₁₇H₂₉NO_5, C₂₄H₆₂O₆, C₃₀H₅₀O₄ and C₁₅H₂₄O in the metabolites of C. falcatum. Among the identified compounds C₁₇H₂₉NO₅ (methoxy phenylpyruvic acid) appeared consistently (at Rt 10.1 min of GC/MS analyzed TIC) in all the biologically active fractions tested. These seven identified compounds constitute phenolics, sugars, acids and few esters.