international society of sugar cane technologists...
TRANSCRIPT
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INTERNATIONAL SOCIETY OF SUGAR CANE
TECHNOLOGISTS
10th Pathology Workshop
‘Understanding Sugar Cane Diseases for their Efficient
Management’
ABSTRACTS
Hosted by
Guangxi Academy of Agricultural Sciences (GXAAS)
State Key Laboratory of Subtropical Bioresources Conservation and Utilization (Guangxi University)
NANNING, CHINA
7 – 11 May, 2012
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Keynote address
GENOME SEQUENCING OF PATHOGENS FOR A BETTER UNDERSTANDING
AND MANAGEMENT OF SUGARCANE DISEASES
P. ROTT
CIRAD, UMR BGPI, F-34398 Montpellier, France
Keywords: Molecular diagnostic methods, Sugarcane yellow leaf virus, Xanthomonas
albilineans
Sequence data from the whole genomes of over 1000 organisms are currently available in the
NCBI (National Center for Biotechnology Information) data base, including genomes of
several viral and bacterial sugarcane pathogens. Genome sequences of sugarcane viruses have
been extensively used to develop molecular diagnostic methods that are very efficient and
useful for the identification and detection of these pathogens, and their control during
movement of sugarcane germplasm. Furthermore, whole genome comparisons have
improved our knowledge of molecular evolution and molecular ecology of sugarcane
pathogens. For instance, sequencing and annotation of the genome of Xanthomonas
albilineans revealed that this bacterial pathogen experienced genome reduction during its
speciation. Additionally, this xanthomonad is notably missing the Hrp type III secretion
system and the xanthan gene cluster that are commonly found in pathogenic Xanthomonas
species. Transposon mutagenesis of X. albilineans resulted in the identification of new
candidate fitness and pathogenicity factors that could be used to develop genetic-based
control methods. The comparison of several isolates of Sugarcane yellow leaf virus (SCYLV)
resulted in the identification of several genotypes of this virus which vary in their capacity to
infect sugarcane cultivars. Knowing the genetic diversity of SCYLV in a given location is
therefore important for efficient screening of resistant plant material. However, although
several virus pathogens are already known and extensively studied, it is assumed that
unknown pathogens are still to be discovered in sugarcane, especially if these pathogens do
not cause symptoms that can be easily observed, such as SCYLV. Sequencing of unknown
genomes will facilitate implementing of a new strategy of diagnostics, the so-called
sequence-independent approach which aims at deciphering the virome (= the genomes of all
the viruses that inhabit a particular organism). It is believed that this metagenomics approach
will drastically improve routine quarantine diagnostics in the future and gather dozens, if not
hundreds, of genome sequences that will eventually contribute to the understanding of the
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diversity and evolution of these pathogens, and the pathogenicity mechanisms used to cause
disease.
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RESISTANCE TO SUGARCANE BROWN RUST: OPPORTUNITIES AND
CHALLENGES IN USING THE BRU1 RESISTANCE GENE
N. C. GLYNN, J. C. COMSTOCK AND K. MCCORKLE
1USDA-ARS, Sugarcane Field Station, Canal Point, FL, USA
Keywords: Puccinia melanocephala, disease resistance, molecular screening
Brown rust of sugarcane caused by, Puccinia melanocephala, is a serious problem in the US
sugarcane industry since its introduction in 1978. A major resistance gene, Bru1 was
identified and methodology for detecting it was developed by French scientists at CIRAD.
The majority of the research resulting in the discovery of Bru1 resistance gene was funded by
the International Consortium for Sugarcane Biotechnology (ICSB). Our objective was to
determine the frequency of Bru1 in parental clones, historical clones and all the clones in
Stage II (CP 10 Series) at Canal Point, Florida and to determine its usefulness in the CP
Cultivar Development Program. Of the 1072 parental clones used at Canal Point, Bru1 was
detected in 27 % (287) of the clones. The frequency of Bru1 differed among the parental
clones used for the Florida, Louisiana and Texas crossing programs with 42 % of the Florida
clones and 6 % of the Louisiana clones containing the resistance gene. There was an increase
in the Bru1 frequency in CP clones through time indicating a selection of the gene based on
selection of resistance by lack of rust development in the field. Interestingly brown rust
resistant commercial cultivars, CP 80-1743, CP 88-1762, and CP 89-2143 have Bru1 gene
while susceptible cultivar CP 96-1252 does not. Thus, well over 75 % of the commercial
sugarcane acreage in Florida relies on Bru1 to control brown rust. Bru1 was detected in 44 %
of the 1515 clones in Stage II. The molecular screening for the Bru1 resistance gene in the
CP development program will increase the frequency of brown rust resistance. However, a
serious threat is the breakdown of Bru1 resistance as has happened with the dependence of
similar major genes in other crops. Fortunately, there appears to be other resistance genes
present in the germplasm since some clones are resistant but Bru1 was not detected.
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SCREENING FOR SUGARCANE SMUT IN AUSTRALIA – SMUT TO
SMUTBUSTER
1
S.A. BHUIYAN, 2
B.J. CROFT, 1
M.C. COX, AND 3
R.C. MAGAREY
1BSES Limited, Bundaberg, Australia,
2BSES Limited, Woodford, Australia,
3BSES Limited,
Tully, Australia
Keywords: Economic loss, smut incursion, screening trials, smut-resistant progeny
Sugarcane smut was first reported in Queensland in June 2006. Since then there has been a
rapid disease escalation and smut has now been found in most sugarcane regions. It was
estimated that smut would be found in every farm in Bundaberg-Isis, Mackay and Herbert
region in 2009 and 2010, and economic loss would be reported in crops with susceptible
varieties. BSES commenced screening Australian varieties for resistance to sugarcane smut in
Indonesia in 1998, a few months after the disease was found in the Ord River Irrigation Area
(ORIA) of Western Australia. The screening trials have shown that 69% of the Australian
clones were susceptible to smut. These smut resistance ratings have been used to select
resistant varieties for release to growers, select parent clones for smut specific crosses and to
advise growers of the risk they faced from a smut incursion. The ratings obtained from this
program played a major role in the emergency response, when smut was first found in
Queensland. BSES commenced a smut screening program in Bundaberg, Queensland in
2006. Since then, approximately 10,000 clones from various stages of selection programs
have been screened for smut resistance. Majority of our productive parent clones are highly
susceptible to smut, and a program, called SmutBuster, has commenced to recover the genes
for high productivity found in these parents by identifying smut-resistant progeny from
crosses between susceptible parents and from crosses between susceptible and resistant
parents. Also, it will maintain the current rate of genetic gain by doubling the size of the
selection program. The progress and prospect of current screening program against sugarcane
will be discussed.
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INTROGRESSION BREEDING – DEVELOPMENT OF NEW GERMPLASMS
WITH ENHANCED RESISTANCE TO NEMATODE, PACHYMETRA ROOT ROT
AND SMUT IN AUSTRALIA
1 2
S. A. BHUIYAN, 1
B. J. CROFT, 3
P. JACKSON, 4
R. MAGAREY, 5
G. STIRLING AND 2
M. COX
1
BSES Limited, Woodford, 2 BSES Limited, Bundaberg,
3 CSIRO Townsville,
4 BSES Limited,
Tully, 5 Biological Crop Protection, Brisbane
Keywords: Variety screening, introgression crosses, valuable traits
Smut (Sporisorium scitamineum), pachymetra root rot (Pachymetra chaunorrhiza) and
nematodes (mainly Meloidogyne spp. and Pratylenchus spp) are three of the most important
diseases of sugarcane in Australia. These three diseases currently cause losses of $100-
200M/year to the Australian sugar industry. After the incursion of smut in Queensland in
2006 it was evident that most of the commercial pachymetra root rot resistant varieties were
susceptible to smut. Currently no Australian commercial varieties are resistant to nematodes.
Recently, an Australian collaborative introgression program with Chinese institutes has used
new sources of germplasm, Erianthus spp. and a different set of Saccharum spontaneum
clones to generate over 100 new families. E. arundinaceus and some S. spontaneum clones
have a number of valuable traits, including high vigour, drought and waterlogging tolerance,
resistance to diseases, particularly pachymetra root rot. Preliminary testing of some clones
from crosses using these wild species has shown that they have potential for resistance to
nematodes. BSES-CSIRO has undertaken a project to test the clones from the introgression
crosses for resistance to smut, pachymetra root rot and nematodes. The first screening trials
for smut, pachymetra root rot and root lesion nematodes (P. zeae) were established using 120
clones selected from the introgression crosses in September 2011. A number of research trials
have been established to improve the current nematode screening methods. The results from
the screening and research will be presented.
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SHADES OF ORANGE AND BROWN: TOWARDS EXPLOITING QUANTITATIVE
RUST RESISTANCE IN SUGARCANE
R.S. RUTHERFORD, T. MHORA AND S.A MCFARLANE
South African Sugarcane Research Institute, Private Bag X02, Mount Edgecombe, 4300, RSA
Keywords: AFLP, near infrared, marker assisted breeding.
Most research resulting in the identification of disease resistance genes focuses on major
gene resistance where genetic effects are large and detection is straightforward. In contrast,
genes and mechanisms associated with quantitative resistance, controlled by multiple loci of
small effect, are poorly understood. Due to the effects of individual genes being small,
phenotyping must be performed with high levels of precision. To this end we are exploring
the use of whorl inoculation and detached leaf bioassays in conjunction with chlorophyll
fluorescence and near infrared reflectance spectroscopy for rapidly determining Puccinia
melanocephala (brown rust) reaction phenotype. These techniques are being assessed for
their ability to distinguish between major gene resistance, quantitative resistance and
susceptibility within a SASRI linkage disequilibrium (LD) mapping population. One-third of
the LD genotypes lack a known major gene for resistance to brown rust. These genotypes
have been used to detect AFLP markers associated with quantitative resistance. Results
suggest that quantitative resistance is present, and that it could be further improved and/or
combined with major gene resistance through marker assisted breeding. In combination with
rapid phenotype determination, a high throughput breeding and selection methodology is
under development.
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SUGARCANE ORANGE RUST: ITS HISTORY AND IMPACT IN FLORIDA
J.C. COMSTOCK1, N.C. GLYNN
1 AND R.W. DAVIDSON
2
1USDA-ARS Sugarcane Field Station, Canal Point, FL 33438
2Florida Sugarcane League, Clewiston, FL 33440
Keywords: Puccinia kuehnii, disease resistance, breeding programme
Sugarcane orange rust, caused Puccinia kuehnii, was confirmed in Florida in 2007 by
morphological and molecular techniques. Since then orange rust has been found in numerous
countries in the Western Hemisphere as well as in Africa. In Florida, orange rust has
impacted commercial production and cultivar development programs. Initial observations in
June 2007, severe symptoms of orange rust were observed on CP 80-1743, a cultivar that
occupied 25 % of the acreage and moderate symptoms were present on CP 72-2086,
occupying 4 % of the acreage. Yield losses occurred in 2008 on CP 80-1743. Although
orange rust can develop in warmer temperatures than brown rust the severity usually
decreases in the warmer summer months and then, reappears as cooler temperatures occur in
the fall. An orange rust resistance screening program was initiated and improvements in the
level of resistance have occurred in the Cultivar Development Program. Resistant cultivars
have been developed to control the disease and are being adopted by growers. Changes in the
breeding and the selection programs to develop rust resistant progeny will be discussed.
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IMMUNOLOGICAL AND MOLECULAR DETECTION OF COLLETOTRICHUM
FALCATUM WENT CAUSING RED ROT DISEASE IN SUGARCANE
(SACCHARUM SPECIES HYBRIDS)
R. VELAZHAHAN, K. NITHYA, K. A.I.M. BUKHARI, V. VALLUVAPARIDASAN AND
V. PARANIDHARAN
Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu
Agricultural University, Coimbatore- 641 003, Tamil Nadu, India
Keywords: ELISA, SCAR marker, PCR
Red rot disease caused by Colletotrichum falcatum Went is one of the most destructive
diseases of sugarcane (Saccharum species hybrids) worldwide. The pathogen spreads
primarily through infected sugarcane setts and hence the use of disease-free planting
materials is essential for preventing disease development in the field. In the present study, a
sequence characterized amplified region (SCAR) marker for accurate detection of C.
falcatum was developed in planting materials by using PCR. The SCAR primers were highly
specific to C. falcatum and the expected 442-bp fragment was amplified only from DNA of
isolates and races of C. falcatum but not from any other Colletotrichum spp. tested. The
detection sensitivity of C. falcatum was 0.1 ng for genomic DNA of C. falcatum and 5 ng for
DNA extracted from infected sugarcane tissue. The SCAR primers developed in this study is
highly specific and reproducible and proved to be a powerful tool for detection of this
important pathogen in seed canes.
In order to develop immunological tests for detection of C. falcatum two proteins with
molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum
and used as antigen source to raise polyclonal antibodies. The developed polyclonal
antibodies were tested for detection of C. falcatum by ELISA. The ELISA results revealed
that the developed polyclonal antibodies were highly specific to C. falcatum. The developed
antibodies were very sensitive and could detect C. falcatum proteins even at a dilution of
1:50000. The high specific reactivity and sensitivity of the antisera indicate their potential
suitability for ELISA-based detection of C. falcatum.
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GENETIC VARIABILITY AMONG THE ISOLATES OF COLLETOTRICHUM
FALCATUM WENT CAUSING RED ROT OF SUGARCANE IN TAMIL NADU,
INDIA
K. KARUNANITHI*, K. NITHYA, V. VALLUVAPARIDASAN, V. PARANIDHARAN,
R. VELAZHAHAN AND T. JAYARAJ+.
Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore- 641 003
Tamil Nadu, India
*Regional Research Station, Tamil Nadu Agricultural University, Vridhachalam- 606 001,
Tamil Nadu, India +Tamil Nadu Rice Research Institute, Tamil Nadu agricultural University,
Aduturai- 612 101, Tamil Nadu, India
Keywords: Toxin production, RAPD analysis, phylogenetic analysis
Red rot, caused by Colletotrichum falcatum Went, is one of the most important limiting
factors of sugarcane production in India. The pathogen shows a great diversity in virulence as
a number of pathotypes are known to occur in nature. In the present study, the toxin
producing ability and genetic variability among isolates of C. falcatum collected from major
sugarcane growing areas of Tamil Nadu, India were analyzed. The C. falcatum isolates
differed significantly in their ability to produce toxin in vitro. The toxin from C. falcatum
isolate Cf 671a induced the maximum electrolyte leakage (300 μS) from sugarcane leaf
tissues. The genetic relatedness of the isolates of C. falcatum differing in toxin production
potential was investigated by using RAPD analysis. Analysis of the genetic coefficient matrix
derived from the scores of RAPD profiles showed that minimum and maximum per cent
similarities among the tested C. falcatum isolates were in the range of 19 to 95% respectively.
The phylogenetic analysis by the UPGMA identified two main clusters. Cluster A contains
only one isolate (Cf 98061) and all the other isolates were placed in Cluster B confirming
high genetic diversity among the isolates. No correlation was observed between clustering of
the C. falcatum isolates in the dendrogram and their toxin producing abilities.
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MORPHOLOGICAL AND MOLECULAR DIVERSITY AMONG
COLLETOTRICHUM FALCATUM ISOLATES CAUSING RED ROT DISEASE OF
SUGARCANE IN UTTAR PRADESH, INDIA
D. SINGH1, A.K. TIWARI
2*, S. MALL
3, B. SHUKLA
3, I. Z. AHMAD
1 AND G.P. RAO
4
1Integral University, Lucknow, 226026 ,UP, India
2Sugarcane Research Institute, UPCSR, Shahjahanpur 242001, Uttar Pradesh, India
3Sugarcane Research Station, Kunraghat, Gorakhpur 273008, U.P. India
4Division of Plant Pathology, Indian Agricultural Research Institute, Pusa Campus,
New Delhi-110012, India.
Keywords: Mycelium growth, biological differentials, ITS gene
An extensive survey was conducted in sugarcane growing areas of eastern, central and
western regions of Uttar Pradesh during 2007-2009 to record the incidence of red rot disease
in different commercial varieties. Ten red rot isolates from infected symptomatic cane
samples were collected from six commercial sugarcane varieties viz. CoS 8436, CoS 91269,
CoS 95422, CoSe 98231, CoJ 64 and CoLK 8102 from different places. The most
characteristic symptoms of red rot disease in the fields were discoloration of stalk, reddening
of internodal tissue with white bands, alcoholic smell and yellowing of the crown leaves. The
isolates of red rot were studied for their cultural characteristics namely mycelial colour,
texture and sporulation. All the isolates were able to produce conidia in culture after 8-9 days
of inoculation with the average length diameter ranging from 34 µm to 38 µm. The
morphological culture characteristics revealed a clear cut variation in mycelial growth among
the tested isolates with different patterns and colour such as concentric rings, fluffy growth,
moderately slant and vertical growth, uneven growth with dark coloured mycelium at centre
with white, muddy and dull white colour. Isolate Cf- Kushinagar (variety CoSe 95422)
produced the highest mycelial dry weight while Cf-17 (variety CoSe 95422) recorded the
least. Regarding other characteristics, Cf-1B (variety CoS 8436), Cf-Kushinagar (variety
CoSe 95422) and Cf- 20 (CoSe 92423) produced thicker matty mycelium and were darker in
colour as compared to other isolates. Among the isolates, Cf-20, Cf-19, Cf-18 and Cf-1B
were faster in initiating conidia production and had higher conidial germination. Severity of
isolates to cause disease was studied on the basis of biological differentials (0-9 scale) which
revealed that Cf- Kushinagar isolate was the most virulent one followed by Cf-2B, Cf-20, Cf-
18 and Cf-17 in Uttar Pradesh. Molecular characterization of six virulent isolates (Cf-06, Cf-
08, Cf-04, Cf-10, Cf-17, Cf- Kushinagar) was effected by using ITS-1 and ITS-4 universal
primers specific to the fungus ITS gene to investigate the genetic diversity among them. A
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~550 bp amplicon with prominent intensity obtained from these isolates was cloned and
sequenced. The consensus sequence data of six Cf isolates was deposited in GenBank with
the accession numbers HQ833658, HQ833659, HQ833660, HQ833661, HQ833662 and
HQ833663. The BLASTn analysis revealed 99-94% identity among themselves and with Cf
isolates from different regions of the world. Isolates HQ833661 (Cf-10), HQ833662 (Cf-17)
& HQ833658 (Cf-06) shared maximum identity of 99-98% with Glomerella tucumanensis
(HM627380, HM592294, AY9447748, AY944753, AY944747). Less similarity was shared
by the isolate Cf-4 (HQ833660) with other selected isolates. Phylogenetic analysis revealed
three major clusters with the six isolates along with the other selected isolates used in this
study. The first cluster comprised of HQ833658 (Cf-06), HQ833662 (Cf-17) & HQ833661
(Cf-10) and these isolates showed maximum similarity with Cf isolates reported from Tamil
Nadu, India (AY944742, AY944753, AY944775, AY944748, AY944747, HM637380,
HM592294). Isolates HQ833659 and HQ833663 composed of another cluster while the
isolate HQ833660 (Cf-04) was found in a distinct one. Our results revealed that genetic
diversity exists among red rot isolates in India and further investigation is required with many
more isolates from different part of India to establish their relationship.
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GENETIC DIVERSITY OF SPORISORIUM SCITAMINEUM IN SOUTHERN CHINA
REVEALED BY COMBINED ISSR AND RAPD ANALYSIS
W. SHEN 1,2
, P. XI2, M. LI
2, R. LIU
1, L. SUN
2, Z. JIANG
2 AND L. ZHANG
2
1Guangzhou Sugarcane Industry Research Institute/ Guangdong Key Laboratory of
Sugarcane Improvement and Biorefinery, Guangzhou 510316, China
2Department of Plant Pathology, South China Agricultural University, Guangzhou 510642,
China
Keywords: Sugarcane, smut, ISSR, RAPD, genetic diversity
Sugarcane smut disease, caused by Sporisorium scitamineum, is a major disease of sugarcane
worldwide. In China, smut was reported for the first time in 1932 and has caused serious
problems to the sugarcane industry. Investigation of S. scitamineum genetic diversity is
essential for developing control strategies and for breeding new resistant cultivars. This study
investigated the genetic diversity of 35 mating-type isolates of S. scitamineum, collected from
16 sugarcane varieties in Southern China, using the inter-simple sequence repeat (ISSR) and
random amplified polymorphism DNA (RAPD) techniques. The results showed moderate
genetic diversity among the isolates of S. scitamineum that was associated to some degree
with geographic origins and host origins. Although host correlation was significantly lower
than geographical origins, the genetic diversity was significantly associated with the
physiological races of S. scitamineum. This study provided a strong molecular evidence for
the existence of a new race named race 3, which appeared to be a dominant in Southern
China.
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IDENTIFICATION OF GENES INVOLVED IN EPIPHYTIC SURVIVAL OF
XANTHOMONAS ALBILINEANS, THE CAUSAL AGENT OF LEAF SCALD OF
SUGARCANE
M. IMÈNE AND P. ROTT
CIRAD, UMR BGPI, F-34398 Montpellier, France
Keywords: Molecular factors, wild-type strains, pathogenic mutants, aerial transmission
Xanthomonas albilineans is a bacterial plant pathogen that is mainly spread by infected
cuttings and contaminated harvesting tools. However, at least some strains of the pathogen
are spread by aerial means and are able to colonize the phyllosphere before entering the host
plant. The objective of this study was to identify molecular factors that are involved in the
epiphytic phase of the disease. Several wild-type strains of X. albilineans and a related non
pathogenic species, as well as putative pathogenicity mutants of X. albilineans, were studied
for their capacity to survive on sugarcane leaves. Four week-old tissue-cultured plantlets of
cultivar CP68-1026 were immersed in bacterial suspensions at 107 cfu/ml. Foliar imprints of
the plantlets were performed on selective growth medium 14 days after inoculation. The
wild-type strain of X. albilineans that was not associated with aerial transmission of the
pathogen, and that belongs to genetic group PFGE-A, had a lower epiphytical survival
capacity than the wild-type strains that are aerially transmitted and that belong to genetic
group PFGE-B. An outer-membrane A (XaOmpA1) mutant and surface polysaccharide
mutants completely lost their capacity to survive on the leaf surface. In contrast, a motility
mutant, a mutant affected in production of small molecules, and the non pathogenic species
related to X. albilineans were all able to colonize the phyllosphere similarly to wild-type
strains that are transmitted by aerial means. Mutants of the rpf gene cluster, involved in
bacterial communication, showed different behaviors depending on the mutated gene.
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COMPARATIVE PROTEOME ANALYSIS OF THE REACTION TO INFECTION
BY XANTHOMONAS ALBILINEANS IN LEAF SCALD RESISTANT AND
SUSCEPTIBLE CULTIVARS
F. GARCES, J. HOY, AND Z. Y. CHEN
Department of Plant Pathology and Crop Physiology, Louisiana State University
Agricultural Center, Baton Rouge, Louisiana, USA
Keywords: Induced defence responses, induced systemic resistance, defence mechanisms
Leaf scald, caused by Xanthomonas albilineans (Xa), is an important disease of sugarcane
that has been reported in more than 66 countries causing losses in susceptible cultivars of 10-
34% in cane yield and up to 30% in juice quality. Host plant resistance is the main control
method; however, the molecular mechanisms of resistance are not well understood.
Therefore, a comparative proteomic analysis was carried out in a time-course experiment to
identify proteins differentially expressed in response to Xa infection of three cultivars, Ho 95-
988 (resistant), LCP 85-384 (highly resistant), and HoCP 89-846 (highly susceptible).
Numerous up- and down-regulated spots were detected in resistant cultivars during systemic
infection, whereas differential spot expression was greatest in inoculated leaves of the
susceptible cultivar. The majority of identified proteins (67%) are putatively located in the
chloroplast, followed by the mitochondrion (14%), the cytosol (14%), and the apoplast (5%).
Based on their potential biological functions, most of the identified proteins are involved in
photosynthesis (48%) followed by plant defense (20%), metabolism (12%), development
(12%), transcription (4%), and unknown (4%). Identified proteins were highly homologous to
cyclophilin, translationally controlled tumor protein (TCTP), thylakoid ascorbate peroxidase
(tAPOD), germin-like protein (GLP), and thioredoxins, all of which are known to be
associated with induced defense responses. For example, down-regulation of tAPOD and the
thioredoxins and up-regulation of GLP could result in the accumulation of reactive oxygen
species, particularly H2O2, in the cytoplasm and the apoplast, to restrict pathogen colonization
after initial infection. This speculation is supported by the differential induction of proteins
involved in ethylene biosynthesis, another key signaling molecule in induced systemic
resistance (ISR). The induced expression of proteins involved in primary metabolism could
provide the energy needed for ISR. Differences in induced protein identity in the two
resistant cultivars compared to the susceptible one suggest the occurrence of inducible, yet
different defense mechanisms in the resistant cultivars.
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IDENTIFICATION OF DIFFERENTIALLY EXPRESSED TRANSCRIPTS IN
SUGARCANE UPON COLLETOTRICHUM FALCATUM INFECTION BY
SUPPRESSION SUBTRACTIVE HYBRIDIZATION, SSH
R. VISWANATHAN, M. SATHYABHAMA, A. R. SUNDAR AND P. MALATHI
Sugarcane Breeding Institute, ICAR, Coimbatore 641007, India
Keywords: Red rot of sugarcane, host resistance, defense mechanism, subtractive libraries
Red rot caused by the fungal pathogen Colletotrichum falcatum Went is the major constraint
for sugarcane production in India and other Asian countries. Inheritance of red rot resistance
is not completely known due to the genetic complexities in sugarcane. Hence, we have
initiated detailed studies to understand the defense mechanism operating against red rot in
sugarcane through suppression subtractive hybridization (SSH) libraries. Three sets of cDNA
libraries were created to identify differentially expressed transcripts in stalk tissue in red rot-
resistant cv Co 93009, 12, 36 and 72 h after C. falcatum infection by subtraction with the red
rot susceptible cv CoC 671. A total of 850 transcripts were found to be differentially
expressed in the three libraries. The transcripts were assigned to their functional categories
after sequencing and vector trimming. The transcripts involved in protein structure folding
and fatty acid metabolism were induced relatively more in the 12 h library whereas,
transcripts associated with carbohydrate metabolism, signal transduction and cell wall
strengthening were observed in the 36 h and 72 h libraries. Lipoxygenase, which catalyzes
the biosynthesis of jasmonic acid from fatty acid and WD repeat protein involved in protein
folding were found abundant only in 12 and 36 h libraries. This suggests the possible role of
signaling events which are very much essential in the early hours of pathogen infection.
Similarly carbohydrate metabolizing transcripts were found to be abundant in the 36 and 72 h
libraries that links carbohydrate metabolism with disease resistance in sugarcane stalk tissue.
Defense responsive transcripts like chitinase, β-1, 3-glucanase, peroxidase and superoxide
dismutase were found to be induced in all time intervals. Validation of the previous
differentially expressed transcripts is being quantified by qRT-PCR in a set of sugarcane
varieties.
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AN INSIGHT INTO THE GENOME OF SPORISORIUM SCITAMINEUM
Z. WANG1,
Y. LIAO1,2
, G. ZHU3
, J. MENG1, C. ZOU
1,4, Y. REN
5,
Z. ZHOU5
, Y. FENG5
, QI CHEN1,4
, L. SHI1,4
, R WEN1,4
, J. SHANG1,4
, L. WANG5
, B.
CHEN1,2,4*
1State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,
Guangxi Univieristy，Nanning, 530005, China
2College of Agriculture, Guangxi University，Nanning, 530005, China
3Guangxi Academy of Agricultural Sciences, Nanning, 530007, China
4College of Life Science and Technology，Guangxi University, Nanning, 530005, China
5TEDA School of Biological Sciences and Biotechnology, Nankai University, Tianjin 300457,
China
Keywords: Mechanism of pathogenicity, smut, phylogenetic analysis
Smut caused by Sporisorium scitamineum is a major disease of sugarcane worldwide. In
order to better understand the mechanism of pathogenicity of this fungus, the whole genome
of S. scitamineum strain JG36, which was isolated from Guangxi, China was sequenced. The
genome of the haploid JG36 was assembled into 19 scaffolds with a total length of
18,315,206 bp and 6522 cds were predicted. Tanscriptome of JG36 was determined from
both haploid and the diploid stages. Of 20,940 clustered transcripts from the infected tissues,
7138 transcripts were identified to belong to fungi. Phylogentic analysis of the actin gene
showed that S. scitamineum is very close to Ustilago maydis, and ortholog genes are
conserved in these two fungi. The genome of an opposite mating type strain, JG35, was also
sequenced.
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YIELD LOSS CAUSED BY THE TOP ROT FORM OF RED STRIPE ON
SUGARCANE AND FACTORS AFFECTING INCIDENCE AND SEVERITY
M. P. GRISHAM AND R. M. JOHNSON
United States Department of Agriculture, Agriculture Research Service, Sugarcane Research
Unit, 5883 USDA Road, Houma, Louisiana70360, USA
Keywords: Acidovirax avenae subsp. avenae, soil properties, seed cane
Over the past 25 years in Louisiana, symptoms of red stripe caused by Acidovirax avenae
subsp. avenae on sugarcane were limited to the red stripe form of the disease with no
apparent yield loss. However, the more severe top rot symptom of the disease was observed
in commercial sugarcane fields in 2010, most commonly in cultivar HoCP 00-950. Two
commercial fields of cultivar HoCP 00-950, one plant cane (PC) and one first ratoon (FR),
were subdivided into 113 and 84 plots, respectively. In the PC field, tonnes cane/hectare, kg
sugar/tonne, and kg sugar/hectare were reduced in plots with >20% of stalks with top rot
symptoms by 5%, 10%, and 14%, respectively, when compared to FR field plots with lower
disease incidence; and 4%, 6%, and 9%, respectively. A disease incidence, nitrogen fertility
rate, and soil texture interaction was noted in plots of a nitrogen fertility rate experiment on
cultivar HoCP 00-950. Incidence was higher among plots in heavy clay soils verses lighter,
more silty soils. Disease incidence increased with increasing rates of added nitrogen in the
heavy clay soil compared to the control, no added nitrogen plots. In the lighter soil, disease
incidence was higher in plots with added nitrogen, but did not differ among the different
nitrogen rates. To determine the effect of using seed cane infected with red stripe, plots were
planted with stalks cut randomly from a field in which top rot symptoms of red stripe were
observed. Shoot counts were reduced by 15% compared to the plots planted with
symptomless stalks only. Use of stalks with top rot symptoms resulted in a 60% reduction of
shoots per plot, while shoot counts in plots planted with stalks showing only leaf striping
symptoms did not differ from plots planted with stalks showing no symptoms.
19
15
POLYCLONAL ANTISERUM SPECIFITY AGAINST LEIFSONIA XYLI SUBSP.
XYLI, CAUSAL AGENT OF SUGARCANE RATOON STUNT DISEASE
X. N. XIE1,2
, M. H. CHEN1,2
, D. ZHOU1,2
, L. T. YANG1,2,3, *
, Y. R. LI2,3,4*
1 Agricultural College, Guangxi University, Nanning 530005, China;
2 State Key Laboratory of Subtropical Agriculture Bioresources Conservation and
Utilization, Nanning 530005, P.R. China; 3 Sugarcane Research Center, Chinese Academy of Agricultural Sciences/ Sugarcane
Research Center, Guangxi Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture/ Guangxi Key
Laboratory of Sugarcane Genetic Improvement, China; 4
Guangxi Crop Genetic Improvement and Biotechnology Lab, Nanning 530007, China
Keywords: sugarcane, ratoon stunting disease (RSD), tissue blot immunoassay (TBIA)
To evaluate the presence of the bacterium Leifsonia xyli subsp. xyli (Lxx) in propagation
material of sugarcane for the control of ratoon stunt disease (RSD), tissue blot immunoassay
(TBIA), which is a simple, rapid and sensitive method for large scale screening, is employed.
The objective of the present study was to develop a polyclonal antiserum against Lxx and to
evaluate its specificity. The bacterium Lxx was isolated from the juice of RSD- infected
sugarcane, cultured on modified SC medium, and observed under transmission electron
microscopy (TEM). The morphology and PCR detection results of the bacterium were similar
to those reported in the literature. The rDNA-ITS sequence of the isolated bacterium was the
same those Lxx reported from Brazil (accession number AE06822), Australia (accession
number AF034641) and China (accession number EU723209). The antigen was prepared by
resuspending pure culture in PBS and inactivating it in 0.1% formaldehyde in PBS. The
immunization schedule in a rabbit constituted of two intramuscular injections of 1:1 mixture
of the antigen: Freund adjuvant (complete and incomplete, with interval of 21 days) and two
subcutaneous injections of the antigen, within ten-day intervals. The antiserum was tested by
ELISA to determine: (i) titre of the antibody, (ii) reaction and specificity against Lxx. The
highest dilution of antiserum (1:256000) presented strong positive reaction and high
specificity against Lxx. The results of TBIA were consistent and comparable with RSD
polyclonal antiserum imported from the USA. It is confirmed that the isolated pathogen is
Lxx and the polyclonal antiserum is reliable to the bacterium.
20
16
QUANTIFICATION OF XANTHOMONAS ALBILINEANS IN SUGARCANE BY
QPCR FOR CULTIVAR RESISTANCE SCREENING
F. GARCES AND J. HOY
Department of Plant Pathology and Crop Physiology, LSU Agricultural Center, Baton
Rouge, Louisiana, USA
Keywords: Leaf scald, bacterial population, vascular infection
Leaf scald caused by Xanthomonas albilineans (Xa) is an important bacterial disease
affecting sugarcane in many different countries. Host plant resistance is the principal method
of control, but leaf scald screening based on visible symptom evaluation following
inoculation is problematic because of erratic symptom expression. Resistance is associated
with a lower bacterial population during systemic infection. Therefore, real-time, quantitative
polymerase-chain-reaction (qPCR) assays were developed utilizing SYBR Green and a
TaqMan probe to quantify Xa populations in infected plants. Samples were collected from the
youngest fully emerged leaf (TVD), the second younger leaf above the TVD (TVD-2), and
the third older leaf below (TVD +3) along with stem sap. All were collected in pooled
samples of five. DNA was extracted from sap and leaf diffusates by a boiling and alkaline
disruption method. Two resistant (LCP 85-384 and Ho 95-988) and two susceptible cultivars
(HoCP 85-845 and HoCP 89-846) gave similar Xa populations with vascular infection and
disease severity also similar from both greenhouse and field experiments. Differences in Xa
population levels between resistant and susceptible cultivars were greatest in systemically
infected TVD-2 leaf samples. Xa populations in the resistant cultivars were 102
- 103
bacteria
per ml compared to 106
- 107
bacteria per ml in the susceptible cultivars. These preliminary
experiments under greenhouse and field conditions suggest that Xa population quantification
by qPCR has the potential to provide an alternative method to screen for resistance to leaf
scald. However, initial experiments with larger numbers of clones suggest that Xa
populations in infected plants of susceptible genotypes may decline under some conditions,
and assay timing will be important. Additional testing of larger numbers of sugarcane
genotypes with variable levels of resistance is in progress.
21
17
GREEN GRASSY SHOOT DISEASE (GGSD) IN VIETNAM
R.C. MAGAREY1, P.J. NIELSEN
2, L.W. BURGESS
3 AND NGO VAN TU
2
1BSES Limited, Tully,
2NAT&L Sugar Factory, Nghe An Province, Vietnam,
3University of
Sydney, New South Wales
Keywords: yield losses, phytoplasmas, epidemic
Green grassy shoot disease (GGSD) has reached major disease status in Nghe An Province,
Vietnam. The disease was first recognised in Thailand in the 1990s and is known only from
SE Asia (Thailand, Vietnam). The disease has rapidly escalated in commercial crops in the
Quy Hop sugar factory and is spreading to surrounding areas. GGSD is caused by a
phytoplasma with similarities to the causal agents of white leaf (WLD) and grassy shoot
(GSD) diseases. The first finding of the disease was in Nghe An in 2006; affected varieties
include MY55-14 and ROC10. Severe stunting of affected stools may occur with the
characteristic grassy appearance, especially in ratoon crops. Symptoms include small green
tillers arising from the base of stools late in the crop growth cycle. This is followed by
multiple small, green tillers in subsequent ratoon crops with very few well-grown stalks of
harvestable cane. Failed ratoons and the early replanting of crops contribute to the very
significant economic effects of the disease. With limited identified varietal disease resistance,
there is a great need to develop resistance screening techniques, in conjunction with the
introduction of a greater range of germplasm. Immediate controls include the
roguing/termination of infested crops coupled with the replanting of disease-free seedcane.
Further research is needed to determine whether there are insect vectors of the disease, to
confirm the causal agent, and to survey the distribution of the disease – both within Vietnam
and in neighbouring countries.
22
18
GRASSY SHOOT PHYTOPLASMA BECOMING A SERIOUS THREAT TO
COMMERCIAL SUGARCANE VARIETIES IN CENTRAL-EASTERN UTTAR
PRADESH, INDIA
A. K. TIWARI1, S.K. VISHWAKARMA
1 AND G.P. RAO
2*
1 Sugarcane Research Institute, Shahajahanpur 242 001, Uttar Pradesh, India
2 Division Plant Pathology, Indian Agricultural research Institute, New Delhi 110012
Keywords: Nested-PCR, crop growth and yield, leafhoppers
Sugarcane grassy shoot (SCGS) disease caused by a phytoplasma has become a serious threat in both
tropical and subtropical parts of India. During a recent survey in two consecutive years (2010-2011 &
2011-12) in five districts of the Central & Eastern Uttar Pradesh, (Pilibhit, Balarampur, Sultanpur,
Shahjahnapur, Gola) SCGS incidence from 10 to 56% were recorded in seven promising commercial
varieties of sugarcane (CoS 07250, CoS 95255, CoSe 01434, CoS 99259, CoS 98259, CoS 96275,
CoS 97264). The maximum recorded incidence of the disease was in variety CoS 97264 (56%) in
Pilibhit district in the year 2010-2011 during the monsoon period. The maximum incidence of SCGS
disease in all the varieties at all locations was observed at 25.7 (max) to 22.9 (min) temperature and
83 per cent average humidity during the month of August in both the consecutive years. The
association of phytoplasmas in all the seven symptomatic sugarcane varieties was confirmed by
nested PCR by utilizing the primer pairs P1/P7 and R16F2n/R16R2. SCGS was found to adversely
affect the crop growth and yield of the affected varieties. Detailed studies were performed on two
major affected varieties grown in the central part of Uttar Pradesh, India viz., CoS 95255 and CoS
07250. In variety CoS 07250 a reduction of 14.38%, 27.5% and 26.8% in brix, Pol % and sucrose %
respectively, was observed. However in variety CoS 95255 the reduction was 22.26%, 27.9% and
26.4%, in brix, Pol % and sucrose % respectively. The cane weight of the SCGS disease-affected cane
varieties viz. CoS 07250 and CoS 95255 was also significantly reduced. The extraction per cent of
juice was also drastically affected and it was nearly 40% less in both the infected varieties as
compared to the healthy ones. Leafhoppers in the vicinity of surveyed sugarcane fields and nearby
growing weeds were collected for further identification and association of phytoplasma in these
suspected vector species. The studies on the exact cause of secondary spread of the disease in the
surveyed area are in progress.
23
19
PROGRESS IN MOLECULAR CHARACTERIZATION OF SUGARCANE
GRASSY SHOOT AND SUGARCANE LEAF YELLOWS PHYTOPLASMAS IN
INDIA
G.P. RAO*, MADHUPRIYA AND A. KUMAR
Division Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012
Keywords: Nested-PCR, 16SrDNA, phylogenetic relationship, housekeeping genes
Sugarcane Grassy Shoot (SCGS) disease has been recorded in most sugarcane-growing areas
of India and is characterized by the production of a large number of non-productive thin,
slender, adventitious chlorotic tillers from the base of the affected stools. The disease is
particularly pronounced in the ratoon crop where the clusters of slender tillers with reduced
leaves usually growing erect giving the appearance of a field full of perennial grass, and from
which it has derived its popular name "grassy shoot". Since diseased plants exhibit various
phenotypic symptoms under field conditions, most of the time the diagnosis of the disease
becomes difficult. Universal phytoplasma-specific primer pairs P1 and P7 were used for
nested PCR assays that successfully detected the SCGS phytoplasma in sugarcane and in its
reported leafhopper vector, Deltocephalus vulgaris in India. Nucleotide sequence analysis of
16S rRNA genes revealed that SCGS phytoplasma affecting sugarcane crops in India is very
closely related to the Sugarcane White Leaf (SCWL) agent and is, thus, a member of the rice
yellow dwarf (RYD) phytoplasma group. SCGS and SCWL phytoplasmas shared a 16S
rDNA sequence similarity of 97.5 to 98.8%. Of the phytoplasmas that cluster in other
phylogenetic groups, those most closely related to SCGS phytoplasma are the BGWL
(=‘Candidatus Phytoplasma cynodontis’) and brachiaria grass white leaf (BraWL) agents,
which share 97.3 and 97.1% 16S rDNA sequence similarity, respectively. In addition to
SCGS phytoplasmas association with sugarcane, leaf yellows phytoplasma (SCYLP), a
recently recognized disease of sugarcane, in India was also detected using PCR-based
methods. In some cases, mixed infection of SCGS and SCLYP were also reported. In a recent
study in India, the phylogenetic relationships of SCGS phytoplasma isolates among
themselves and related phytoplasmas based on 16S rRNA gene sequences showed 100%
identity with no variation among 16S rRNA gene sequences. Although there are significant
variations in phenotypic expression of SCGS phytoplasmas on sugarcane, no genotypic
variations could be established so far. Further studies are therefore needed to locate other
genes in phytoplasma genome which could determine variations in phenotypic expression of
the disease. Such information is lacking and further characterization of SCGS-phytoplasma
24
based on their pathogenicity genes is required. Along this line, other housekeeping genes
such as secA, gryA and gyrB were explored and primers were designed. Sequencing of these
genes from phytoplasma would allow designing specific primers for SCGC in India. In our
present work, a 480 bp of Sec A, a 1473 bp of gyrA and a 1535bp of gyrB DNA fragment
from SCGS isolates were sequenced and compared. The nucleotide and amino acid sequence
analysis of all these novel genes allowed us to clarify the phylogenetic position of SCGS
disease phytoplasma. Our results indicate the existence of strainal diversity among SCGS and
SCLYP isolates in India, for which the work is in progress. The new gene sequences have
also been used to redefine the taxonomic relationships among the existing SCGS and SCYLP
isolates in India. This would form the basis for development of strain specific diagnostics for
SCGS disease affecting sugarcane in India.
25
20
PREPARATIONS TO HELP PROTECT THE AUSTRALIAN SUGARCANE
INDUSTRY FROM EXOTIC DISEASES AND PESTS
R.C. MAGAREY, L.S. KUNIATA, K.S. BRAITHWAITE, P.R. SAMSON, N.P.
THOMPSON, R. KOMBUKON2
1BSES Limited, Tully,
2Ramu Agri-Industries, PNG,
3BSES Limited, Mackay,
4BSES Limited,
Indooroopilly
Keywords: downy mildew, Ramu stunt, sugarcane streak mosaic
There are a number of threats to the Australian sugarcane industry located in Indonesia and
Papua New Guinea, including various moth borers (Scirpophaga excerptalis, Chilo
sacchariphagus, C. auricilius, Sesamia grisescens, Chilo terrenellus), sugarcane streak
mosaic, downy mildew (Peronosclerospora sacchari), Ramu stunt and others. In recent
years the Australian sugarcane industry has witnessed the disruption caused by an exotic
disease incursion, with smut affecting crops in all districts. BSES Limited has a policy of
preparing for potential exotic pest or disease incursions; part of this strategy led to sourcing
Federal Government (ACIAR, SRDC) funding for research into key Indonesian and PNG
threats. This paper reports on work being undertaken in the following areas: i) field
resistance screening, ii) assessing the incidence/strains of disease pathogens in commercial
crops and in wild canes, iii) investigating rapid shade-house resistance screening techniques
for Sesamia grisescens, DM and Ramu stunt, and iv) investigations into the Ramu stunt
causal agent. Research is progressing and a number of Australian commercial varieties are
included in current resistance screening trials. Difficulties have been experienced in the
application of sufficient pest or disease pressure in some field trials. Intensive shade house
research is providing new information on pest and disease biology and ways in which the
resistance screening tests may be accelerated. Pathogen research is highlighting the nature
and variation in the causal agents.
26
21
SCREENING FOR SUGARCANE YELLOW LEAF VIRUS IN
QUARANTINE IN MAURITIUS
N. JOOMUN, Y. PARMESSUR, M. ANTOINE AND A. DOOKUN-SAUMTALLY
Biotechnology Department, Mauritius Sugarcane Industry Research Institute, Réduit,
Mauritius
Keywords: RT-PCR, Real-Time PCR, virus elimination
The Mauritius Sugarcane Industry Research Institute (MSIRI) relies on both its germplasm collection
and the introduction of foreign genetic material to improve varieties in its breeding programme. In the
past, varieties imported in the form of cuttings underwent a two year quarantine cycle during which
they were assessed for major diseases through conventional techniques and infected ones were
destroyed. One improvement to this procedure is the application of molecular tools in the detection of
Sugarcane yellow leaf virus (SCYLV) in symptomless plant cane during the first year and the
elimination of the virus using tissue culture techniques. Thus in the second year, at the end of the
quarantine cycle, disease-free plantlets are already available for field transfer. Conventional RT-PCR
coupled with a Real-Time RT-PCR confirmatory test is being applied for SCYLV screening. Both
techniques are equally reliable for screening plants of about six months old. From data compiled for
the period 1991-2011, it had been observed that more than one third of the varieties introduced (106
out of 331) from different countries of diverse geographical regions cultivating sugarcane were
infected by the virus. Using tissue culture techniques, the virus was eliminated from most varieties
that were regenerated in vitro, with a success rate reaching 100 % in most cases. The diagnostics
together with the tissue culture techniques are now contributing to the introduction of SCYLV virus-
free varieties in Mauritius.
27
22
DEVELOPMENT OF MOLECULAR DIAGNOSTICS FOR
SUGARCANE DOWNY MILDEW
N. THOMPSON1, R. MAGAREY
2, L. KUNIATA
3 AND B. CROFT
4
1 BSES Limited, 50 Meiers Road, Indooroopilly, QLD 4068, Australia;
2 BSES Limited, PO
Box 566 Tully, QLD 4854, Australia; 3 Ramu Agri Industries Limited (RAIL), Gusap Downs,
PO Box 2183 Lae, Morobe Province, Papua New Guinea; 4 BSES Woodford, 90 Old Cove
Road, Woodford, QLD 4514, Australia
Keywords: Peronosclerospora, PCR, biosecurity
Sugarcane downy mildew is caused by several species in the Peronosclerospora genus
including P. sacchari and P. philippinensis. The disease has been found throughout many of
the sugarcane growing areas of the world, but was eradicated from commercial growing areas
in Australia in the 1950s. The disease is widespread in the Ramu Agri Industries (RAIL)
sugarcane plantation in Papua New Guinea and is estimated to cause yield losses of up to
40% in susceptible varieties. This presents a biosecurity risk to Australia and research is
being done to understand the extent of this threat. Diagnostics of sugarcane downy mildew by
symptoms is relatively straightforward as the classic symptoms of chlorotic stripes on the
leaves and the presence of down on the underside of the leaf and/or the leaf shredding
symptoms can be easily identified. However it can be difficult to determine the species
identification by traditional taxonomy. A molecular diagnostic test would be useful for
studies of species diversity, positive identification in case of a disease incursion, and to
support traditional taxonomy. Peronosclerospora specific diagnostic primers were designed
to amplify sections of several genes including Cytochrome oxidase I (COX-1) and Beta-
tubulin (B-tub). The COX-1 primer pair was designed to amplify across a region of variable
size which could theoretically act as an indicator of species variation. These primers were
tested on known Peronosclerospora species from the Qld Herbarium collection and the
predicted variation in amplicon size and sequence supported the traditional taxonomy. COX-I
PCR analysis of downy mildew samples collected from Papua New Guinea show the
presence of at least 2 size variants of Personosclerospora: one variant dominating the
commercial varieties; and at least one variant in non-commercial sugarcanes including garden
canes (Saccharum officinarum and S. edule), wild canes (S. robustum and S. spontaneum) as
well as in other species including Miscanthus spp. and maize. Sequencing and further
analysis of downy mildew samples in Papua New Guinea and other countries will reveal the
28
composition of the population of Peronosclerospora species able to infect Saccharum and its
relatives.
23
MOLECULAR IDENTIFICATION OF VIRUSES INFECTING SUGARCANE IN
NANNING AND PREPARATION OF ANTISERUM AGAINST
SORGHUM MOSAIC VIRUS
R. WEN 1,2
, J. LI 2, Z. WANG
1, X. XIAN
2, H. LIU
3,
Y. LIAO 1,3
AND B. CHEN 1,2*
1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-
Bioresources, Guangxi Univieristy Nanning, 530005, China;
2 College of Life Science and Technology Guangxi University Nanning, 530005,
China
3 College of Agriculture, Guangxi University Nanning, 530005, China
Keywords: TR-PCR, multiplex-PCR, RT-PCR, yellow leaf, mosaic
Guangxi is the most important province producing sugarcane in China, providing more than 65% of
the sugar source of the nation. Viral diseases are among the most important constraints to the
sugarcane industry. Recently, we used RT-PCR, Multiplex RT-PCR, and small RNA sequencing to
survey viruses from sugarcane plants with symptoms in the Nanning region. Sorghum mosaic virus
(SrMV), Sugarcane yellow leaf virus (SCYLV), and Sugarcane mosaic virus (SCMV) were found in
some sugarcane samples. The primary data showed that SrMV and SCYLV are the two most
prevalent viral diseases in this region. Co-infection of SCYLV and SrMV were observed in several
cases. Most co-infected sugarcane leaves showed mosaic symptom, however, leaves infected with a
single virus (SCYLV or SrMV) showed mild symptomatic or was asymptomatic. A positive sample of
SrMV was further selected for full genome sequencing. A high titer (1:32000) antiserum specific to
SrMV was generated by using the E. coli-expressed SrMV-CP to immune rabbits. These
developments are a good foundation for sugarcane virus detection, production of virus-free seedling,
virus epidemiology, and basic virology research.
29
24
THE RUST SITUATION IN THE SOUTH AFRICAN SUGARCANE INDUSTRY
S.A. MCFARLANE, L.A. MARTIN, P. RAMOUTHAR, A.C. KOCH AND R.S.
RUTHERFORD
South African Sugarcane Research Institute, 170 Flanders Drive, Mount Edgecombe, 4300,
KwaZulu-Natal, South Africa
Email: [email protected], [email protected],
[email protected], [email protected],
Keywords: Puccinia melanocephala, fungicides, rust risk model
Brown rust (Puccinia melanocephala H. & P. Sydow) is prevalent in the South African sugarcane
industry. While there has been an increased focus on releasing varieties with adequate resistance to
the disease in recent years, many of the older releases have exhibited moderate to severe symptoms
when conditions have favoured brown rust development. A fungicide (a.i. epoxyconazole/
pyraclostrobin) was found to be effective against brown rust and has received conditional registration
for use in the industry. Two applications reduced infection in the brown rust-susceptible variety N29
by 64% and resulted in a 68% increase in yield (tons cane/hectare). To assist growers in the decision
to use fungicides as well as the timing of application, a simple linear model based on temperature and
relative humidity has been developed. The model is currently being validated in the industry. In
addition, a new species of rust has been observed on a number of South African varieties. General
Internal Transcribed Spacer (ITS) region sequences confirmed that the unknown species belongs to
the Puccinia genus and primers from the ITS1, ITS2 and 5.8S rDNA regions confirmed that it is not
P. melanocephala or P. kuehnii. Gene sequencing of the 28S gene region is in progress and
preliminary results suggest that the unknown rust has not been reported on sugarcane previously.
30
25
PRESENCE OF ORANGE RUST (PUCCINIA KUEHNII EJ BUTLER) OF
SUGARCANE IN THE CAUCA VALLEY IN COLOMBIA AND MANAGEMENT
STRATEGIES
JUAN CARLOS ÁNGEL S., MARCELA CADAVID O. AND JORGE I. VICTORIA K
Colombian Sugarcane Research Center, CENICAÑA
P. O. Box. 9138. Cali-Colombia
Keywords: Evaluations, diagnosis, resistance
Orange rust is caused by the fungus Puccinia kuehnii. Initial symptoms of the disease are
small lesions, long and yellow, forming a pale yellow-green halo that increases in size. The
lesions grow and become orange to brown in colour. In 2000 orange rust caused losses in
sugarcane tonnage in Australia. By 2007 it was also recorded in other countries namely
Papua New Guinea, Indonesia and Philippines. Between 2007 and 2010 orange rust was
disseminated in Florida (USA), Guatemala, Costa Rica, Nicaragua, Mexico, Salvador,
Panama, Cuba and Brazil. In July 2010, it was detected in the Cauca Valley in Colombia, and
its presence was confirmed through visual observation, optical microscopy, electron scanning
microscopy and molecular diagnostics. Since it was found in Colombia, orange rust was
evaluated in nurseries, commercial lots and research lots of different varieties in the Cauca
Valley. The most susceptible varieties identified were CC 01-1884, CC01-1866 and CC 01-
1305 and were immediately eliminated. Among 82 Cenicaña Colombia (CC) varieties
evaluated, 22 were susceptible to orange rust and three varieties were affected by both brown
rust and orange rust diseases. Evaluation of the Cenicaña sugarcane germplasm bank showed
that 9.3 % of the 1354 cultivars tested were susceptible. Commercial varieties CC85-92 and
CC84-75 that are mostly grown are resistant to orange rust. Due to the presence of orange
rust in Colombia, Cenicaña has taken the decision to include resistance to this disease as a
selection criterion in its selection process for new varieties. Sugar mills and growers were
alerted on the presence of orange rust in sugarcane in Colombia through conferences, field
trips, brochures and articles in magazines. There is now a continuous orange rust disease
monitoring in the valley of the Cauca River and in other areas growing sugarcane in the
country.
31
26
YIELD LOSS INCITED BY ORANGE RUST (PUCCINIA KUEHNII) ON A
HIGHLY SUSCEPTIBLE SUGARCANE CULTIVAR IN FLORIDA
R. N. RAID1, J. C. COMSTOCK
2, AND N. GLYNN
2
1Everglades Research and Education Center, University of Florida, Belle Glade,FL
2USDA Sugarcane Field Station, Canal Point, FL
Keywords: Fungicide, Pyraclostrobin
Sugarcane orange rust, incited by Puccinia kuehnii, was initially reported in the Western
Hemisphere in 2007, when it was first observed in Florida. Since that time, it has affected
several commercial cultivars, notably CP80-1743, CP72-2086, and CL85-1040. Experiments
were conducted during 2008 and 2009 to quantify yield losses on the latter, the most
susceptible of these cultivars. Varying levels of orange rust were established by applying the
fungicide pyraclostrobin at 7, 14, 21, and 28 day intervals. Experimental units measured five
rows by 15 m and were arranged in a randomized complete block design with eight
replications. Non-sprayed plots served as controls. Disease severities ranged from near zero
in the 7-day treatments to over 35% in the controls when assessed on the terminal third of the
fourth leaf below the top visible dewlap. During 2008, millable stalk populations were
reduced by 12% and stalk biomass by 32% when controls were compared with the 7-day
treatments. Together, these accounted for reductions of 43% in tons of cane per hectare.
Sucrose concentrations were significantly reduced by nearly 18% on this cultivar in 2008.
During 2009, differences in millable stalk populations were less than 2%, this difference
(12% vs 2%) from year–to-year presumably being due to the relatively late epidemic in 2009.
Stalk biomass, in contrast was reduced by 33% during 2009, and cane tonnage by 32%.
Cumulative losses in terms of sugar per unit area of cane measured 53% and 43% for 2008
and 2009, respectively. These findings demonstrate the highly destructive nature of this
disease under favorable conditions and underscore the urgency to breed for host plant
resistance.
32
27
EVALUATION OF FUNGICIDES FOR CONTROL OF ORANGE
RUST ON SUGARCANE
R. N. RAID1, J. C. COMSTOCK
2, AND N. C. GLYNN
2
1University of Florida, Everglades Research and Education Center, Belle Glade, FL
2USDA-ARS, Sugarcane Field Station, Canal Point, FL
[email protected], [email protected]
Keywords: Puccinia kuehnii, strobilurin, triazole
Orange rust of sugarcane, incited by Puccinia kuehnii, was first observed in Florida during
June 2007 on one of the industry’s most important commercial cultivars, CP80-1743. This
was the first report of this disease in the Western Hemisphere. It has since been reported in
several other Central American and Caribbean Countries. Trials were initiated to investigate
the feasibility of fungicides serving as an interim or supplementary management strategy
until replacement of CP 80-1743 was completed. Thirteen different fungicide treatments were
examined for their efficacy in controlling orange rust during the 2008/2009 growing season.
Experimental units consisted of two rows of cane 15 m in length, replicated four times.
Fungicide treatments consisted of select candidates from two major classes of fungicides, the
strobilurins (FRAC group 11) and triazoles (FRAC group 3), alone, and in combination or
alternation. Fungicide applications were made using a CO2 backpack sprayer and were
initiated following canopy closure (approx. 1.5-m ht) at 21 day intervals. Rust severity in the
trial area was moderately severe, with severities in excess of 30% on the distal third of the
fourth leaf beneath the top-visible-dewlap leaf in the untreated check. Results indicate that
the strobilurin fungicides provided the highest level of control, followed by
strobilurin/triazole combinations, and finally, the triazole fungicides alone. In separate trials
using the strobilurin fungicide pyraclostrobin, fungicide treatments were demonstrated
capable of reducing orange rust to levels sufficient to significantly reduce yield losses by as
much as 40%. While economic factors will ultimately be an important consideration, levels
of orange rust control obtained in these studies show promise regarding prospects for
fungicides as a potential management tool.
33
28
INCIDENCE OF YELLOW SPOT IN THE SUPERHUMID ZONE OF MAURITIUS
S. DHAYAN AND S. SAUMTALLY
Mauritius Sugarcane Industry Research Institute (MSIRI), Réduit, Mauritius
Keywords: Mycovellosiella koepkei, sugarcane, resistant varieties
Yellow spot (Mycovellosiella koepkei) is the most important foliar disease of sugarcane in the
superhumid zone (> 2500 mm annual rainfall) of Mauritius. It reached epidemic proportion in
1977-78 and since then has maintained a high inoculum pressure. Infection is monitored
monthly from January to August in the highly susceptible reference variety B 3337 in trials
established in the same zone. The percentage infection recorded is used to estimate the
importance of the disease incidence during the year. Figures obtained in the last decade
showed a consistent pattern. Infection started from trace level during summer in January,
increased markedly in March to reach a peak in April or May before dropping off in June at
the onset of winter. The peak infection recorded varied from 23% to 56% with an average of
44%. Years 2003, 2004 and 2005 were considered to be periods of severe yellow spot
incidence reaching 55%, 56% and 54% respectively on variety B 3337. The level is
correlated with rainfall pattern. Some 60-120 promising clones are screened every year by
exposure to the disease in plant cane and two ratoon crops. Results of the last 10 trials
showed that the number of susceptible clones rejected was high and ranged from 40-70%. A
new breeding programme using only resistant and slightly susceptible parents has been
initiated with the objective to develop resistant varieties for the superhumid zone of
Mauritius.
34
29
MOLECULAR IDENTIFICATION AND SEQUENCE ANALYSIS OF SUGARCANE
YELLOW LEAF VIRUS ISOLATES FROM CHINA
S.J. GAO1, Q.N. WANG
2, Y. H LIN
1, Y.B PAN
3 AND R.K CHEN
1*
1Key Laboratory of Sugarcane Biology and Genetic Improvement, Ministry of Agriculture,
Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China 2 Guangzhou Sugarcane Industry Research Institute, Guangzhou 510316, Guangdong, China
3 USDA–ARS, Southern Regional Research Center, Sugarcane Research Unit, P.O. Box 470,
Houma, LA 70361, USA
Keywords: Genomic sequence, phylogenetic analysis, sugarcane (Saccharum hybrids), viral
genotype
The Sugarcane yellow leaf virus (SCYLV-Polerovirus) that causes yellow leaf (YL) disease
in sugarcane was recently reported in China. In this paper, a survey of sugarcane clones
(Saccharum spp. hybrids) for the existence of SCYLV in Chinese main sugarcane-growing
provinces was carried by reverse transcriptase PCR and serological methods. To study the
SCYLV phylogenetic diversity, a 5879 nt complete genomic sequence of CHN-FJ1 isolate
and partial genomic sequence (2915 nt) in 13 Chinese isolates were amplified, cloned and
sequenced. Homological sequence alignment in six ORFs of SCYLV genomes showed that
the genetic variation was ordered by ORF0 > ORF1 > ORF5 > ORF2 > ORF3 > ORF4. The
genetic diversity of SCYLV was studied in 14 isolates from China and 29 reported isolates
from GenBank presenting worldwide origins based on either the full or partial genomic
nucleotide sequences. Phylogenetic analysis demonstrated that the 14 Chinese isolates and 7
isolates from the GenBbank clustered together in one large group; genotype BRA.
Additional, five distinct genotypes (HAW, PER, REU, CHN1, and COL) were formed in
different phylogenetic clusters. Five Chinese published isolates clustered in genotype PER
and CHN1. Therefore, we speculated that at least three genotypes were associated with YL in
China. Interestingly, a deletion of 39 nts was found in the sequence of CHN-GD3 isolate
collected from a susceptible cultivar. The gap of nucleotides was present in the middle region
35
of ORF1 that closely precedes the overlap between ORFs 1 and 2, where is one of the
recombination breakpoints in the Luteoviridae family.
30
HOW DOES SCYLV GENETIC DIVERSITY IMPACT ON SUGARCANE DISEASE
RESISTANCE AND BREEDING?
J.H. DAUGROIS1 AND S. DÉBIBAKAS
1,2
1UMR BGPI, CIRAD, Station de Roujol, 97170 Petit-Bourg, Guadeloupe F.W.I. ;
2 Université des Antilles et de la Guyane, Département de Biologie, UFR Sciences Exactes et
Naturelles, Campus de Fouillole, 97159 Pointe-à-Pitre, Guadeloupe F.W.I.
Keywords: Yellow leaf, virus incidence, tissue blot immunoassay, genotype incidence
To date, six distinct genotypes of the Sugarcane yellow leaf virus have been described in the
literature (BRA, CUB, CHN1, IND, PER and REU). However, little information is available
regarding the impact of virus genetic diversity on disease resistance. Three SCYLV genetic
groups that can be identified with specific primers by RT-PCR are present in Guadeloupe:
BRA-PER, CUB and REU. The impact of the virus genotype diversity on rating for
resistance to yellow leaf was studied on a sub-population of 40 sugarcane clones issued from
a population of 200 clones planted in a complete three blocks design. The sub-population was
screened for virus incidence by sampling 10 leaves per plot and detecting SCYLV by tissue
blot immunoassay (TBIA). For each plot, SCYLV genotypes were diagnosed by RT-PCR in
a bulk of the TBIA positive leaves. The experiment was repeated in a second crop cycle. The
results gave an estimation of genotype incidences for each clone. Of the 185 samples used for
SCYLV genotyping, BRA-PER was found in 105, CUB in 170 and REU in 99 samples. CUB
was the most frequent genotype encountered and had the largest cultivar host range,
overlapping cultivar host ranges of BRA-PER and REU. Additionally, CUB incidence gave
the best correlation with SCYLV incidence in each cultivar (R² 0.87). In addition, the
incidences of virus genotypes in each cultivar compared by regression analysis showed that
the incidence of CUB was correlated to the incidence of REU (R² 0.46), whereas the
correlations between the incidence of BRA-PER and the two other genotype incidences were
low (R² of 0.12 and 0.14). In view of these results, it is suspected that plant resistance
mechanisms for yellow leaf disease depend on the genotype involved and that, in
36
Guadeloupe, we are mainly looking for resistance to CUB genotype as its incidence overlaps
with the two other ones, masking resistance to REU and BRA-PER.
31
INVESTIGATIONS ON YELLOW LEAF DISEASE IN SUGARCANE
GROWING AREAS OF GUANGXI
M.X. YAN, W.H. HUANG, C.H. HUANG, Z.Y. DENG, J.J WEI, X.H. PAN,
J.L. WEI, X.K. SHANG
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/ Sugarcane
Research Center, Chinese Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, P.R. China/
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi,
P.R. China
Keywords: Sugarcane yellow leaf virus (SCYLV), healthy seed cane, RT-PCR
The present study was conducted to investigate the occurrence of Sugarcane yellow leaf virus
(SCYLV), causal agent of yellow leaf disease in sugarcane, and to provide the scientific basis
for healthy seed cane production of sugarcane in Guangxi, China. Sugarcane leaf samples
with SCYLV symptoms of different stages were collected from different sugarcane growing
areas of Guangxi. The virus was detected using one-step RT-PCR method with SCYLV
specific primers (P1:5′-AATCAGTGCACACATCCGAG-3′/P2:5′-
GGAGCGTCGCCTACCTATT-3′) and its amplified PCR products were sequenced. Forty-
two out of 84 samples screened were positive for SCYLV by RT-PCR and comprised 50% of
cultivars grown. The sugarcane crop was seriously infected by SCYLV in central Guangxi
including Liuzhou, Liucheng, Yizhou and Luzhai where 19 out of 25 (76%) sugarcane
cultivars were found infected. The incidence was most serious in Liucheng, where 100%
cultivars were infected by the virus. In western Guangxi, 50% of the cultivars were infected
whereas in southern Guangxi, and in eastern Guangxi 28.2% and 16.6% of cultivars were
infected respectively. The results also showed that SCYLV infection was variety-dependent
as variety Liucheng 03-182 was highly susceptible. The susceptibility of varieties followed
the infection pattern: Liucheng 03-182 > GT21 > YT55 > YT93-159. The major commercial
sugarcane variety ROC22 was also affected by SCYLV in Liucheng, Yizhou and Jinguang
37
state farm, the major sugarcane areas in Guangxi. It is concluded that yellow leaf has been
spreading and is now well established in sugarcane growing areas of Guangxi.
32
INVESTIGATION ON SCMV INFECTING SUGARCANE IN GUANGXI, CHINA
W.H. HUANG, M.X. YAN, Z.Y. DENG, C.H. HUANG AND J.J. WEI
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/ Sugarcane
Research Center, Chinese Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, P.R. China/
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi,
P.R. China
Keywords: RT-PCR, mosaic symptoms, sequencing
In order to understand the occurrence of sugarcane mosaic virus (SCMV), leaf samples with
mosaic symptoms were collected from sugarcane growing in different areas of Guangxi,
China. The virus was detected by one-step RT-PCR using SCMV- specific primers
(SCMVF4: 5′-GTTTTYCACCAAGCTGGAACAGTC-3′; Y = C or T, SCMVR3: 5′-
AGCTGTGTGTCTCTCTGTATTCTC-3′). The results showed that 69.4% samples were
positive. Eight amplified PCR products were chosen for direct sequencing analyses. BLAST
analysis revealed that they were all homologous to reported SCMV-CP gene. The
investigation indicated that ROC22, the major sugarcane variety in Guangxi was seriously
infected by SCMV, and other varieties such as ROC16, ROC28, ROC95-8899, Taiyou,
Liucheng 03-182, Liucheng 03-1137 and several Guitang (GT) clones bred by the Sugarcane
Research Institute, Guangxi Academy of Agricultural Sciences, were also positive for SCMV
when detected by RT-PCR.
38
C1
STUDY ON SOME PLANT PARASITIC NEMATODES OF SUGARCANE IN
KHUZESTAN PROVINCE OF IRAN
H. M. REZAMAHALLEH1, E. SHOKOOHI
2, N. NASIRPOOR
1 AND A. M. NASAB
2
1Sugarcane and by Products Development Research and Training Institute, Khuzestan, Iran
2Plant Protection Department, College of Agriculture, Shahid Bahonar University of
Kerman, Iran
Keywords: taxonomy, Meloidogyne javanica, Pratylenchus zea, Tylenchorhynchus
annulatus, Helicotylenchus exallus
Sugarcane is one of the main crops in Khuzestan province, Iran. In order to study plant
parasitic nematodes of the order Rhabditida (Sub order: Tylenchina) of sugarcane in
Khuzestan province, 120 soil and root samples were collected from sugarcane fields in 2009
and 2010. The samples were collected from chlorotic and stunted plants with yellow stripes
on the young leaves. The nematodes were extracted by centrifugal flotation technique.
Permanent slides were prepared and morphological and morphometric characters were
studied using an Olympus CH2 light microscope attached to a drawing tube. In the
preliminary analysis of this survey, Meloidogyne javanica, Pratylenchus zea,
Tylenchorhynchus annulatus and Helicotylenchus exallus were identified. The number of M.
javanica and H. exallus in some sugarcane fields were more than 400 nematodes per 100 g of
soil. This is the first report of occurrence of nematode species in sugarcane fields in Iran.
39
C2
FIRST REPORT OF LEIFSONIA XYLI SUBSP. XYLI, CAUSAL AGENT OF
RATOON STUNT DISEASE OF SUGARCANE IN IRAN
K. TAHER-KHANI1, A. C. MATTIN
2, H. H. SOHI
3
AND N. NASSIR-POUR1
1- Institute of Sugarcane Research & Training, Sugarcane Development & By-product Co.,
Ahwaz, Iran
2- National Sugarcane Research Institute (INICA), Sugar Ministry, Havana, Cuba
3-National Research Center for Genetic Engineering & Biotechnology, Tehran, Iran
Keywords: Ratoon stunt disease, isolation, molecular identification
Ratoon stunt disease (RSD) is often cited as the most important disease of sugarcane in the
world. For evaluating the incidence of RSD in Iran, ten mature stalks of 60 cultivars were
collected randomly. Functional and non-functional vessels of each stalk were determined by
staining following the stalk transpiration method (STM). Among the 60 cultivars sampled,
the percentage non-functional vessels due to plugging of xylem vessels resulting from
colonization by the bacteria ranged between 11% in CP48-103 to 69% in NCo293. For
isolating the bacterium, a modified sugarcane (M-SC) axenic medium was used. Colonies
were observed after two weeks following aerobic incubation at 28˚C. The colonies were 0.1-
0.3 mm in diameter, circular with entire margins, convex and non-pigmented. Taking into
account the morphological and biochemical characteristics, the bacterium was identified as
Leifsonia xyli subsp. xyli. The isolate from cultivar CP50-28 was named LxxIr. Further
identification of the bacterium was confirmed using polymerase chain reaction (PCR) with
samples from STM-positive stalks of cv. CP48-103, CP63-588, CP50-28, and CP45-3 and
from LxxIr lysate and LxxIr non-lysate samples. Primers used for PCR were Cxx1 (5'-
CCGAAGTGAGCAGATTGACC-3') and Cxx2 (5'-ACCCTGTGTTGTTTTCAACG-3') and
the thermocycler parameters were as follows: denaturation at 94°C for 5 min, 30 cycles at
95°C for 1 min, 57°C for 1min, 72°C for 30s, and a final extension at 72°C for 10 min. The
PCR product (437 bp) of each sample was cloned and sequenced. It showed 98-99 % identity
40
with the 16S-23S intergenic spacer region of L. xyli subsp. xyli, thus confirming occurrence
of ratoon stunt disease in Iran. This is the first report of the disease in Iran
C3
SUGARCANE DISEASES AND THEIR CONTROL IN BANGLADESH
M. S. RAHMAN1 AND M. K. BASHAR
2
1. Senior Scientific Officer, Pathology Division, Bangladesh Sugarcane Research Institute,
Ishurdi-6620, Pabna, Bangladesh
2. Director General, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Pabna,
Bangladesh
Keywords: Major diseases, red rot, control measures
Sugarcane is an important cash and industrial crop in Bangladesh. Every year it covers on an
average 2% of total cultivable land. The yield of sugar cane and its recovery in Bangladesh is
quite low compared to other cane growing countries. Diseases play an important role for not
only yield reduction but also for a low recovery. It is estimated that annually at least 20% cane
losses are associated with different diseases. In Bangladesh, 40 diseases and disorders of
sugarcane have been reported of which 22 are caused by fungi, 4 by bacteria, 2 by
mycoplasma, 1 by virus, 2 by nematodes, 2 by parasitic weeds and 7 by physiological
disorders. However, based on severity, only 10 are considered as major (red rot, wilt, smut,
sett rot or pineapple, white leaf, red stripe and top rot, leaf scald, ratoon stunt, root knot and
striga) while the remaining are considered as minor ones. Of the main diseases, red rot is
considered as a major threat to sugarcane production in Bangladesh. Due to its incidence,
many potential varieties have been withdrawn from commercial cultivation. It is a major
challenge for the breeders in the introduction and development of high sugar content
varieties. The prevailing management practices to control sugar cane diseases and other
disorders include: selection of medium-high to high altitude for sugar cane cultivation; use of
resistant varieties; use of heat treated healthy seed material for the production of foundation
seed and their successive generation to obtain certified seed; sett treatment with fungicide
carbendazim before plantation, roguing of diseased clumps as and when they are detected;
timely control of insect pests like top shoot borer, stem borer, rootstock borers; avoiding
41
water logging condition in the field; avoiding ratooning of diseased crops and crop rotation
with green manure crops.
C4
DISEASE EPIDEMICS IN QUEENSLAND AND THE INFLUENCE OF VARIETAL
DOMINANCE ON THEIR DEVELOPMENT
R.C. MAGAREY AND J.I. BULL
BSES Limited, Tully
Keywords: Orange rust, smut, Fiji leaf gall, susceptible varieties
The dominance of commercial varieties within districts and regions is examined in the light
of the development of disease epidemics. Disease epidemics that have developed in Australia
within the last 60 years are brown rust, eye spot, Fiji leaf gall, leaf scald, orange rust,
Pachymetra root rot, smut and sugarcane mosaic. The influence of a single variety is
illustrated very well by the very rapid onset of orange rust when the Australian industry was
dominated by the highly productive Q124. This resulted in the most widespread, severe
disease occurrence and largest, single-year financial loss of any of the epidemics. There was a
strong correlation between area under one variety and the occurrence of a disease epidemic
(brown rust, Fiji leaf gall, leaf scald, orange rust and Pachymetra root rot) for some diseases.
This was not always the case as the noble cane Badila (S. officinarum) was grown for over 40
years as a large proportion of commercial production in several areas of northern Queensland
with no disease epidemic occurring. A brief financial analysis showed that profitability was
maximised by allowing the high-yielding variety to dominate regional production.
42
C5
INVESTIGATION ON THE MAJOR FUNGAL DISEASES OF
SUGARCANE IN GUANGXI, CHINA
J.J. WEI, Z.Y. DENG*, F.X. FANG, W.H. HUANG, M.X. YAN, X.H. PAN, X.J. LIU
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/ Sugarcane
Research Center, Chinese Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, P.R. China/
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi,
P.R. China
Keywords: Fungal diseases, surveys, damages
Guangxi is the largest sugarcane and sugar producing province in China and accounts for
more than 65% of Chinese sugar production. Besides various abiotic stresses, sugarcane
diseases have become a major constraint and one of the limiting factors for sugarcane
production in Guangxi. Surveys on fungal diseases were conducted in sugarcane growing
areas of Guangxi, China during the period 2008-2010 in order to understand their types,
occurrence, distribution and damages caused by them to sugarcane. Thirteen major fungal
diseases namely; pineapple, smut, top rot, rust, leaf scorch, red rot, ring spot, sheath blight,
white spot, eye spot, yellow spot, brown stripe and brown spot were identified following field
investigations, microscopic observations and pathogenicity determination. The 13 fungal
diseases occurred in almost all sugarcane growing areas with smut, top rot, rust and leaf
scorch among the more serious ones. Smut and top rot diseases were destructive to sugarcane
production. Rust tended to spread with the cultivation of new susceptible sugarcane varieties
in recent years.
43
C6
SUGARCANE DISEASES IN EGYPT RESEARCH AND DEVELOPMENT
MOSTAFA M.A.EL-KHOLI AND AYMAN M.H.ESH
Sugar Crops Research Institute, ARC, Giza, Egypt
[email protected], [email protected]
Keywords: Disease forecasting, molecular techniques
Sugarcane is an important cash crop cultivated on approximately 125 000 ha including eight
factories areas with an average national yield of 120 tonnes/ ha. Despite its consideration as
one of the main sugar crops, in terms of yield per area unit, there are several constraints
facing its production in Egypt and they can be summarized as follows: long dependency on
cultivar GT54-9 (known for short as C9) which was introduced to farmers on a wide scale
since 1980 and now occupies almost 97 % of the cane plantation area. Current strategies
undertaken for better quality production include the introduction of certified seed cane free of
diseases. Vegetative propagation of sugarcane results in the accumulation and carryover of
different pathogens in seed pieces. Pathogens affecting sugarcane have been increasing in
recent years and currently about 130 different diseases and pests, affecting the crop, have
been reported worldwide. In Egypt, 10 diseases (eye spot, mosaic, pineapple, pokkah boeng,
ratoon stunt, red rot, rind disease, ring spot, smut, and streak) has been detected and smut
caused by Sporisorium scitaminea is widely prevalent. Roguing and disease forecasting are
applied for smut control and sugarcane fields with ≥ 5% disease incidence are discouraged
for ratooning. In recent years, biotechnology approaches for management of sugarcane
diseases have been applied and the development of molecular techniques for identifying and
characterization of sugarcane pathogens is making rapid progress.
44
C7
PROGRESS OF SUGARCANE DISEASE RESEARCH IN CHINA
Y. LIAO1,2
, Z. WANG1, G. ZHU
3, R. WEN
1,4 AND B. CHEN
1,4*
1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,
Guangxi Univieristy, Nanning, 530005, China;
2 College of Agriculture, Guangxi University，Nanning, 530005, China;
3 Guangxi Academy of Agricultural Sciences, Nanning, 530007, China
4 College of Life Science and Technology，Guangxi University，Nanning, 530005, China
Keywords: Smut, viral diseases, ratoon stunt, Ceratovacuna lanigera
Smut and viral diseases are the two most important problems among more than 30 sugarcane
diseases occurring in China. For smut, the whole genome sequence and transcriptome of the
fungal pathogen Sporisorium scitamineum were determined recently with a combination of
high throughput sequencing coupled with Sanger’s method. Response of sugarcane to smut
fungus infection was investigated and it was revealed that 36 genes were up-regulated and 20
proteins involved in several metabolism pathways were differentially expressed. Transgenic
sugarcane expressing chitinase and β-1,3-glucanase was shown to be resistant to S.
scitamineum. Five viruses including Sugarcane mosaic virus (SCMV), Sorghum mosaic virus
(SrMV), Sugarcane yellow leaf virus (SCYLV), Sugarcane streak mosaic virus (SCSMV)
and Sugarcane bacilliform virus (SCBV) have also been reported in China. SCYLV is now
sporadic in many sugarcane planting regions in southern China. SCBV prevails mainly in
Yunnan province while SCSMV is detected only from a sugarcane variety imported from
India. Ceratovacuna lanigera was shown to be a transmission vector of SCYLV. Transgenic
sugarcane with introduced SCMV-CP was developed and shown to be resistant to SCMV
infection. Eradication of viruses by heat treatment to generate virus-free plants have been in
application and other measures such as water and fertilizer management were found to be
45
effective in delaying or abating the symptom development of yellow leaf disease. Ratoon
stunt disease caused by Leifsonia xyli subsp. xyli (Lxx), is now becoming increasingly
important in China and a PCR based protocol was developed and is widely used for detection
of Lxx in sugarcane seed stem. Bacterium-free sugarcane plants have been obtained by tissue
culture.
C8
ESTABLISHMENT OF AN INTERNATIONAL QUARANTINE FACILITY FOR
THE EXCHANGE OF SUGAR CANE GERMPLSM AMONG ACP COUNTRIES
S. SAUMTALLY, S. DHAYAN, G. TRITON, M. ANTOINE, N. JOOMUN AND A.
DOOKUN-SAUMTALLY
Mauritius Sugacane Industry Research Institute (MSIRI), Réduit, Mauritius
Keywords: Biosecurity, disease elimination, tissue culture
In the framework of increasing the competitiveness of their sugar industries, an African,
Caribbean and Pacific (ACP) Sugar Research Programme has been established with the
objectives of promoting new sugarcane varieties, reducing costs of production and limiting
the negative impact of production on the environment. ACP states have been actively
involved in germplasm exchange and this is likely to be enhanced. In many instances, the
movement of sugarcane clones is effected without formal quarantine or by superficial
observations in open quarantine by unqualified personnel. It is recognized that safeguards for
preventing the introduction of damaging pathogens in ACP countries are essential. So as to
avoid duplication of facilities in these countries, a project funded by the European Union was
approved to set up an international quarantine station in Mauritius. An existing quarantine
and buildings were refurbished and equipped to be operational in 2012. The facility will be
able to accommodate some 150 varieties at any one time. A phytosanitary planning will be
undertaken on the diseases to be tested for, depending on the country of origin. Conventional
and molecular diagnostic methods will be applied in disease detection while elimination of
pathogens would be undertaken by heat therapy and tissue culture. It is expected that the
exchange of disease-free sugarcane germplasm between ACP countries will be enhanced
through this facility.
46
C9
MANAGEMENT OF SYSTEMIC DISEASES OF SUGARCANE IN TEXAS AND
LOUISIANA
J. M. AMADOR1 AND J. L. FLYNN
2
1 Retired, Texas AgriLife Research, Texas AgriLife Research and Extension Center, Weslaco,
Texas, USA 2
Kleentek, a Division of Certis USA, Baton Rouge, Louisiana, USA
Keywords: mosaic, ratoon stunt, yellow leaf, leaf scald
Several systemic diseases seriously impact the sugarcane industries in both Texas and
Louisiana. Diseases such as mosaic (SCMV), ratoon stunt disease (RSD), yellow leaf
syndrome (YLS), and leaf scald have caused much economic damage. The main line of
defense against these diseases has been the development of resistant varieties, an important
effort in both states. Several breeding programs operated by public institutions, one in South
Texas conducted at the Texas AgriLife Research experiment station at Weslaco, and two in
Louisiana, conducted by the Louisiana State University Agricultural Center at St. Gabriel,
near Baton Rouge, and the other by the United States Department of Agriculture, Agriculture
Research Service at Houma Louisiana, respectively, have been successful in providing a
steady stream of new varieties with improved resistance to these diseases. However, when
resistance is not available, or not strong enough in commercially desirable varieties, other
means are used. Planting disease free seed has been important in the management of
systemic diseases. The use of hot air and hot water treatments has been tried over the years
with mixed results. Currently, the use of tissue cultured plants derived from meristematic
tissues extracted from disease indexed source plants has gained wide acceptance by the
industry. This effort is conducted mainly by commercial entities that produce, grow, and sell
seedcane to producers. Disease occurrence in the industry has been reduced drastically. As
an example, a recent RSD incidence survey showed that from 57.7 % of infected fields and
47
16.9% of infested stalks, the disease was reduced to 0% in each case, respectively. In
Louisiana, current RSD levels are near 0% industry wide. Results are so positive that almost
75 % of the seedcane currently used is from tissue cultured sources.
P1
DEVELOPMENT OF AN INOCULATION METHOD FOR TRUE SEEDLINGS OF
SUGARCANE AGAINST SMUT
1 2
S. A. BHUIYAN, 1 B. J. CROFT,
3 R. S. JAMES,
2 AND
M C COX
1 BSES Limited, Woodford, QLD 4514, Australia,
2 BSES Limited, Bundaberg, QLD 4670
Australia, 3 Australian Quarantine and Inspection Services (AQIS), Darwin, NT 0820
Australia
Keywords: Sporisorium scitamineum, dip inoculation, natural spread
Sugarcane smut, caused by Sporisorium scitamineum, was first reported in Queensland, the
main sugarcane growing region in Australia, in 2006. At this time, approximately 80% of the
commercial varieties in the Australian sugarcane industry were susceptible to smut. Previous
studies have shown that 5 to 15% of progeny from two highly susceptible parents are
resistant to smut. BSES has undertaken a program to recover smut resistant clones from high
value smut susceptible crosses. As part of the program, approx. 40,000 seedlings from
susceptible crosses were screened for smut resistance in two phases, as true seedlings
(germinated seeds from a cross) and as vegetatively propagated clones from true seedlings.
There is no accepted method for inoculating true seedlings with smut. The aim of the research
was to develop an inoculation method for true seedlings that would provide a reliable
indication of field resistance. Two experiments were established at the BSES smut research
farm, Bundaberg in 2007 and 2009 to examine four methods for inoculating true seedlings.
The treatments were, (i) dip four-week-old seedlings (dip inoculation) in smut spore
suspension (106 spores/mL water) , (ii) trim and spray six-week-old seedlings with smut
spore suspension, (iii) natural spread by planting the seedlings between smut infected
spreader rows, and (iv) spray germinating seedlings (two days after germination) with a smut
spore suspension. Fifty seedlings from 12 families with three resistance categories
48
(susceptible, intermediate and resistant), were included in each method. The results from both
years’ trials indicated that dip inoculation was most effective for screening true seedlings
against sugarcane smut. There were highly significant correlations (r = 0.75 – 0.91, P<0.001)
between percent smut infected seedlings and mid-parent rating in the dip inoculation method.
Natural infection was effective but this method requires more time and considerably larger
areas. The dip inoculation method has been adopted for the routine screening of sugarcane
seedlings for smut.
P2
DEVELOPMENT OF A NEW METHOD OF STATISTICAL ANALYSIS FOR
DISEASE SCREENING TRIALS
1 J. STRINGER,
2 B. J. CROFT,
3 S. A. BHUIYAN,
1 E. DEOMANO,
4 R. MAGAREY,
3 M.
COX AND 5 X. XU
1BSES Limited Indooroopilly,
2 BSES Limited Woodford,
3 BSES Limited Bundaberg,
4 BSES
Limited Tully, 5East Malling Research, Kent, United Kingdom
Keywords: disease screening trials, linear mixed models
Many endemic diseases are controlled through the cropping of resistant sugarcane varieties.
Routine resistance screening trials for a range of diseases are a core activity of BSES. Central
to this is the appropriate statistical analysis of data from these disease screening trials; this is
important since potential high-yielding varieties could be discarded from the breeding
program if they are incorrectly rated too susceptible, resources are wasted on varieties that
are incorrectly rated resistant or varieties incorrectly rated resistant are released to industry
and the variety suffers serious losses. The method used by BSES for rating varieties for
disease resistance since the 1970s was based on a regression equation for the relationship
between the disease score of a set standard varieties in the trial and the historical ratings of
the standard. This regression equation was then used to assign a rating to the test clones. In a
recent review of the analysis of disease resistance trials, a new technique based on analysis
with a linear mixed model was developed. The standard varieties are separated into groups
based on the least significant difference test and the test varieties are placed in the group to
which they best fit. A correlation is performed on the score of the standards in the new trial
against the average score from all previous trials to determine if the current trial can be
considered to provide a reliable estimate for the standards. In this paper, these new methods
49
are applied to smut screening data and the impact on disease ratings and selection of new
varieties is discussed.
P3
MECHANISM OF RESISTANCE OF AUSTRALIAN SUGARCANE VARIETIES
AGAINST SMUT
S. A. BHUIYAN1, B. J. CROFT
1 AND K. AITKEN
2
1
BSES Limited, Brisbane, Australia, 2 Commonwealth Scientific and Industrial Research
Organisation (CSIRO), Brisbane, Australia
Keywords: Sporisorium scitamineum, internal resistance, external resistance
The available literatures suggested that there are at least two types of resistance mechanisms
in sugarcane plants to smut (Sporisorium scitamineum); i) bud scale or external resistance
and ii) internal resistance. The external resistance is believed to be chemical or physical
barriers on sugarcane buds which prevent smut teliospores from penetrating and establishing
infection. On the other hand, internal resistance is governed by the interaction of plant and
fungus within the invading plant tissue. The aims of this study were to (i) understand the
resistance mechanisms of some important commercial varieties, (ii) understand the
relationship between age of bud and smut resistance, and (iii) identify the gene(s) involve in
the resistance. A series of experiments were conducted in BSES Limited Bundaberg and
CSIRO from 2008 to 2010. Research suggested that the majority of the Australian
commercial varieties possessed internal resistance mechanisms. Major varieties such as,
Q208A, Q200A and KQ228A possessed external resistance and Q183A, Q171A and Q232A
possessed internal resistance mechanism. Age of the bud has significant effects on smut
infection. Younger buds were more susceptible to smut compared to older bud. A study of
progeny from a cross between a cultivar that contained internal resistance and a cultivar with
only external resistance suggested major genes were responsible for internal resistance.
50
Future work is being planned using new molecular and histopathological techniques to
elucidate the external and internal resistance mechanisms identified in the current studies.
P4
EFFICACY OF BIOCHEMICALS IN RELATION TO RED ROT
RESISTANCE IN SUGARCANE
A. SINGH, S. K. VISHWAKARMA, P. KUMAR*, A. K. TIWARI AND M. L. SHARMA
U.P. Council of Sugarcane Research, Shahjhanpur-242001 (U.P.), India
Keywords: Colletotricum falcatum, Phenylalanine ammonia lyase, Tyrosine ammonium
lyase, phenolics, gum content
Red rot, caused by Colletotrichum falcatum Went., is a major threat of sugarcane and directly
affects the production of the crop in India and worldwide. Four sugarcane varieties namely
SES 594, Co 312, Co 1148 and CoJ 64 were inoculated with two pathotypes, Cf-01 & Cf-08,
of C. falcatum during 2008-09 and 2009-10 to identify biochemical markers in relation to red
rot-resistant genotypes. Phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase
(TAL) are key enzymes of the phenylpropanoid pathway that are induced in response to
biotic and abiotic stresses. In the resistant red rot genotype SES 594, a significant increase in
the activity of PAL and TAL was observed following inoculation with the pathogen. These
enzymes therefore play an important role and are directly correlated with the variety
resistance. It was also observed that the total phenolic content in the resistant variety SES 594
was higher than in the most susceptible variety Co 312 and it may be possible that a
particular phenolic compound is responsible for resistance. The increase in total phenolic
content indicates that it may be associated with the induction of PAL and TAL activities,
which facilitate the formation of several phenolic compounds. Free proline contents were also
increased in plants inoculated with C. falcatum. The effect of proline however appears to be
primarily quantitative as it accelerates and increases the existing defence reaction. Higher
51
amount of the presence of gum in the resistant genotype SES 594 indicated that gum content
could also play a role in resistance or susceptibility of the genotypes.
P5
DEVELOPMENT OF A SENSITIVE NESTED-PCR ASSAY FOR THE DETECTION
OF SPOROSORIUM SCITAMINEUM
W. SHEN 1,2
, P. XI2, M. LI
2, L. SUN
2, Z. JIANG
2 AND L. ZHANG
2
1Guangzhou Sugarcane Industry Research Institute/ Guangdong Key Laboratory of
Sugarcane Improvement and Biorefinery, Guangzhou 510316, China
2Department of Plant Pathology, South China Agricultural University, Guangzhou 510642,
China
Keywords: Sugarcane, smut, ITS
A species-specific polymerase chain reaction (PCR) assay was developed for rapid and
accurate detection of Sporosorium scitamineum, the causal agent of sugarcane smut disease.
Based on nucleotide differences in the internal transcribed spacer (ITS) sequences of S.
scitamineum, a pair of species-specific primers, SL1/SR2, was designed by using a panel of
fungal and bacterial species as controls. The primers SL1/SR2 specifically amplified a unique
PCR product about 530 bp in length from S. scitamineum strains, with a sensitivity of 200 fg
of the fungal genomic DNA in a 25 μL reaction solution. To increase sensitivity, a nested-
PCR protocol was further established, which used ITS4/ITS5 as the first-round primers
followed by the primer pair SL1/SR2. This protocol increased the detection sensitivity by
10,000-fold compared to the PCR method and could detect the fungal DNA as low as 20 ag.
The nested-PCR detected S. scitamineum from young sugarcane leaves with no visible smut
disease symptoms. The findings from this study provide a sensitive and reliable technique for
the early detection of S. scitamineum, which would be useful for sugarcane quarantine and
production of smut-free seed canes.
52
P6
EFFECT OF PLANTING HEALTHY SEED CANE ON SOME AGRONOMIC
CHARACTERISTICS IN SEVERAL NEW SUGARCANE VARIETIES
Z.-YUN DENG*, H.-BIN LIU, F.-XUE FANG, W. XIAN, S.-YUN TANG, L.-WANG
WANG, X.-JING LIU, M. LI, J.-XUN LU AND L. XU
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/ Sugarcane
Research Center, Chinese Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, P.R. China/
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi,
P.R. China
Keywords: Ratoon stunt disease, healthy seed cane, yield
Healthy seed cane (HS) could effectively control ratoon stunt disease (RSD) of sugarcane. In
order to investigate the efficiency of HS, a comparative plant and ratoon crop trials using HS
from 6 sugarcane varieties was conducted during 2006-2008. Healthy seed canes were hot
water treated at 50°C for 2 h. Before planting the HS were checked for RSD using PCR
detection method. The results indicated that the cane and sugar yield increased significantly
(p<0.05; p<0.01) when using HS compared to common seed canes (CS) non-treated controls.
The positive effects of HS planting continued in the ratoon crop. The cane and sugar yields
were 16.4% and 15.9% higher respectively in the HS plantation as compared to CS controls.
The sucrose content in cane was not found to be affected, but the cane yield components were
influenced by planting the HS. Healthy seed canes enhanced the seedling emergence rate,
promoted tillering rate, plant height, stalk diameter and number of millable stalks. Therefore
HS planting is recommended for commercial sugarcane production.
53
P7
THE EFFECTS OF RATOON STUNT DISEASE ON XYLEM STRUCTURE AND
ENDOGENOUS HORMONE LEVELS IN SUGARCANE STALK
M. H. CHEN1,2
, X. N. XIE1,2
, L. T. YANG1,2,3
AND Y. R. LI2,3,4,*
1 Agricultural College, Guangxi University, Nanning 530005, China
2 State Key Laboratory of Subtropical Agriculture Bioresources Conservation and
Utilization, Nanning 530005, P.R. China 3 Sugarcane Research Center, Chinese Academy of Agricultural Sciences/ Sugarcane
Research Center, Guangxi Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, China/ Guangxi
Key Laboratory of Sugarcane Genetic Improvement 4
Guangxi Crop Genetic Improvement and Biotechnology Lab, Nanning 530007, China
Keywords: Leifsonia xyli subsp. xyli, pathogen-free plants, transmission electron microscopy
Ratoon stunt disease (RSD) of sugarcane (Saccharum spp. hybrids), caused by the bacterium
Leifsonia xyli subsp. xyli (Lxx), is one of the most economically damaging diseases
worldwide. The objective of the present study was to determine the effects of RSD on
sugarcane growth, endogenous hormone levels and the xylem structure in stalk. Two
varieties, ROC22 and the chewing variety Badila, were selected as planting material and RSD
infection was determined using PCR reaction. The cane seeds were grown in pots and
consisted of three treatments: (i) pathogen-free setts as control; (ii) thermal-treated infected
RSD setts at 52oC for 30 min and (iii) infected RSD setts. The plant height and contents of
endogenous hormones in cane stalk were analyzed after 90, 120, 150 and 180 days of
plantation. The results showed that (1) the plant height resulting from RSD infected setts was
54
significantly lower in the pathogen-free control and in thermal treated plants; (2) the contents
of GA3 and IAA were lower in the RSD infected plants than those in the control and thermal
treated plants, in contrast to the content of ABA which was higher in the RSD infected plant
as compared to the other two treatments. Both varieties showed similar plant height and
endogenous hormone levels pattern and there was no significant difference between the
pathogen–free control and the thermal treatment. Difference was observed in the xylem under
TEM between the healthy and infected cane stalk. A large amount of electron-dense
substances accumulated in the infected cells that could be associated with bacterial presence.
In addition the xylem cell walls were degraded and broken at different degrees in the infected
stalk. The results indicate that yield loss in sugarcane due to RSD infection may be associated
with water and nutrition transportation disorders and endogenous hormone balance in the
diseased plants.
P8
DETECTION OF RATOON STUNT DISEASE IN DISEASE-FREE SEED CANE OF
SUGARCANE BY REAL-TIME FLUORESCENCE QUANTITATIVE PCR
M. DAN, S. LI, K. X. YU，J.H. YOU AND C.H. HUANG
Guangxi Sugarcane Research Institute, Guangxi Academy of Agricultural
Sciences/Sugarcane Research Center, Chinese Academy of Agricultural Sciences/Key
Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of
Agriculture, Nanning 530007, China
Keywords: Leifsonia xyli subsp. xyli, tissue culture, disease detection
The aims of this study were i) to detect Leifsonia xyli subsp. xyli, (Lxx) causal agent of ratoon
stunting disease (RSD) in disease-free seed cane of sugarcane by real-time fluorescence
quantitative PCR and ii) to establish and improve the real-time PCR technology system for
RSD detection. Six sugarcanes plants of variety ROC22 infected with ratoon stunting disease
were, were put in tissue culture to free them from the Lxx bacterium. Conventional PCR and
Real-time fluorescence PCR were used to detect the Lxx pathogen in 10 of the tissue culture
plantlets. No Lxx bacterium was detected by both PCR techniques, indicating that tissue
culture technique could be efficiently applied for the production of healthy plantlets, free
from RSD.
55
P9
ACIDOVORAX AVENAE SUBSP. AVENAE ASSOCIATED WITH RED STRIPE
SYMPTOMS OF SUGARCANE IN PAKISTAN*
S. ZIA-UL-HUSSNAIN1, M. I. HAQUE
2, S. AFGHAN
3 AND A. SHAHAZAD
4
1 PhD (Scholar), Department of Plant Pathology, Pir Mehr Ali Shah (PMAS) Arid
Agriculture University Rawalpindi, Pakistan 2 Chairman, Department of Plant Pathology, Pir Mehr Ali Shah (PMAS) Arid Agriculture
University Rawalpindi, Pakistan 3 Director Research, Shakarganj Sugar Research Institute, Toba Road Jhang, Pakistan
4 Nematologist, Shakarganj Sugar Research Institute, Toba Road Jhang, Pakistan
[email protected], [email protected], [email protected],
Keywords: DAC-ELISA, yeast dextrose chalk agar
Symptoms of watery green stripe near the midrib of leaves were observed on promising
clones of sugarcane, namely, NSG-49, CPSG-2453, CP-NIA-82-223, CSSG-2402 and US-
114, planted during autumn 2009-2010. Bacteria were isolated on yeast extract dextrose chalk
agar (YDC) media from the infected leaf of variety CSSG-2402. Off-yellow colour and
convex bacterial colonies of 1.8 to 2.0 mm diameter were observed. Morphological
appearance and biochemical characterizations identified the bacterium as Acidovorax avenae
subsp. avenae. The strain was detected by direct antibody coating Enzyme-Linked
Immunosorbent Assay (DAC-ELISA) using antiserum raised against Acidovorax avenae
subsp. avenae. Out of 27 varieties tested by DAC-ELISA, the results showed that sugarcane
clone CSSG-2402 and US-114 had a higher titer than the varieties tested CP-NIA-82-223,
56
CPSG-2453 and NSG-49. Varieties GT-11, SPF-213, SPF-238 and CP77-400 were weakly
positive at 405 nm. A bacterial suspension (5 × 107 CFU/ml) was prepared from freshly
grown pure culture of A. avenae subsp. avenae and inoculated into the growing point of 25
varieties to assess their resistance to the disease. Sugarcane clones NSG-555, SPSG- 79,
CPF-237, HSF-240, CSSG-668, CSSG-676, NSG-311, CPSG-3481, NSG-59, CPSG-2923,
CSSG-212, CPSG-104, HoSG-1257, CPSG- 25, CSSG-239, CPSG-2713 were resistant; SPF-
238, CP77-400, SPF-213, GT-11 showed moderate resistance; NSG-49, CPSG-2453 and CP-
NIA-82-223 were moderately susceptible and CSSG-2402 and US-114 were susceptible to
the red stripe pathogen. The pathogen was re-isolated from the inoculated plants and
identified as Acidovorax avenae subsp. avenae.
*A first report was published in African Journal of Biotechnology vol 10 (37), pp 7191-7197, 20 July 2011
P10
INVESTIGATION OF FUSARIUM PATHOGENS INFECTING
SUGARCANE IN KHUZESTAN, IRAN
H. MOAZZEN-REZAMAHALLEH1 AND R. F. NEZHAD
2
1Department of Plant Protection, College of Agriculture, University of Chamran, Iran
2 Sugarcane Development & By-Products Research & Training Institute, Khuzestan, Ahvaz,
Iran
Keywords: Pathogenicity, knife-cut symptoms, pokkah boeng, Fusarium verticillioides
(moniliforme), Fusarium subglutinans, Fusarium proliferum
During 2006-2007, infected seed cane was collected from several sugarcane agro-industries.
Knife-cut symptoms on one or both sides of the stalk were observed. Approximately 270
isolates of Fusarium were isolated from the knife-cut infection and after subculturing on
specific culture medium and conducting macroscopic observations (color, morphology and
colony growth rate) as well as microscopic evaluation (phyalid type, appearance or absence
of microconidia chain), the causal agents associated with the symptoms were identified to
belong to three Fusarium spp.; F. verticillioides (moniliforme), F. subglutinans and F.
proliferatum. The former two species are known to cause pokkah boeng in sugarcane. The
occurrence of the three species was as follows: F. verticillioides (moniliforme), 57%; F.
proliferatum, 30% and F. subglutinans, 13%. A detached stem technique showed that all
isolates were pathogenic. The dominant species in the agro-industries area located in the
north of Khuzestan was F. subglutinans while F.verticillioides was mainly found in the south.
57
P11
BIOLOGICAL CHARACTERISTICS OF BROWN RUST IN BEIHAI, CHINA AND
ITS CONTROL STRATEGY
J.J. WEI, Z.Y. DENG, W.H. HUANG, X.H. PAN, B.H. WANG AND X.J. LIU
Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences/ Sugarcane
Research Center, Chinese Academy of Agricultural Sciences/ Key Laboratory of Sugarcane
Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture, P.R. China/
Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi,
P.R. China
Keywords: uredospores germination, spore viability, optimum pH, optimum temperature
Sugarcane brown rust is an important disease causing damage to leaves. The biological
characteristics of the brown rust pathogen found in Beihai, Guangxi, China were studied. The
results showed that the germination of uredospores were promoted under conditions of
darkness or when cultured with 1.5% glucose. Germination of the uredospores took place at
temperature ranging from 20 oC to 25
oC, but the optimum was at 25
oC. The suitable relative
humidity for germination of the uredospore was 80% or higher, and the optimum condition
was 100% which resulted in the germination rate of 28.3%. The uredospores germinated in a
pH ranging from 4.1 to 11.1, but the optimum was at 7.0. Under normal conditions, the
viability of the uredospores was 120 days at the most. Selection of resistant varieties and
appropriate cultivation management were the key measures for control of the disease.
58
P12
OCCURRENCE OF SUGARCANE LEAF SPOT DISEASE CAUSED BY BIPOLARIS
SPICIFERA IN CHINA
S. H. LIN, C. H. HUANG*, Z. Y. DENG, M. X. YAN, W. H. HUANG, J. J. WEI AND Z. Q.
QIN
Guangxi Sugarcane Research Institute, Guangxi Academy of Agricultural
Sciences/Sugarcane Research Center, Chinese Academy of Agricultural Sciences/Key
Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of
Agriculture, Nanning 530007, China/ Guangxi Key Laboratory of Sugarcane Genetic
Improvement *[email protected]
Keywords: Pathogenicity, conidial morphology, rDNA-ITS nucleotide sequences,
A new leaf spot disease of sugarcane, caused by Bipolaris spicifera was observed for the first
time in Luoman Township of Liuzhou City, Guangxi, China in 2011. The disease occurrence
and symptom development were investigated in the field. Three pathogenic isolates (L1, L2
and L3) were obtained and their pathogenicity was assessed by direct inoculation based on
Koch’s postulates. The conidial morphology and internal transcribed spacer (rDNA-ITS)
sequences confirmed that the pathogenic isolates were B. spicifera as the nucleotide
sequences of the isolates showed 99 – 100 % identity with those of the GenBank-derived B.
spicifera isolates. The accession numbers in GenBank database of these three pathogenic
isolates are JN695634, JN695635 and JN695636, respectively, while their collection numbers
in the China General Microbiological Culture Collection Center in Beijing, China are
59
CGMCC 3.14922 and CGMCC 3.14923, respectively. This is the first report on B. spicifera
causing sugarcane leaf spot disease in China.
P13
WIDESPREAD OCCURRENCE AND BIOLOGICAL CHARACTERIZATION OF
SUGARCANE STREAK MOSAIC VIRUS INFECTING SUGARCANE IN INDONESIA
L. K. PUTRA1,*
, A. KRISTINI1, E. M. ACHADIAN
1, T. A. DAMAYANTI
2,
R. MAGAREY3 AND N. THOMPSON
3
1Indonesian Sugar Research Institute, Jl. Pahlawan 25 Pasuruan67126 Indonesia
2Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University
(IPB), Jl. Kamper, Darmaga, Bogor 16680 Indonesia 3BSES Limited, PO Box 86, Indooroopilly, Qld 4068, Australia
Keywords: disease incidence, detection, transmission, host range, yield losses, control
Streak mosaic is a new disease of sugarcane in Indonesia caused by Sugarcane streak mosaic
virus (SCSMV). Since the first report in 2005, the disease is now widely distributed in the
commercial sugarcanes in Java and may be also outside Java. A survey was conducted during
the milling season of 2008/2009 at 30 sugar factories areas across Java. The results revealed
that about 30% of the sugarcane farms at 28 sugar factories were affected by the disease.
Most of commercial cane cultivars were infected by the virus and it predominantly infected
cultivar PS 864. Reverse transcriptase PCR detection using a pair of SCSMV specific to coat
protein (CP) gene primers, SCSMV-547F and SCSMV AP3, successfully amplified a 500 bp
DNA fragment, suggesting the positive identity of SCSMV. Protein analysis of the virus
confirmed a coat protein size of approximately 40 kDa and under electron microscope a
flexuous, filamentous particle about 890 nm in length was observed. The nucleotide sequence
60
of the CP gene of the virus had the highest identity with that of a SCSMV isolate from
Pakistan. The virus is easily transmitted by infected cane cuttings, mechanically transmitted
through wounds made by carborundum, abrasive pad rubbing or cutting knife and could also
be transmitted by Melanaphis sacchari and Toxoptera citricidus. Host range test on 23 plant
species revealed that maize, sorghum and Dactyloctenium aegyptium were alternative hosts
of SCSMV, while others were not. A preliminary yield loss assessment on cultivar PS 864
revealed that the disease incidence at ≥ 50% reduced sugar yield by about 20%. Hot water
treatment of cane cuttings was not able to eliminate the virus in cane stalks but only
postponeed the appearance of the symptom. Response of 16 commercial cane cultivars to
artificially inoculation of SCSMV showed that only 5 cultivars were resistant to the disease.
P14
DEVELOPMENT OF THE LOOP MEDIATED ISOTHERMAL AMPLIFICATION
METHOD FOR DETECTION OF SUGARCANE YELLOW LEAF VIRUS
E. FERNANDEZ 1, R. AMATA
2, P. ROTT
1 AND P. ROUMAGNAC
1
1 CIRAD, UMR BGPI, F-34398 Montpellier, France
2 Kenya Agricultural Research Institute (KARI) P.O. Box 57811 Nairobi, 00200 Kenya
Keywords: Yellow leaf, tissue blot immunoassay, diagnostic methods
Sugarcane yellow leaf is a disease caused by Sugarcane yellow leaf virus (SCYLV). It is a
major emerging disease of sugarcane that has been reported in numerous locations including
Brazil, Mauritius, Reunion Island, French West Indies, South Africa, Swaziland, Malawi and
Zimbabwe. The development of efficient tools to diagnose this disease is important,
especially diagnostic methods that are effective both at the laboratory and at field level. This
study aims at developing a rapid, sensitive and accurate method for the detection of SCYLV,
using the Loop Mediated Isothermal Amplification (LAMP) technology. The presence of
SCYLV was tested in sugarcane leaf samples by both tissue blot immunoassay (TBIA) and
LAMP method. The efficiency of the LAMP technology to detect SCYLV in plants that were
grown in CIRAD’s quarantine greenhouses and in leaves that were collected from several
locations including Reunion Island, Kenya and the French West Indies will be reported.
61
P15
DIVERSITY OF ENDOPHYTIC DIAZOTROPH BACTERIA ASSOCIATED WITH
SUGARCANE IN EGYPT AND THEIR ANTIFUNGAL ACTIVITY
A. M. H. ESH1, M. M. A. EL-KHOLI
1, M. GRISHAM
2 AND W. PAUL
2
1.
Sugar Crops Research Institute, ARC, Giza, Egypt. 2.
Sugarcane Research Unite Houma, LA. USA
Keywords : Nitrogen fixing bacteria, acetylene reduction assay, sequence analysis
A screening for nitrogen fixing bacteria in sugarcane grown in Egypt was preformed. A
nitrogen free medium, LGI-P, was used to isolate bacteria from cane stalks. Among 52
isolates subjected to acetylene reduction assay (ARA) to determine nitrogenase activity, 20
were found to have a remarkably high activity. Such isolates were identified and
characterized through 16S rDNA sequence analysis and blasted in gene bank NCBI. Using
the sequencing data of the 16S rDNA genes, the 20 isolates were grouped in 7 genera:
Pantoea spp. (3 isolates: P. agglomerans and P. anthophila), Enterobacter spp. (4 isolates: E.
ludwigii and E. cloacae), Pectobacterium sp. (3 isolates: P. cypripedii), Pseudomonas sp. (4
isolates: P. putida), Frateuria or Dyella sp. (1 isolate), Burkholderia spp. (2 isolates: B.
gladiol and B. plantarii) and Aneurinibacillus sp. (3 isolates: A. aneurinolyticus). The in vitro
antagonistic study showed a noticeable antagonistic effect of certain genera against sugarcane
smut or red rot pathogens.
62
P16
STUDIES ON SUGARCANE NEMATODE SPECIES AND THEIR DISTRIBUTION
IN GUANGXI, CHINA
J.L. WEI, C.H. HUANG*, X.K. SHANG, X.H. PAN, D.F. HUANG AND B.H. WANG
Guangxi Sugarcane Research Institute, Nanning Guangxi, 530007, China
Keywords: Helicotylenchus, Tylenchorhynchus, Pratylenchus, paddy fields
An investigation on nematode species of sugarcane and their distribution in Guangxi, China
was carried out. Soil samples from major sugarcane areas were collected and nematodes
identified using the Baermann shallow dish funnel separation method and optical microscopy.
Sixteen sugarcane parasitic nematodes were identified and they belonged to Helicotylenchus,
Tylenchorhynchus and Pratylenchus species. Nematodes were more frequently encountered
in paddy fields compared to dry sloping fields and in ratoon crops. Tylenchorhynchus and
Pratylenchus species were identified in paddy ratoon sugarcane crops while in the dry
sloping plant cane fields, Helicotylenchus species were present.