Sri Hendrastuti Hidayat{ XE "Hidayat, S.H." }1, Sri Sulandari2, and Sriani Sujiprihati1
1
Bogor Agricultural University, Campus Darmaga, Bogor 16680, Indonesia
2
Gadjah Mada University, Yogyakarta 55281, Indonesia
INTRODUCTION
Whitefly‐transmitted geminiviruses (WTGs) has been reported to
infect several crops in Indonesia including tobacco, tomato, chilli
pepper, ageratum, and cucumber. Infection of WTGs in chilli
pepper causes severe crop damage and becoming a major threat
since early 2000. The most unique symptoms associated with the
virus infection involved yellowing and leaf curling, therefore it
was known as pepper yellow leaf curl (PYLC) disease. Biological
and molecular characterisation of the causal agent reveals that
several WTGs are associated with the disease. Disease spread
was very fast due to activity of its insect vector, Bemisia tabaci,
which grows very prominently in the tropic climate. Therefore,
disease control is becoming very difficult. Breeding program for
WTGs resistance varieties is one of major activities in regard to
disease control strategy in Indonesia since commercial cultivars
carrying resistance to the diseases have not yet been released.
Evaluation of chilli pepper genotypes showed that some
germplasms are very promising for development of cultivars
with resistance or tolerance to the disease.
MATERIALS AND METHODS
Analysis of Genetic Diversity. Pepper plant showing typical
symptoms of PYLCV infection were collected from several chilli
pepper production areas in Indonesia. Extraction of total DNA
and PCR amplification was done according to procedure
explained previously (1, 2). Sequence data obtained following
nucleotide sequencing of the PCR product was analysed using
ClustalW program version 1.83 EMBL‐EBI.
Evaluation of Chilli Pepper Genotypes. Inoculation of PYLCV by B. tabaci was conducted as explained previously (3). Response of
different chilli pepper genotypes was classified into three groups
i.e. resistant, moderately resistant, and susceptible based on
symptoms expression and disease incidence.
RESULTS AND DISCUSSION
Identity of geminivirus infecting chilli pepper in Indonesia was
determined based on their hairpin loop structure and repetitive
sequence found in the common region. These hairpin loop
structure was found in all geminivirus sequences so far (4).
Variability in the structure as well as the length of hairpin loop
region was observed among PYLCV isolates. This may indicate
the possible genetic diversity among WTGs infecting chilli pepper
in Indonesia. Phylogenetic analyses involving 32 sequences
showed that PYLCV isolates can be differentiated into several
clusters. Interestingly, they are all quite different from WTGs
infecting tomato in Indonesia but more closely related to tomato
yellow leaf curl virus from Thailand.
Evaluation of 11 commercial cultivars and 27 genotypes of chilli
pepper showed that the symptoms were developed within 2 to 3
weeks after inoculation, although some genotypes required
longer incubation period. Disease incidence was varied among
different genotypes, i.e. in the range of 12 up to 100%. Selection
of potential genotypes was proceeded for further breeding
activity in order to develop resistant varieties for PYLCV.
ACKNOWLEDGEMENTS
This research was supported in part by ACIAR—AVRDC Chilli
Integrated Disease Management Project.
REFERENCES
1. Doyle JJ, Doyle JJ (1999) Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
2. Rojas MR, Gilbertson RL. Russel DR, Maxwell DP (1993) Use of degenerate primers in the polymerase chain reaction to detect whitefly‐transmitted geminiviruses. Plant Disease 77, 340–347. 3. Aidawati N, Hidayat SH, Suseno R, Sosromarsono s (2002)
Transmission of an Indonesian isolate of tobacco leaf curl virus by Bemisia tabaci Genn. (Hemiptera:Aleyrodidae). Plant Pathol J 18,
231–236.
4. Ikegami M, Morinaga T, Miura K (1988) Potential gene product of bean golden mosaic virus have higher sequence homologies to those of tomato golden mosaic virus than those of cassava laten virus. Virus Genes 1,191–203.
Session
2D—Disease
management
Fungicide resistance in cucurbit powdery mildew
G. MacManus, C. Akem{ XE "Akem, C." }, K. Stockdale, D. Lakhesar, E. Jovicich and P. BoccalatteHorticulture and Forestry Science, Queensland Primary Industries and Fisheries, P.O. Box 15, Ayr, Qld 4807
INTRODUCTION
Powdery mildew, caused by Podosphaera xanthii, (Castagne) is a
major constraint to commercial cucurbit production in Australia
and worldwide. Management of this disease has relied primarily
on the use of foliar fungicides sprays. The development of strains
of the pathogen with resistance to systemic fungicides is
becoming increasingly widespread with the excessive use of
these fungicides. Resistance problems were first reported in
Queensland in the late 1980s (1) and since then there has been
no resistance monitoring program.
The aim of this study was to determine if resistance had
developed to the four systemic fungicides (Amistar, Bayfidan,
Nimrod and Spinflo) registered in Australia for the control of
powdery mildew in cucurbit crops in the Burdekin region of
north Queensland.
MATERIALS AND METHODS
In 2008, 21‐day old seedlings of a powdery mildew susceptible
zucchini variety (Congo, SPS) were used in bioassays to test for
resistance against current registered fungicides. Plants with good
vigour were sprayed with the four systemic fungicides at half,
full and double the recommended label rates of application and
water as controls, 24 h prior to overnight field exposure in
various cucurbit crops at seven different locations in the
Burdekin production region. Actively growing apical shoots were
removed from each plant leaving two cotyledons and three to
four true leaves.
The exposed treated seedlings were randomly placed on
benches in a glasshouse where night temperatures averaged
about 20°C and day temperatures 30°C. Three lower true leaves
of each seedling, which served as replications for each plant
were rated for disease severity 11 and 15 days after field
exposure. Disease severity (% leaf area infected) was estimated
to the nearest 5%. The data collected was analysed using
Genstat 11 to determine treatment differences.
RESULTS
Powdery mildew infection was first noticed on the water‐
sprayed control seedlings 7 days after field exposure. Disease
severity at the second rating was always higher than the first (Fig
1; A‐C; based on the recommended label rate for each of the
fungicides). There was low disease severity (≤15% on the
controls) at four of the locations with no significant treatment
differences for the first and second ratings. At the other three
locations; Clare, Rocky Ponds and Guthalungra, disease severity
on the controls was quite high (~60%). All the fungicide
treatments were effective against the disease, except at Rocky
Ponds and Guthalungra where they were not significantly
different from the controls.
DISCUSSION
The results from Rocky Ponds and Guthalungra clearly show that
there is a fungicide resistance problem in some areas in the
Burdekin region. This is a major cause for concern. Similar results
were recorded on seedlings exposed to isolates from the
Bundaberg region of Central Queensland.
These results reinforce the need for monitoring of isolate
sensitivities to the main systemic fungicides on a seasonal basis
in the main production regions. Promoting integrated crop
management strategies is vital. These include spraying only
when needed, using resistant varieties and using fungicide
alternatives as substitutes for protectant fungicides, as well as
destroying old crop residues in finished strips.
C lare (Wate rme lon)
0 20 40 60 80 Am istar Bay fidan Cont rol Nim rod Spin flo Tre atme nt % L ea f A rea I n fe ct ed Rating 1 Rating 2 Guthalungra (Rockme l on)
0 20 40 60 80 Am ista r Bay fidan Con trol Nim rod Spin flo Tre atme nt % L ea f A r ea I n fe ct ed Rat ing 1 Rat ing 2 Rocky Ponds (Hone yde w)
0 20 40 60 Am istar Bayf idan Cont rol Nim rod Spin flo Tre atme nt % L ea f A re a I n fe ct ed Rating 1 Rating 2
Figures 1. A‐C: Effect of systemic fungicides on powdery mildew disease
severity in cucurbit crops in the Burdekin region of north Queensland.
ACKNOWLEGEMENTS
Funding for this work was provided by Horticulture Australia
Limited (HAL) for which we are grateful.
REFERENCE
1. O’Brien, R.G., Vawdrey, L.L. and Glass, R.J. (1988). Fungicide resistance in cucurbit powdery mildew (Sphaerotheca fuliginea) and its effect on field control. Australian Journal of Experimental
Keynote
speaker
Population genetic analyses of plant pathogens: new challenges and opportunities
C.C. Linde{ XE "Linde, C.C." }Botany and Zoology, Research School of Biology, College of Medicine, Biology and Environment, Bldg. 116, Daley Rd, Australian National
University, Canberra, ACT 0200, Australia
INTRODUCTION
The study of population genetics attempts to investigate
evolutionary forces such as mutation, migration, genetic drift,
selection and recombination, and how gene frequencies change
in populations to shape their genetic structure. These
evolutionary forces and the interaction amongst them are
particularly important in plant pathogens where, combined with
the pathogen’s life history characteristics, they determine the
evolutionary potential. The population genetics of plant
pathogens has been investigated for at least 30 years. Early
studies on population genetics of plant pathogens concentrated
on the effect sexual reproduction has on levels of genetic
diversity in populations (Burdon and Roelfs, 1985a, b) and what
impact that had on disease control. Similar studies have
continued with investigations of pathogen capacities to rapidly
adapt to new environments such as developing resistance
against a fungicide or overcoming a resistance gene in the plant
host (McDonald and Linde, 2002).
Although the questions we ask in the population genetics of
plant pathogens has not changed significantly, advances in DNA
sequencing and analytical approaches have significantly
improved the accuracy of parameter estimates. In particular,
coalescent based approaches are a powerful extension of
classical population genetics because it is a collection of
mathematical models that can accommodate biological
phenomena as reflected in molecular data. The emphasis in
coalescent thinking is to view populations backwards in time,
using the divergence observable in a population to estimate the
time to a most recent common ancestor. This ancestor is the
point where gene genealogies `coalesce’, in a single biological
organism.
The barley scald pathogen, Rhynchosporium secalis, will be used
as an example to illustrate the importance of some of these
evolutionary forces and how coalescent based methods
significantly improved our understanding of the pathogens’
biology.
MATERIALS AND METHODS
Populations of R. secalis were characterised with 14
microsatellite loci (Linde et al., 2009) and several sequence loci
(Zaffarano et al., 2009). Several population genetic parameters
were investigated, including migration among populations. This
was investigated with a coalescent method in the program IM
(Hey and Nielsen, 2004) and results were compared to estimates
derived from traditional FST estimates (Weir and Cockerham,
1984).
RESULTS AND DISCUSSION
The results of this comparison revealed that coalescent based
approaches offer several advantages over other analytical
methods to estimate parameters such as migration and genetic
drift. Traditional measures of the translation of FST into gene
flow assume that subpopulations have the same size, population
sizes are constant, or that there are infinitely many populations,
and that migration rates are all symmetric. Due to these
underlying assumptions, migration estimates derived from FST
are almost always flawed and incorrect estimates are achieved
when these assumptions are not met. This is often the case since
pathogen populations are constantly influenced by the host
populations or human‐mediated migration.
With coalescent methods, the direction of migration is obtained.
This means the major source and sink populations for migration
can be determined which is useful in determining breaches of
quarantine or major migration routes due to eg prevailing wind
currents. In R. secalis, unusually high migration rates in both
directions between Australia and South Africa and Australia and
New Zealand cause particular concern for disease management.
A comparison of the results revealed that migration estimates
based on coalescent analyses were frequently non‐symmetric,
meaning one population contributed significantly more migrants
than the other. This contributed to migration estimates derived
from Fst being over‐ or under‐estimated. Furthermore, Fst derived
migration estimates were usually underestimated when the
migration was high, and/or when population sample sizes were
low.
Coalescent analyses provided population genetic parameter
estimates that are more reliable, are less dependent on
population sizes being stable and are affected less by
populations with small sample sizes. Improved analyses and
their usefulness in plant pathology are discussed.
REFERENCES
1. Burdon, J.J., Roelfs, A.P., 1985a. Isozyme and virulence variation in
asexually reproducing populations of Puccinia graminis and
Puccinia recondita on wheat. Phytopathology 75, 907–913. 2. Burdon, J.J., Roelfs, A.P., 1985b. The effect of sexual and asexual
reproduction on the isozyme structure of populations of Puccinia
graminis. Phytopathology 75, 1068–1073.
3. McDonald, B.A., Linde, C., 2002. Pathogen population genetics,
evolutionary potential, and durable resistance. Annu. Rev.
Phytopathol. 40, 349–379.
4. Linde, C.C., Zala, M., McDonald, B.A., 2009. Molecular evidence for recent founder populations and human‐mediated migration in the
barley scald pathogen Rhynchosporium secalis. Molecular
Phylogenetics and Evolution 51, 454–464.
5. Zaffarano, P.L., McDonald, B.A., Linde, C.C., 2009.
Phylogeographical analyses reveal global migration patterns of the barley scald pathogen Rhynchosporium secalis. Molecular Ecology,
279–293.
6. Hey, J., Nielsen, R., 2004. Multilocus methods for estimation population sizes, migration rates and divergence times, with application to the divergence of Drosophila pseudoobscura and D.
persimilis. Genetics 167, 747–760.
7. Weir, B.S., Cockerham, C.C., 1984. Estimating F‐statistics for the analysis of population structure. Evolution 38, 1358–1370.