anthracnose by Agrobacterium
A.S.M. Auyong{ XE "Auyong, A.S.M." }, R. Ford and P.W.J. TaylorBioMarka/Centre for Plant Health, Melbourne School of Land and Environment, The University of Melbourne, 3010, Victoria
INTRODUCTION
Anthracnose disease of chilli pepper is caused by a complex of Colletotrichum spp. with C. capsici being the most severe in
South East Asia (1). Knowledge of the mechanisms for host
resistance and pathogenicity is crucial for developing effective
and durable disease control. Several putative pathogenicity
genes involved in C. capsici infection of chilli pepper have been
identified and partially cloned (Auyong, unpublished). A fungal
transformation system is required to prove the function of these
putative genes in the infection process. In this study an efficient
transformation system was successfully developed to serve as a
platform towards understanding chilli pepper‐C. capsici
interactions. Agrobacterium tumefaciens carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein
(GFP) gene was used to transform the conidiospores of C.
capsici. Transformation efficiency was correlated with
conidiospores density, ratio of conidiospores to bacterial cells,
type of Agrobacterium strains and plasmid, presence or absence
of acetosyringone, co‐cultivation time and co‐cultivation
temperature.
MATERIALS AND METHODS
Fungal culture. Colletotrichum capsici, BRIP 26974 isolated from
Capsicum annuum was supplied by the Department of Primary
Industry (DPI), Queensland, Australia, and maintained on potato
dextrose agar (PDA).
Fungal transformation. Conidial suspension was prepared and
adjusted to 102, 104, 106 and 108 conidiospores per ml and mixed
at different ratio (1:3, 1:5, 1:1, 3:1 and 5:1) with Agrobacterium
(AGL1 or LBA4404) containing either pJF1, pPK2 or pKHt plasmid.
The mixture was plated onto filter paper on solid induction
medium, either amended or non‐amended with 200 μM
acetosyringone. Following co‐cultivation for 1, 2, 3, 4, and 5 days
at co‐cultivation temperature of 24°C, 28°C, 32°C or 36°C, the
fungal‐bacterial cells on the filter paper were transferred to PDA
amended with hygromycin B and cefotaxime to eliminate the A.
tumefaciens cells. Individual transformants were transferred
after 4 to 6 days to PDA amended with hygromycin B. In all
experiments, C. capsici conidiospores co‐cultivated with
uninoculated induction medium were included as a negative
control. All experiments were replicated and results were
analysed using ANOVA.
Analysis of transformation events. The frequency and
randomness of T‐DNA integration in the fungal genome was
determined by PCR and Southern blot.
RESULTS AND DISCUSSION
Agrobacterium was successfully used to transform C. capsici
conidiospores and mycelium (Figure 1). The biological
differences among fungal species can influence transformation
efficiencies in different filamentous fungi (2). Following
optimisation, high transformation efficiencies were routinely
obtained for C. capsici.
Conidiospore density. Transformation efficiency was consistently
found to be optimum at the conidiospores density of 106 and 108
conidiospores per ml. Hence, subsequent transformations were
carried out using 106 conidiospores per ml.
Ratio of fungal spores to bacterial cells. The highest
transformation efficiency was obtained with equal volume of the
mixture.
Agrobacterium strains. A. tumefaciens AGL1 strain produced
more transformants (16.2% more) than LBA4404 regardless of
the binary vector used.
Plasmid type. pJF1 and pPK2 plasmids provided similar
transformation efficiencies. In contrast, pKHt plasmid produced
significantly less transformants (p<0.01).
Acetosyringone. Transformants were only obtained when the
medium was supplemented with the wound respond molecule,
acetosyringone.
Co‐cultivation time. The number of transformants increased
depending on the increased period of co‐cultivation. Prolonged
co‐cultivation period however, brought about excessive growth
of the fungus and resulted in difficulty in isolating single
colonies.
Co‐cultivation temperature. The optimal co‐cultivation
temperature for C. capsici transformation was 24°C. The number
of transformants dramatically decreased when the co‐cultivation
temperature was increased to 28°C and at 32°C no fungal
colonies were observed. Lower temperatures appeared to be
beneficial towards the T‐DNA transfer (2).
PCR and Southern blot analysis. PCR of transformants detected
the T‐DNA insertions. Southern analysis indicated that 87.5% of
the transformants had a single copy of the T‐DNA integrated
randomly in the fungal genomes.
Figure 1. Expression of GFP in conidiospores and in hyphae colonising,
intramurally, infected parenchyma cells of chilli pepper, as visualised with green fluorescent filter.
Based on an optimised protocol, Agrobacterium transformation
approach has proven to be an efficient method in transforming C. capsici.
ACKNOWLEDGEMENTS
We thank Professor L. Vaillancourt (University of Kentucky),
Professor S. Covert (University of Georgia) and Professor S. Kang
(The Pennsylvania State University) for supply of pJF1, pPK2 and
pKHt plasmids, respectively.
REFERENCES
1. Montri P, Taylor PWJ, Mongkolporn O (2009) Pathotypes of Colletotrichum capsici, the Causal Agent of Chili Anthracnose, in Thailand. Plant Disease 93: 17–20.
2. Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ (2008) Agrobacterium‐mediated transformation of the filamentous fungus Aspergillus awamori. Nature Protocols 3: 1671–1678.
Posters
60 Infection process of endophytic Colletotrichum gloeosporioides on cacao leaves
C. Blomley{ XE "Blomley, C." }A, E.C.Y LiewB and D.I. GuestA
A
Faculty of Agriculture Food and Natural Resources, The University of Sydney, 2006, NSW
B
Royal Botanic Gardens Trust, The Royal Botanic Gardens, Sydney, NSW, 2000
INTRODUCTION
Colletotrichum species are commonly isolated as endophytes
from leaves and fruits of tropical plants. In preliminary surveys Colletotrichum spp. accounted for 29–48% of isolates sampled as
endophytes from leaves of the cacao tree (Theobroma cacao) in
four sites in Australia and Papua New Guinea. By definition
endophytes do not cause disease symptoms at the time they are
isolated from plant tissue. The infection process and subsequent
tissue colonisation has been elucidated for only a few endophyte
host interactions, none of which include tropical plants.
Colletotrichum species can penetrate plant tissue through
wounds, natural openings such as stomata or by penetration of
the plant cuticle1. They are often categorised into three groups:
intracellular hemibiotrophs, subcuticular intramural colonisers
and those that display a combination of the two infection
strategies1. Intracellular hemibiotrophs first grow biotrophically
in host tissue before switching to a necrotrophic stage which
results in symptom development. Subcuticular intramural
pathogens grow beneath the cuticle and cause dissolution of the
epidermal cell walls. The aim of this research was to investigate
the infection process of an endophytic isolate of C. gloeosporioides on T. cacao leaves.
MATERIALS AND METHODS
An isolate of C. gloeosporioides was isolated from apparently
healthy leaf tissue of T. cacao in Far North Queensland,
Australia. Young and mature leaves of cacao were sprayed with a
1x105 conidia/mL suspension to runoff. Leaf tissue was sampled
for observations every 2h for 16h, at 24h and then every 24h for
6 days. Tissue was cleared for 4h at 60˚C followed by 20h at
room temperature in a solution of 0.15% trichloroacetic acid in
3:1 ethanol:chloroform. Tissue was immersed in 0.025% aniline
blue in lactoglycerol for 1h at 60˚C followed by 23h at room
temperature in order to stain fungal hyphae. Experiments were
repeated at least three times.
RESULTS
Conidia began germinating within 6 hours post inoculation (hpi),
usually giving rise to one and rarely two germ tubes. Appressoria
were produced at 8–10 hpi, either directly or at the end of a
short germ tube and became melanised by 12 hpi. Infection pegs
were produced predominantly over cell walls at 12–16 hpi in
both young and mature leaves. Stomatal penetration was never
observed. Infection vesicles were visible at 3 days post
inoculation (dpi) in young leaves and appeared as thick, highly
lobed hyphae which filled the epidermal cell directly beneath the
infection peg. At 4–5 dpi infection vesicles had branched into
narrow secondary hyphae which penetrated cell walls and grew
inter‐ and intra‐cellularly in young leaves (Fig. 1). Infection
vesicles formed in mature leaves 4–5 dpi and had a similar
appearance to those in young leaves (Fig 2). In mature leaves,
infection was restricted to the initial cell in which the infection
vesicle formed over the 6 days of observation.
Figure 1. Primary hyphae of C. gloeosporioides colonising epidermal cells
of T. cacao leaves 4 dpi. Bar = 10µm
Figure 2. Primary hyphae of C. gloeosporioides restricted to the
epidermal cell directly below appressoria (5 dpi). Bar = 20um).
DISCUSSION
Endophytic C. gloeosporioides on T. cacao leaves can be
categorised as an intercellular hemibiotroph. Infection was
observed in epidermal cells directly beneath the appressorium
and no subcuticular intramural growth was observed. The
infection process did not differ on young and mature T. cacao
leaves in the first 3 dpi. Following this, colonisation was more
rapid in young leaves and led to the production of disease
symptoms. Infection in mature leaves remained biotrophic and
fungal growth appeared to cease after infection and colonisation
of one epidermal cell. The length of the biotrophic,
asymptomatic phase has been correlated the redox state2 and
pH of the host tissue3 in other Colletotrichum‐host interactions.
Factors affecting the infection process in T. cacao are currently
being investigated.
REFERENCES
1. Bailey JA, O'Connell RJ, Pring RJ & Nasby C (1992). Infection strategies of Colletotrichum species. In ‘Colletotrichum: Biology, Pathology and Control’ (Eds JA Barley & MJ Jeger) pp. 88–120. (C.A.B. International: Wallingford)
2. Wei YD, Byer KN, Goodwin, PH (1997) Hemibiotrophic infection of round‐leaves mallow by Colletotrichum gloeosporioides f.sp. malvae in relation to leaf senescence and reducing agents. Mycological Research 101, 357–364
3. Kramer‐Haimovitch H, Servi E, Katan T, Rollins J, OkonY, & Prusky,
D. (2006) Effect of Ammonia production by Colletotrichum
gloeosporioides on pelB activation, pectate lyase secretion and fruit pathogenicity. Applied and Environmental Microbiology 72,1034– 1039.
Posters
2 A Phytophthora sp. is the cause of jackfruit decline in the philippines
L.M. Borines{ XE "Borines, L.M." }A, R. DanielB and D. GuestB
A
Department of Pest Management, Visayas State University, Visca, Baybay, 6521 Leyte, Philippines
B
University of Sydney, Sydney, NSW 2006 Australia
INTRODUCTION
Jackfruit is a very popular domestic fruit in the Philippines. It has
a wide distribution and is cultivated throughout the country. A
survey of jackfruit growers in the Eastern Visayas indicated that
wilt disease was the main constraint to improved productivity. In
some areas up to 90% of jackfruit trees are affected by wilt
disease, manifested by leaf yellowing, defoliation, girdling stem
lesions and rot. Previous attempts to identify the pathogen
yielded a range of fungal, nematode or bacterial isolates, none
of which proved pathogenic. Accurate identification of the cause
of the decline syndrome is imperative for the control of the
disease. This study seeks to isolate and identify the pathogen
causing jackfruit wilt and to evaluate a range of disease
management strategies through participatory action research.
MATERIALS AND METHODS
Affected roots, stem canker lesions and soil from near infected
trees was suspended in water and baited with flower petals1.
Lesions that developed within 2 days were surface sterilised and
plated on Potato Dextrose Agar, Carrot Agar and Onion Agar,
supplemented with benomyl, nystatin and streptomycin. Pure
cultures were re‐introduced to flower baits to induce sporangia,
zoospore and chlamydospore formation for inoculation of
detached jackfruit leaves and seedlings.
RESULTS
Wilt disease was recorded in all the fields examined within Leyte
and Samar islands, at an incidence of 5–90% of trees. Areas with
very high incidence were typically subject to periodic heavy
flooding, particularly during the rainy season. Yield losses were
estimated to be range from 5–80%. Field visits and farmer
interviews showed that almost all of the farmers were unaware
of the cause of the disease or appropriate management
strategies.
A Phytophthora species was consistently isolated from affected
jackfruit roots and canker lesions, and from soil collected near
infected plants. Pathogenicity was confirmed when the isolates
produced typical wilting symptoms on inoculated plants (Fig. 1a)
and leaf lesions (Fig.1b).
a b
Figure 1. a. Un‐inoculated (leftmost) and inoculated jackfruit seedlings
showing different degrees of wilting. b. The isolated pathogen causes leaf lesions.
In pure culture the mycelium of the pathogen is white with a
stellate growth pattern. Sporangia are seldom produced in PDA,
Carrot or Onion Agar, but readily produced when pure cultures
were re‐introduced to flower petal baits. The pathogen
produced spherical to ovoid sporangia with an average length of
41.5 µm and breadth of 26.5 µm. Sporangia have a relatively
long pedicels, are semi‐papillate to papillate and release
zoospores through a vesicle before they separate and swim
away (Figs 2a and 2b). The isolated Phytophthora species
produces abundant intercalary and terminal chlamydospores
when re‐introduced to flower baits.
a b
Figure 2. a) Phytophthora sporangia, b) zoospores exiting via spherical
vesicles.
Participatory action research (PAR) disease management trials
are being established by researchers, extension officers and
jackfruit farmers in Leyte and Samar Islands. Nine PAR trials have
been established in Leyte and Samar to test a range of
management options including field sanitation, organic
amendments, improved drainage, good nursery practices and
chemical control in managing wilt disease.
The identification of the pathogen associated with the symptoms
of decline and wilt will enable the development of more
effective, targeted management strategies.
ACKNOWLEDGMENTS
This research is funded by ACIAR HORT/2006/067/2
REFERENCES
Drenth A. & Guest DI. 2006. Biology and Management of Phytophthora
diseases in the tropics. ACIAR Monograph 114.
Posters
3 The effects ofof calcium Colletotrichum chloride acutatumand calcium and carbonate Penicillium on expansum germination
and growth
K.S.H. Boyd‐Wilson{ XE "Boyd‐Wilson, K.S.H." }A and M. WalterA
A
The New Zealand Institute for Plant and Food Research Limited, PO Box 51, Lincoln 7640, New Zealand
INTRODUCTION
Calcium chloride (CaCl2) and calcium carbonate (CaCO3) have
been found to enhance the biocontrol activity of yeasts against a
range of diseases. A number of factors including inhibition of the
pathogen by the compounds may account for this (1). The aim of
this research was to investigate whether these compounds
inhibited germination and growth of Penicillium expansum, the
causal agent of blue mould of apples, and of Colletotrichum acutatum, which causes bitter rot of pome fruit (2,3).
MATERIALS AND METHODS
Germination (%) and germ‐tube length (µm) were assessed after
20–24 h at 20ºC for three C. acutatum isolates made up in 0, 10,
or 20 mg/ml CaCl2. P. expansum requires exogenous nutrients to
germinate well, thus germination assays were conducted in 0,
12.5 and 25% apple broth with 20 mg/ml CaCl2 for three P.
expansum isolates. For each combination, the per cent
germination of 150 conidia and germ‐tube length of 30 conidia
was recorded.
Because suspended CaCO3 made it difficult to observe conidia in
germination tests, the effects of CaCO3 and CaCl2 (each 20
mg/ml) on the three isolates of P. expansum and two of C. acutatum were also investigated by dilution plating on 0, 12.5
and 25% apple broth agar with and without CaCl2 and counting
colony forming units (cfu) after 4–7 days.
All experiments were conducted twice and data were analysed
using analysis of variance. Germ‐tube lengths were log10
transformed and colony counts were square‐root transformed
before analysis. P=0.05 was used to assess significance.
RESULTS AND DISCUSSION
Increasing concentrations of CaCl2 increased the germination and
germ‐tube length of C. acutatum, although there was a
significant interaction between the factors studied (Table 1).
Table 1. Mean per cent germination and germ‐tube length (log10
transformed) for each Colletotrichum acutatum isolate and CaCl2 concentration.
Germination (%) Germ‐tube length (µm log10)
CaCl2 (mg/ml) CaCl2 (mg/ml) Isolates 0 10 20 0 10 20 C4 57 57 85 0.56 0.45 0.34 C7 41 69 100 0.39 0.67 0.70 C8 71 51 85 0.45 0.27 0.70
s.e.d. interaction 17.2 s.e.d concentration 0.097
P. expansum conidia did not germinate in water alone. In apple
broth, the addition of CaCl2 significantly reduced mean
germination of P. expansum from 37 to 16% and germ‐tube
length from 0.382 to 0.160 compared with the control. There
was no difference in germination and germ‐tube length between
the two concentrations of apple broth.
On apple broth agar, no differences between treatments were
observed and therefore results are given only for agar with no
apple broth.
For all P. expansum isolates, the addition of CaCO3 to the agar
resulted in significantly fewer cfu than in the CaCl2 and water
only treatments. The addition of calcium carbonate did not
significantly affect cfu counts of C. acutatum isolates.
In conclusion, neither CaCl2 nor CaCO3 reduced germination,
germ‐tube growth or cfu counts for C. acutatum. This suggests
that some other mode of action contributes to enhancement of
bitter rot control when these compounds are combined with
yeasts. In contrast, the germination, germ‐tube growth and cfu
counts for P. expansum were all reduced by the addition of CaCl2
and CaCO3 to the growth medium suggesting that this direct
inhibition could contribute to the improved blue mould control
in apples when yeasts are combined with these compounds.
ACKNOWLEDGEMENTS
Thanks to the New Zealand Foundation for Research, Science
and Technology for funding this project (C06X0302).
REFERENCES
1. Everett KR, Vanneste JL, Hallett IC, Walter M (2005) Ecological alternatives for disease management of fruit rot pathogens. New
Zealand Plant Protection 58, 55–61.
2. Rosenberger DA (1997) Blue mold. In ‘Compendium of apple and pear diseases’. (Eds A L Jones, HS Aldwinckle) pp 54–55. (APS Press: USA)
3. Sutton TB (1997) Bitter rot. In ‘Compendium of apple and pear diseases’. (Eds A L Jones, HS Aldwinckle) pp 15–16. (APS Press: USA)