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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 1
Biological control of pest snails in Australia using native nematodes
Background
Four major pest species of snails in Australia
Introduced from Mediterranean area almost one century ago Theba pisana
(white Italian snail or sand dun snail)
Cochlicella barbara(conical or pointed snail)
Cochlicella acuta(small conical or pointed snail )
Cernuella virgata(vineyard snail or common white
snail )2
Summer(Dec. to Feb)(Dec. to Feb)
Autumn(Mar. to May)
Spring(Sep. to Nov.)
Winter(June to Aug.)
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 2
5
Current control strategies for snails
stubblestubble management over summer (grazing, rolling, slashing)
burning baiting
windrowing header modifications
post harvest grain cleaning
biological control (parasitic fly)
concerns with health and
environmental issues
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Current status of Biological Control of Mediterranean Snails in Australia
Parasitoid fliesParasitoid flies
Sarcophaga uncicurva and S. balanina•For C. vigata and T. pisana•Host range too wide
Sarcophaga penicillataSarcophaga penicillata•For C. virgata•Released at 20 sites on the Yorke Peninsula•10% establishment• Spread one km over a two year period
7
Current status of Biological Control of Mediterranean Snails in Australia
Attempts in 1990s found six nematode isolates able to parasitise C. virgata, T.
pisana and C.
Rhabditidnematode R954 was found to be
best.
Field trials inconclusive due to adverse
weather
acuta
Charwat et al. (2000). Biocon Sci & Tech 10, 147‐1558
June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 3
Field collections of snails
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Laboratory culture of snails
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Collection of nematode isolates
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New South WalesSouth Australia
Wagga Wagga
Victoria
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 4
Isolation from larvae and snails
13
h
PRIMARY PATHOGENICITY TESTS
Screening the promising nematode strains
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Pathogenicity Testing
15
Summary of Pathogenicity Tests
I l N113 N212 N431Isolates N113, N212, N431, N432 and N622 killed C.
virgata
Isolate N512 killed C. barbara
Nematodes were recovered from all
cadavers
Further testing showed isolate N431 killed all 4
snail species in 4 to 8 days
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 5
Pathogenicity test for nematode isolates against pest snail of C. virgata under laboratory conditions
Isolate Density Species Reps Time Mortality Recovery
N431
500
500
500
500
C. barbara
T. pisana
C. virgata
C. acuta
2
2
4
2
8 d
8 d
8 d
8 d
100%
100%
100%
100%
+++
+++
+++
++
400 C barbara 4 8 d 75% +++
N512
400
400
400
400
C. barbara
T. pisana
C. virgata
C. acuta
4
4
4
4
8 d
8 d
8 d
8 d
75%
0%
0%
0%
+++
‐
‐
‐
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Pathogenicity test for nematode isolate N431 against pest snail of C. virgata under field‐like conditions
Isolate Density Species Reps Time Mortality Cadaver
N431
600
1300
C. virgata
C. virgata
10
10
8 d
8 d
30%
60%
++
+++
Control Water C. virgata 10 8 d 10%‐
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b f l l h l l h
NEMATODE IDENTIFICATION
A combination of molecular and morphological approaches
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 6
Identification of Nematodes
Morphology using Light and Scanning Electron
Microscopy
Sequencing of the 18S ribosomal RNA gene of the nematodes isolated with primers • SSU18A/SSU26R and• SSU18A/SSU26R and• RHAB1350F/RHAB1868R.
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Light Microscopy
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Morphology of Nematodes (SEM)
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b f l l h l l h
BACTERIAL IDENTIFICATION
A combination of molecular and morphological approaches
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 7
Identification of bacterial isolates
A total of 31 bacteria were isolated from the bodies of four promising nematode
isolates.
The identification of these bacteria isolates was conducted by both fatty acid analysis and DNA barcoding (with two different primers: 0008F/0532R andprimers: 0008F/0532R and
341F/534R).
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FURTHER PATHOGENICITY TESTS
Nematode/Bacterial Interactions
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Pathogenicity test with different nematode/bacteria combinations against C. virgata
Figure : Pathogenicity test with different nematode/bacteria combinations against C. virgata (1#‐‐‐‐‐4711B6; 2#‐‐‐‐‐0431B1; Dark columns‐‐‐‐‐4711B6 involved nematode/bacteria combinations; light columns‐‐‐‐‐0431B1 involved nematode/bacteria combinations)
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Pathogenicity test with different nematode/bacteria combinations against C. barbara
Figure : Pathogenicity tests against large and small C. barbarawith nematode/bacteria combinations involved bacterium 4711B6. D: 4711B6; 1 and 2: large and small snails.
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 8
Pathogenicity test with different nematode/bacteria combinations against C. barbara
Figure : Pathogenicity tests against large and small C. barbara with nematode/bacterial combinations involved bacterium 0431B1. B: 0431B1; 1 and 2: large and small snails.
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SOME POINTS
N4711 + 4711B6 andN0431 + 0431B1 , caused significantly higher snail mortality
over other nematode/bacteria combinations (P <
4711B6 or 0431B1related
nematode/bacterial combinations also caused higher snail mortality among C.
It appears that large C. barbara are more sensitive to the attack
of nematode/bacterial combinations than0.001) against C.
virgata.barbara. combinations than
small C. barbara
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PATHOGENICITY OF OTHER ORGANISMS
Earthworms
Slugs
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Earthworm Pathogenicity tests
Nematode/bacteriDensity (per ml) Earthworm size Repetition Days
Earthworm a combinations
Density (per ml) Earthworm size Repetition Daysmortality (%)
N0431 + 4711B6 2333 Large or small 8 14 0
N4711 + 4711B6 2056 Large or small 8 14 0
N3814 + 4711B6 1722 Large or small 8 14 0
N4712 + 4711B6 3722 Large or small 8 14 0
N0431 + 0431B1 3333 Large or small 8 14 0
N4711 + 0431B1 4056 Large or small 8 14 0
N3814 + 0431B1 1268 Large or small 8 14 0
N4712 + 0431B1 1556 Large 8 14 0
Negative control 0 Large 8 14 0
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 9
Slug Pathogenicity tests
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Pathogenicity on slugs
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Pathogenicity on slugs
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FIELD TRIALSOptions to be considered currently
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WWPPG seminar session 1 by Aisuo Wang 10
Factors affecting the field trials
Snails’ needs Nematodes’ needs
Food Shading Temperature Oxygen Moisture
Moisture
Temperature Density No UV Oxygen Hosts
To ensure the success of field trials
Suitable locations for field trials (natural
conditions)
Nice moisture of the soil
Right temperature of the soil
Right nematode strains
Enough density of nematodes
Right percentage of infective juveniles in
total nematode populations
Right formulation
of nematodes
Enough contact between nematodes and snails
Good management of the field
trials
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Case study: field trial in 2010
Lessons we got
The snail enclosures made with steel could not efficiently enclosesnails. At the end of the trial, the total snail
number (including both dead and alive ones) in each enclosure could
The size of the snail cage (33 cm x 33 cm) is too small to prevent snails from moving out of the
The design of the snail enclosures actually encourage snails to
escape from nematodes’ attack by
not match the original data. More surprisingly, foreign snails were found in some enclosures.
gboundaries.
ycrawling up to the top
of the cages.
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1st option for future field trials
Major points
Expand the plot area (say 10 m x 10 m) so that snails cannot
Use suitable tool (such as plastic screen) to
immobilize snails
Use suitable materials (such as timber) to hold down the screen firmly and thusmove out of the
boundaries easily within the test
areafirmly, and thus prevent snail from
escaping
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June 14, 2011
WWPPG seminar session 1 by Aisuo Wang 11
Weights to hold down the screen
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2nd option for future field trials
Major points
Set up a barrier area (wide enough) with nematodes to separate pest snails
Use snail attractants to lure pest snails crossing over
Examine the reactions of snails when they have l d
p pfrom materials to be protected
crossing over the nematodes
barrier
crawled across the nematode
barriers
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Nematode bait areaPest snails area
Snail attractants area
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