Bio-protection Solutions to
Forest Biosecurity Problems
Travis Glare
Biocontrol & Biosecurity, AgResearch
Biosecurity risks
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Gypsy moth
Tussock moths
Painted apple moth
Vespula wasps
Bark beetles
Gum leaf skeletoniser
Investigation of Beauveria for
control of Hylastes and Hylurgus
Steve Reay, Mike Brownbridge, Tracey Nelson
•Effective pathogens
around the world
•Development of Beauveria
as a potential biopesticide
H. ligniperda killed by B. bassiana
Progress
•
Isolated fungi which kill Hylastes and Hylurgus
– Beauveria spp. (including new pathogen)
•
Genetically typed fungus and beetles-Biosecurity
model
•
Tested Beauveria formulations in laboratory and
field
– effective pathogen in the laboratory
– formulations persists on stumps, but control
only moderate to date
Beauveria bassiana
B. caledonica, a new pathogen of Hylastes and Hylurgus
•Originally described from Scottish soil
•Isolated from both Hylastes and Hylurgus
populations in north island pine forests
•Common, but difficult to separate from B. bassiana.
F528
F155
B. caledonica
F527
F526
F153
B. vermiconia
F45
GenBank AB027381
B. amorpha
B.brongniartii
F470
F480
F305
FI297
Beauveria caledonica
GenBank AB0273832
B. bassiana
F361
GenBank AF16813
Relationship between B. caledonica isolates and some other Beauveria spp.
Research in progress
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Developing novel formulations mixing attractants with bark
beetle active fungus
Investigating application and target of application, such as
directly in seedling potting mix
Examining new strains of fungi from Europe
Typing beetles from Europe
P. radiata seedlings damaged by Hylastes ater
Microbial control of painted apple moth:
the virulence and safety of OranNPV
Ngaire Markwick1, Jo Poulton1 Vernon
Ward2, Vivienne Young2, Nod Kay3 and
Travis Glare4
1 HortResearch,
Auckland
of Microbiology, Otago
University, Dunedin
3 Ensis, Scion, Rotorua
4 AgResearch, Lincoln
2 Department
Goal
• To determine if any microbial pathogens present
in New Zealand or identified in Australia have
potential as control agents to slow the spread of
painted apple moth, Teia anartoides.
The programme was divided into three sections:
1. Collection, identification and importation of
pathogens from Australia
2. Identification of pathogens from painted apple
moth in New Zealand
3. Bioassay of pathogens available in New Zealand
Approach
General insect pathogens
(from other insects)
PAM pathogens
collect and store
Molecular
identification
bioassay against PAM
select best candidate(s)
test host range
determine potential
MAF
identify
ERMA
Started with….
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Virus
– known virus from Lymantriidae and some other
Lepidoptera
– Undescibed virus attacking PAM colonies
– Described NPV of PAM in Australia
Fungus
– generalists
– Beauveria bassiana attacking PAM in NZ
Bacteria
– generalists
– Bacillus thuringiensis
Effectiveness of
Lymantriid viruses
•Gypchek - NPV from Lymantria
dispar from the USDA, ≈1996
Virtuss
100
WSTM
80
% Mortality
•Biolavirus - LD (Lymantria dispar
NPV produced in Czech republic,
≈1995)
•Virtuss, Douglas fir tussock moth,
Orgyia pseudotsugata, MNPV
(produced in O. leucostigma), 1990
•Whitemarked tussock moth, O.
leucostigma SNPV, from New
Brunswick, Canada, 1997
60
40
20
Gypchek
Control
Biola
0
5
10
15
20
25
30
Days after inoculation
35
40
An NPV infecting the Australian PAM
• Orgyia anartoides NPV was located, purified and three isolates were
imported into NZ
• Each isolate showed similar pathogenicity
• 100% mortality at PIB concentrations of 105/larva in 9 days
• Infection confirmed by PCR of polyhedrin gene
Host Range testing of OranNPV
Insect species
Noctuidae
Helicoverpa armigera
Spodoptera litura
Thysanoplusia orichalcea
Uraba lugens
Tortricidae
Epiphyas postvittana
Planotortrix octo
P. excessana (NI)
P. excessana (SI)
P. notophaea
Ctenopseutis obliquana
C. herana
Cnephasia jactatana
Geometridae
Pseudocoremia suavis
Nymphalidae
Danaus plexippus
Plutellidae
Plutella xylostella
Apidae
Apis mellifera - adults
Survival to pupation
(corrected for control
mortality)
Growth rate/ Weight
Significance of
difference (P)
100.00
100.00
95.45
100.00
0.14 ns
0.72 ns
0.29 ns
nd
93.92
82.61
91.67
95.24
95.84
90.91
100.19
90.48
0.35 ns
0.79 ns
0.65 ns
0.05 ns
0.52 ns
0.72 ns
0.51 ns
0.47 ns
102.17
0.17 ns
100.00
ℵ 0.13 ns
↵ 0.16 ns
100.00
nd
not affected
-
OranNPV does not
significantly affect
survival or growth
rate towards insects
tested
Phylogenetic Analysis of OranNPV
Percent Identity
1
Divergence
1
2
3
4
5
6
7
8
9
10
2.2
13.6
26.8
20.0
19.2
28.3
24.5
28.5
28.8
1
2
97.8
13.7
25.0
18.8
17.6
26.4
25.6
25.3
27.4
2
3
87.7
87.7
24.6
16.1
19.2
26.8
20.8
28.5
26.8
3
4
77.6
78.9
79.2
27.9
22.3
14.1
27.5
22.6
14.4
4
5
76.3
77.3
79.2
71.0
19.5
28.5
24.7
31.7
26.9
5
6
83.3
84.5
83.3
80.8
82.9
29.6
25.5
22.3
24.6
6
7
76.7
77.9
77.6
87.4
76.5
75.7
28.0
26.8
16.4
7
8
79.5
78.9
82.0
77.3
79.2
78.9
77.0
35.4
28.0
8
9
76.0
78.2
76.0
79.8
73.7
80.1
77.0
71.9
27.9
9
10
76.3
77.3
77.6
87.1
77.5
79.2
85.5
77.0
77.1
1
2
3
4
5
6
7
8
9
10
OranNPV
OranNPV NZ
OpSNPV polh
OpMNPV polh
OrleNPV seq
AcMNPV
EppoMNPV
HaSNPV polh
LdMNPV polh
HcuniNPV
10
OranNPV confirmed as
group II NPV
OpMNPV polh
EppoMNPV
HcuniNPV
LdMNPV polh
AcMNPV
OranNPV
OranNPV NZ
OpSNPV polh
OrleNPV seq
HaSNPV polh
27.1
25
20
15
10
5
0
Characterisation of an unknown pathogen
• A colony of PAM was established at HortResearch, Mt Albert in
2001
• By 2002 over 50% of the colony was dying at each generation
• Microscopic analysis of cadavers revealed large polyhedral
bodies
Most likely a of Cypovirus (CPV)
CPV infection of PAM larvae
100
4-d-old PAM larvae
% Mortality
80
control
PAMCPV
60
40
20
0
0
10
20
30
40
50
Days after inoculation
• Chronic infection causing ~65% mortality after 25 days
Host Range analysis of PAM CPV
Insect species
Noctuidae
Helicoverpa armigera
Spodoptera litura
Tortricidae
Epiphyas postvittana
Planotortrix octo
P. excessana_NI
P. excessana_SI
P. notophaea
Ctenopseustis obliquana
C.herana
Cnephasia jactatana
Survival
+
-
Majority of insects tested infected by the PAM CPV
Other pathogens
Strains of the fungus Beauveria bassiana against
painted apple moth larvae (107 conidia/ml
10
100
9
90
80
8
control, normal diet
7
control, no antibiotics
6
AgR 10*3
5
AgR 10*5
4
AgR 10*7
3
AgR 10*9
2
Percentage mortality
Cumulative mortality (n = 10)
AgR1 bacterium bioassayed against
painted apple moth larvae (20oC)
Control
70
B17 10*7
60
F265 10*7
50
F305 10*7
40
F470 10*7
F507 10*7
30
3404 10*7
20
1
10
0
0
2
4
6
Day after inoculation
8
10
0
0
5
10
Days after inoculation
15
20
Pathogens of painted apple moth
•Both Virtuss (commercially available) and OranNPV
are acceptably specific and either could be used as a
biocontrol agent should the PAM be reintroduced
•CPVs are rarely used as biocontrol agents due to
there ability to produce a chronic infection with a slow
kill time. Still remain fairly unknown.
•Beauveria bassiana, some potential but broad host
range.
•Bacteria, some potential, but broad host range
Conclusions
• In <10 years
ƒ 3 major lymantriid (forest-eating!) pests found in NZ
ƒ 2 successful campaigns have eradicated them.
• For the future:
• An ‘arsenal’ of control measures - contingency plan for future
incursions:
ƒ No native lymantriids in New Zealand.
ƒ No natural enemies or microbial controls
ƒ Test natural enemies/microbial controls in containment for
effectiveness against these species
ƒ Test non-target effects.
• Develop rapid identification systems