Bio-protection Solutions to Forest Biosecurity Problems

Travis Glare

Biocontrol & Biosecurity, AgResearch Biosecurity risks

• Gypsy • Tussock • 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. amorpha

F45 B. vermiconia

GenBank AB027381 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

• 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 2 Department of Microbiology, Otago University, Dunedin 3 Ensis, Scion, Rotorua 4 AgResearch, Lincoln 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 pathogens PAM pathogens (from other )

collect and store Molecular identification identify

bioassay against PAM ERMA select best candidate(s) test host range

determine potential

MAF Started with….

•Virus – known virus from Lymantriidae and some other – Undescibed virus attacking PAM colonies – Described NPV of PAM in Australia • Fungus – generalists – attacking PAM in NZ • Bacteria – generalists – Bacillus thuringiensis Effectiveness of Lymantriid viruses

•Gypchek - NPV from Lymantria Virtuss dispar from the USDA, ≈1996 100 •Biolavirus - LD (Lymantria dispar NPV produced in Czech republic, WSTM ≈1995) •Virtuss, Douglas tussock moth, 80 pseudotsugata, MNPV (produced in O. leucostigma), 1990 •Whitemarked tussock moth, O. 60 leucostigma SNPV, from New Brunswick, Canada, 1997 40

% Mortality 20 Gypchek Control 0 Biola 5 10152025303540 Days after inoculation 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 Survival to pupation Growth rate/ Weight (corrected for control Significance of mortality) difference (P) Noctuidae Helicoverpa armigera 100.00 0.14 ns Spodoptera litura 100.00 0.72 ns Thysanoplusia orichalcea 95.45 0.29 ns Uraba lugens 100.00 nd Tortricidae OranNPV does not Epiphyas postvittana 93.92 0.35 ns significantly affect Planotortrix octo 82.61 0.79 ns P. excessana (NI) 91.67 0.65 ns survival or growth P. excessana (SI) 95.24 0.05 ns rate towards insects P. notophaea 95.84 0.52 ns Ctenopseutis obliquana 90.91 0.72 ns tested C. herana 100.19 0.51 ns Cnephasia jactatana 90.48 0.47 ns Geometridae Pseudocoremia suavis 102.17 0.17 ns Nymphalidae Danaus plexippus 100.00 ℵ 0.13 ns Plutellidae ↵ 0.16 ns Plutella xylostella 100.00 nd Apidae Apis mellifera - adults not affected - Phylogenetic Analysis of OranNPV

Percent Identity 1 2 3 4 5 6 7 8 9 10 1 97.8 87.7 77.6 76.3 83.3 76.7 79.5 76.0 76.3 1 OranNPV 2 2.2 87.7 78.9 77.3 84.5 77.9 78.9 78.2 77.3 2 OranNPV NZ 3 13.6 13.7 79.2 79.2 83.3 77.6 82.0 76.0 77.6 3 OpSNPV polh 4 26.8 25.0 24.6 71.0 80.8 87.4 77.3 79.8 87.1 4 OpMNPV polh

e 5 20.0 18.8 16.1 27.9 82.9 76.5 79.2 73.7 77.5 5 OrleNPV seq c n

e 6 19.2 17.6 19.2 22.3 19.5 75.7 78.9 80.1 79.2 6 AcMNPV g r e

v 7 28.3 26.4 26.8 14.1 28.5 29.6 77.0 77.0 85.5 7 EppoMNPV

Di 8 24.5 25.6 20.8 27.5 24.7 25.5 28.0 71.9 77.0 8 HaSNPV polh 9 28.5 25.3 28.5 22.6 31.7 22.3 26.8 35.4 77.1 9 LdMNPV polh 10 28.8 27.4 26.8 14.4 26.9 24.6 16.4 28.0 27.9 10 HcuniNPV 12345678910

OranNPV confirmed as OpMNPV polh group II NPV 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

80 control PAMCPV 60

40 % Mortality

20

0

0 1020304050 Days after inoculation

• Chronic infection causing ~65% mortality after 25 days Host Range analysis of PAM CPV

Insect species Survival Noctuidae Helicoverpa armigera - Spodoptera litura - Tortricidae Epiphyas postvittana - Planotortrix octo - P. excessana_NI - P. excessana_SI - P. notophaea - Ctenopseustis obliquana - C.herana + Cnephasia jactatana -

Majority of insects tested infected by the PAM CPV Other pathogens

Strains of the fungus Beauveria bassiana against AgR1 bacterium bioassayed against painted apple moth larvae (107 conidia/ml painted apple moth larvae (20oC)

10 100 90 ) 9 0 1 8 80 =

y Control

n control, normal diet lit 70

( 7 a

t B17 10*7 y

control, no antibiotics r lit 6 o 60

a F265 10*7 t

AgR 10*3 m r o 5 50 F305 10*7 ge

AgR 10*5 a m t F470 10*7 e 4 n 40 v AgR 10*7 e i c t F507 10*7 3 r la AgR 10*9 30 u Pe 3404 10*7 2 m 20 u C 1 10 0 0 0246810 0 5 10 15 20 Day after inoculation Days after inoculation 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