Seona Casonato1,3,4, Freda Anderson5, Ann Lawrie1 and David McLaren2,3 Prospects for biological control of serrated tussock and Chilean needle grass Seona Casonato1,3,4, Freda Anderson5, Ann Lawrie1 and David McLaren2,3 1Biotechnology & Environmental Biology, RMIT, Bundoora, Vic. 3083 2Department of Primary Industries, Frankston Centre 3CRC for Australian Weed Management 4Current address: HortResearch, Auckland, NZ 5CERZOS-UNS, Bahía Blanca, Argentina 14/11/2018 TT biocontrol talk

Prospects for biological control of serrated tussock and Chilean needle grass 1. Biological control – Argentina 1.1 Chilean needle grass (Nassella neesiana) 1.2 Serrated tussock (Nassella trichotoma) 2. Biological control – endemic 2.1 Serrated tussock (Nassella trichotoma) 14/11/2018 TT biocontrol talk

Chilean needle grass http://www.weeds.crc.org.au/main/wom_chilean_needle_grass_0905.html# http://www.humeshire.nsw.gov.au/files/1733/File/CNGWeed2.jpg 14/11/2018 TT biocontrol talk

Chilean needle grass http://www.weeds.org.au/cgi-bin/weedident.cgi?tpl=plant.tpl&state=&s=&ibra=all&card=G12 14/11/2018 TT biocontrol talk

Serrated tussock (Nassella trichotoma) 14/11/2018 TT biocontrol talk NRE 1998

Seed spread – inflorescences break off and blow away http://www.weeds.crc.org.au/documents/wmg_serrated_tussock.pdf 140,000 seeds plant-1 yr-1 14/11/2018 TT biocontrol talk

Prospects for biological control of serrated tussock and Chilean needle grass 1. Biological control – Argentina 1.1 Chilean needle grass (Nassella neesiana) 1.2 Serrated tussock (Nassella trichotoma) 2. Biological control – endemic 2.1 Serrated tussock (Nassella trichotoma) 14/11/2018 TT biocontrol talk

1. BIOLOGICAL CONTROL - ARGENTINA Nassella spp. native to Peru, Chile, Uruguay and Argentina (Healy, 1945; Campbell, 1982) Epicentre Argentina Look for classical biological control organisms in these regions Import to Australia Distribute and leave to establish Natural spread and control of Nassella spp. 14/11/2018 TT biocontrol talk

Pathogens collected from Nassella spp. in Argentina during 1988-99 Fungal pathogen Species Nassella spp. infected Teliomycetes (rusts) Puccinia graminella N. hyalina, N. neesiana, N. tenuis P. interveniens N. tenuis P. nassellae N. neesiana, N. trichotoma P. saltensis var. faldensis N. burkartii, N. clarazii P. saltensis var. saltensis N. neesiana, N. tenuissima Uredo sp. N. neesiana, N. tenuis, N. trichotoma Ustomycetes (smuts) Ustilago hypodytes Basidiomycetes (mushrooms) Corticium sp. N. trichotoma Coelomycetes Ascochyta leptospora Stagonospora sp. Hendersonula sp. Agonomycetes Rhizoctonia sp. 14/11/2018 TT biocontrol talk Briese and Evans, 1998; Briese et al., 1999)

search for biological control pathogens in Argentina Begun in 1998 through CERZOS-UNS Funded by NSW Agriculture RIRDC Consortium of local government and community groups in SE Australia Serrated Tussock Working Party in NSW Serrated Tussock Taskforce in Victoria Latterly WONS 14/11/2018 TT biocontrol talk

Areas surveyed for pathogens in Argentina Plant pathologist in Argentina: Dr Freda Anderson, CERZOS-UNS Australian scientist in Argentina: Dr Bill Pettit, CSIRO http://www.rirdc.gov.au/reports/Ras/01-27.pdf 14/11/2018 TT biocontrol talk

Microorganisms collected from grasses of the genus Nassella in Argentina in 1999 Pathogen type Division Genera/families Fungi Teliomycetes (rusts) Puccinia graminella Puccinia aff. avocensis Puccinia nassellae Ustomycetes (smuts) Ustilago sp. Ascomycetes (leaf spots) Botryosphaeria sp. Leptosphaeria sp. Pleospora sp. Basidiomycetes Corticium sp. 14/11/2018 TT biocontrol talk http://www.rirdc.gov.au/reports/Ras/01-27.pdf

Microorganisms collected from grasses of the genus Nassella in Argentina in 1999 (cont.) Pathogen type Division Genera/families Fungi Coelomycetes (leaf spots) Ascochyta sp. Fusarium sp. Phoma terrestris Pseudoseptoria sp. Stagonospora spp. Zinzipegasa argentinensis Hyphomycetes Asperisporium sp. Pericornia sp. Agonomycetes Rhizoctonia sp. Bacteria (yellow slime) Clavibacter? sp. 14/11/2018 TT biocontrol talk http://www.rirdc.gov.au/reports/Ras/01-27.pdf; Anderson 2004a

RUST LIFE CYCLES Rust needs to be autoecious (have all life stages on host plant) heteroecious species – alternate host I aecia macrocyclic e.g. P. graminis pycnia demicyclic e.g. P. lagenophora microcyclic e.g. P. heterospora IV II basidia uredinia leptoforms e.g. P. malvacearum III telia 14/11/2018 TT biocontrol talk

Rust on ST and CNG ST CNG Sphaerellopsis filum (mycoparasite) Puccinia nassellae abundant at several sites CNG 3 rust species associated with severe leaf damage Sphaerellopsis filum (mycoparasite) frequently associated with P. nassellae pustules ‘significant controlling effect on rust populations’ 14/11/2018 TT biocontrol talk Briese et al. (2001); Anderson (2004a)

Sphaerellopsis (Darluca) filum 14/11/2018 TT biocontrol talk http://www.niaes.affrc.go.jp/inventry/microorg/eng/z65e-Sphaer-fil.html

Prospects for biological control of serrated tussock and Chilean needle grass 1. Biological control – Argentina 1.1 Chilean needle grass (Nassella neesiana) 1.2 Serrated tussock (Nassella trichotoma) 2. Biological control – endemic 2.1 Serrated tussock (Nassella trichotoma) 14/11/2018 TT biocontrol talk

CNG - rust fungi in Argentina 3 spp. of rust fungi found P. nassellae most widespread and damaging Uredinia and telia in field (aecia on alternate host?) P. graminella at only one site Telia, pycnia and aecia in field (no urediniospores) P. aff. avocensis no good x heteroecious (aecia on Malvaceae) Uromyces pencanus very damaging 14/11/2018 TT biocontrol talk Briese et al. (2001)

Puccinia nassellae - CNG Uredinia on N. neesiana (site NT 27) Telia on N. neesiana (site NT 16) 14/11/2018 TT biocontrol talk Anderson (2005a)

Puccinia nassellae - CNG Germinating basidiospores of Puccinia nassellae http://www.rirdc.gov.au/reports/Ras/01-27.pdf 14/11/2018 TT biocontrol talk Anderson (2005a)

P. nassellae - CNG Infection of glasshouse plants with urediniospores No infection with teliospores Problems with germinating teliospores (cold treatment needed) No cross-infection with ST (different) Possibly heteroecious? Aecia ± pycnia on plants close to CNG in field Clematis montevidensis (P. recondita?) Solidago chilensis (no pycnia)(P. stipae?) Cestrum parquii (immature, only one site) Verbena sp. (P. verbeniphila?) 14/11/2018 TT biocontrol talk Anderson (2004a)

P. nassellae - CNG Plants of all 7 Australian provenances inoculated with urediniospores All 7 infected with isolate Pn27 None infected with isolate Pn52 Host-strain specificity Subspecies known in CNG 14/11/2018 TT biocontrol talk Anderson (2004a)

Puccinia graminella - CNG Autoecious Aecia and telia always present in field No urediniospores known from life cycle Germination of freshly collected Aeciospores Teliospores Glasshouse infection trials under way 14/11/2018 TT biocontrol talk Anderson (2005a); Anderson (2005c)

Puccinia graminella - CNG P. graminella. Aecia on N. neesiana P. graminella. Aecia and telia on N. neesiana 14/11/2018 TT biocontrol talk Anderson (2005a)

Puccinia graminella - CNG Aecia on Chilean needle grass at site NN 8 Germinating teliospores with basidiospores 14/11/2018 TT biocontrol talk Anderson (2005c)

Uromyces pencanus - CNG Trap plants of Australian CNG heavily infected known on 2 accessions of N. neesiana in Argentina (Lindquist, 1982) Autoecious No paraphyses in uredinia No germination in teliospores Infection of glasshouse plants with urediniospores No aecia yet in field or glasshouse on ‘Stipa setigera’ in Chile by Holway, 1919-1920 (Arthur, 1925) Aecial description matches P. graminella – mistaken identity? 14/11/2018 TT biocontrol talk Anderson (2004); Anderson (2005)

Uromyces pencanus - CNG Telia on Nassella neesiana (site NN3) Uredinia on inoculated plant Uredinia on inoculated plant (site NN3) 14/11/2018 TT biocontrol talk Anderson (2005a)

Uromyces pencanus - CNG Appresorium over stoma Germinating urediniospore on leaf 14/11/2018 TT biocontrol talk Anderson (2005c)

Uromyces pencanus - CNG Isolates (urediniospores) tested on: 7 provenances from Australia 2 provenances from NZ Australian 6/7 susceptible (not Ballarat) Neither from NZ infected 2 Austrostipa spp. Neither infected ST (ACT) No infection 14/11/2018 TT biocontrol talk Anderson (2005a)

Pathogenicity and virulence of Argentinian fungi on CNG - promising Puccinia nassellae (rust) Autoecious? (teliospore infection not shown) Much obvious damage in field urediospores infect but host-strain specificity Puccinia graminella Autoecious, demicyclic plants currently being tested Uromyces pencanus Autoecious? (no teliospore infection yet) 14/11/2018 TT biocontrol talk

Prospects for biological control of serrated tussock and Chilean needle grass 1. Biological control – Argentina 1.1 Chilean needle grass (Nassella neesiana) 1.2 Serrated tussock (Nassella trichotoma) 2. Biological control – endemic 2.1 Serrated tussock (Nassella trichotoma) 14/11/2018 TT biocontrol talk

Puccinia nassellae - ST http://www.rirdc.gov.au/reports/Ras/01-27.pdf Urediniospores on site Puccinia nassellae - ST Only uredinia and telia in literature (Cummins, 1971; Lindquist, 1982) Only uredospores found at sites Trap plants transplanted into infected sites Most plants infected during autumn, not winter Uredinia do not burst open easily Urediniospores collected by suction Used to infect ST and CNG in glasshouse 14/11/2018 TT biocontrol talk Briese et al. (2001)

Puccinia nassellae (from ST) specificity Inoculation trials ST vs CNG ST - uredinia on all inoculated Argentinian plants CNG - no cross-infection Argentinian ST Rust strains species-specific Argentinian vs Australian ST Argentinian ST – uredinia Australian ST – not infected Rust site-specific 14/11/2018 TT biocontrol talk Anderson (2004a); Anderson (2005b)

Ustilago sp. Ustilago sp. on one population of ST Totally prevented seed production Important effect on plant populations? 14/11/2018 TT biocontrol talk Briese et al. (2001)

Ustilago sp. - ST Might not be U. hypodytes Glasshouse experiments http://www.rirdc.gov.au/reports/Ras/01-27.pdf Might not be U. hypodytes Glasshouse experiments Inoculated germinating seeds No infected plant produced seed (controls did) Little fusion of hyphae in germinating spores 14/11/2018 TT biocontrol talk Briese et al. (2001)

Corticium Corticium ubiquitous on ST and CNG etc. Associated with tussock decline Heavy sporulation (mats) observed at crown on dead and dying culms (pull out easily) Isolated on to PDA – hyphae only Transplant experiment set up on site – ST Under way 14/11/2018 TT biocontrol talk Briese et al. (2001)

Pathogenicity and virulence of Argentinian fungi on ST – much less promising Puccinia nassellae (rust) Some obvious damage in field Problems infecting plants in glasshouse Ustilago sp. As for rust Corticium sp. Dieback in field, plants in field pull out easily Roots covered with white layer of fungus Could not grow easily in media or in glasshouse Only hyphae on PDA (may not be infective) 14/11/2018 TT biocontrol talk

Prospects for biological control of serrated tussock and Chilean needle grass 1. Biological control – Argentina 1.1 Chilean needle grass (Nassella neesiana) 1.2 Serrated tussock (Nassella trichotoma) 2. Biological control – endemic 2.1 Serrated tussock (Nassella trichotoma) 14/11/2018 TT biocontrol talk

Search for local pathogens for inundative biocontrol of ST Plants of serrated tussock surveyed within 50 km radius of Melbourne, Victoria during 1996-7 and 1998-9 6 fungi found Arthrinium sp. (seed epiphyte) Ascochyta sp. (leaf spot) Dinemasporium sp. (leaf spot) Fusarium oxysporum (crown rot) Pleospora sp. (leaf spot) Zinzipegasa argentinensis (leaf spot)* *new record for Australia Previously recorded only on one ‘Stipa sp.’ in Argentina Hussaini, I.P., Lawrie, A.C. and McLaren, D.A. (1998). Fungi in Victoria with biological control potential for Nassella trichotoma (serrated tussock). Plant Protection Quarterly 13, 99-101. 14/11/2018 TT biocontrol talk

Fruiting bodies on leaf (incurved hairs) Fruiting body inside stem and wall detail Zinzipegasa argentinensis Conidia (spores) with tails Hussaini, I.P., Lawrie, A.C. and McLaren, D.A. (1998). Fungi in Victoria with biological control potential for Nassella trichotoma (serrated tussock). Plant Protection Quarterly 13, 99-101. 14/11/2018 TT biocontrol talk

Effects on seedlings of ST Germination (n=20) of Nassella trichotoma seeds inoculated with fungi at 3x106 CFU. Data are meanSE. Treatment No. germinated/20 None (control) 17±3a Dinemasporium 0b Ascochyta 5±3b Fusarium 3±0.3b Unidentified fungus 5±0.3b Hussaini, I.P., Lawrie, A.C. and McLaren, D.A. (2000). Pathogens on and variation in Nassella trichtoma (Poales: Poaceae) in Australia. In: Proceedings of the X International Symposium on Biological Control of Weeds 4-14 July 1999 (ed N. R. Spencer), pp.269-280. (Montana State University, Bozeman). 14/11/2018 TT biocontrol talk

Effectiveness of endemic fungi on ST Host specificity and pathogenicity tested on mature plants for 3 spp.: Arthrinium sp. Dinemasporium sp. Fusarium oxysporum Pot trial in glasshouse 5-6-month-old serrated tussock 30 other grass species incl. Austrostipa Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. 14/11/2018 TT biocontrol talk

Effectiveness of endemic fungi on ST Species No. of plants infected/no. treated Dinemasporium Fusarium Arthrinium Control Nassella trichotoma (V8) 3/3 0/3 N. trichotoma (V10) Dinemasporium sp. most effective/specific fruiting bodies, but little necrosis around them leaf blades died from the tip if fruiting bodies on the same blade no plant died Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. 14/11/2018 TT biocontrol talk

Specificity of endemic fungi on ST Arthrinium sp. also infected: 2/47 plants of other species 1/2 Dichelachne crinita, 1/3 Poa poiformis none died Dinemasporium sp. also infected: 2/53 plants of other species 1/2 A. exilis, 1/3 A. mollis Fusarium oxysporum also infected: 26/29 plants from 16 Austrostipa spp. 5/21 plants from 11 other native grass species 5 Austrostipa species had ≥50% plants die (too wet?) Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. 14/11/2018 TT biocontrol talk

Effectiveness and specificity of endemic fungi on ST fungi not host-specific 2-18 other species infected too Dinemasporium best for specificity did not kill serrated tussock plants But 6 plants of 4 Austrostipa species died with Fusarium oxysporum (not forma specialis) unlikely that any could be used ‘as is’ for inundative biological control Mutate (irradiate spores with UV)? Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. 14/11/2018 TT biocontrol talk

Seed burial trial - ST Hypothesis: Effects of main factors and significant interactions on proportion of Nassella trichotoma seed infected after recovery from burial at 6 sites for 3 burial durations. Hypothesis: Serrated tussock infestation not uniform in area Some areas not/less infested than others Possible soil microbe action on seeds? Bury seeds - see if infected differently at different sites Infection did vary with site, duration and provenance Factor P value Burial site 0.042 Burial duration 0.000012 Burial depth 0.094 Fungicide 0.028 Provenance 0.0054 Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. 14/11/2018 TT biocontrol talk

Infection (%) of retained serrated tussock seed buried in soil for 7 months by the major types of microorganisms. Genus or group Range (%) Mean (%) Alternaria spp. 55.3-92.6 77.5 Bipolaris spp. 2.4-33.1 16.3 Trichoderma spp. 0-23.9 3.6 Fusarium spp. 0-3.8 0.7 Actinomycetes 0-2.7 0.6 Coniochaeta spp. 0-4.5 0.4 Penicillium spp. 0-1.2 0.2 Others 0-1.9 0.3 Gloeosporium spp. 0-0.8 0.1 14/11/2018 TT biocontrol talk Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT.

Germination (%) of serrated tussock seed in the presence of five fungi used as the inoculum Fungus genus Transformed mean Back-transformed mean (%) Alternaria 16.3 8a Gloeosporium 18.5 10a Trichoderma 31.1 28b Fusarium 17.3 9a Penicillium 28.6 23b (sed for transformed data=5.08) Possible use in biological control? Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. 14/11/2018 TT biocontrol talk

Search for more biological control organisms for ST looked in areas with established serrated tussock Bacchus Marsh Class 5 infestation 14/11/2018 TT biocontrol talk

Property near Bacchus Marsh Class 5 tussock 14/11/2018 TT biocontrol talk

Insect larvae on serrated tussock at Bacchus Marsh insect larvae found in serrated tussock plants but no visible damage – protection from predators? unlikely to complete life cycle on serrated tussock 14/11/2018 TT biocontrol talk

Property near Bacchus Marsh Strange green patches seen in Class 5 fields 14/11/2018 TT biocontrol talk

Cause of green patches Mushroom mycelium From ‘fairy rings’ Useful in biological control in future? 14/11/2018 TT biocontrol talk

References - Argentina Briese, D.T., and Evans, H.C. (1998). Biological control of serrated tussock (Nassella trichotoma): is it worth pursuing? Plant Protection Quarterly 13, 94-97. Briese, D.T., McLaren, D.A., Pettit, W.J., Zapater, M., Anderson, F., Delhey, R., and Distel, R. (1999). New biological control initiatives against weeds of South American origin in Australia: Nassella tussock grasses and blue heliotrope. In Spencer, N.R. (Ed.) Proceedings of the X International Symposium on Biological Control of Weeds, 4-14 July 1999, Montana State University, Bozeman, Montana, USA. Briese, D.T., Pettit, W., and Anderson, F. (2001). Biological control of serrated tussock and Chilean needle grass. Report for the Rural Industries Research & Development Corporation. RIRDC Publication No. 01/27 (Project No. UA-48A (http://www.rirdc.gov.au/reports/Ras/01-27.pdf ). Anderson, F. (2004a). Progress report on investigations of fungal pathogens of serrated tussock (Nassella trichotoma) and Chilean needle grass (N. neesiana), May 2003-February 2004. CERZOS-UNS, Bahia Blanca, Argentina. Anderson, F., and McLaren, D. (2004b). Biological control of Chilean needle grass, recent results. CERZOS-UNS, Bahia Blanca, Argentina Anderson, F. (2005a). Progress report on investigations of fungal pathogens of serrated tussock (Nassella trichotoma) and Chilean needle grass (N. neesiana), March 2004-February 2005. CERZOS-UNS, Bahia Blanca, Argentina Anderson, F. (2005b). Progress report on investigations of fungal pathogens of serrated tussock (Nassella trichotoma) and Chilean needle grass (N. neesiana), July 2005. CERZOS-UNS, Bahia Blanca, Argentina Anderson, F. (2005c). Progress report on investigations of fungal pathogens of serrated tussock (Nassella trichotoma) and Chilean needle grass (N. neesiana), August 2005 - October 2005. CERZOS-UNS, Bahia Blanca, Argentina 14/11/2018 TT biocontrol talk

References - Australia Casonato, S.G. (2003). An investigation into the biology and natural enemies of serrated tussock in Australia. Ph.D. thesis, RMIT. Hussaini, I.P., Lawrie, A.C. and McLaren, D.A. (2000). Pathogens on and variation in Nassella trichtoma (Poales: Poaceae) in Australia. In: Spencer, N.R. (Ed.). Proceedings of the X International Symposium on Biological Control of Weeds 4-14 July 1999, pp.269-280. (Montana State University, Bozeman). Hussaini, I.P., Lawrie, A.C. and McLaren, D.A. (1998). Fungi in Victoria with biological control potential for Nassella trichotoma (serrated tussock). Plant Protection Quarterly 13, 99-101. 14/11/2018 TT biocontrol talk