Prospects for biological control of serrated tussock and Chilean needle grass

1
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

2
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)

3
Chilean needle grass
http//www.weeds.crc.org.au/main/wom_chilean_needl
e_grass_0905.html
http//www.humeshire.nsw.gov.au/files/1733/File/CN
GWeed2.jpg
4
Chilean needle grass
http//www.weeds.org.au/cgi-bin/weedident.cgi?tpl
plant.tplstatesibraallcardG12
5
Serrated tussock (Nassella trichotoma)
NRE 1998
6
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
7
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)

8
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.

9
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 N. neesiana, N. tenuis, N. trichotoma
Basidiomycetes (mushrooms) Corticium sp. N. trichotoma
Coelomycetes Ascochyta leptospora N. trichotoma
Stagonospora sp. N. trichotoma
Hendersonula sp. N. trichotoma
Agonomycetes Rhizoctonia sp. N. trichotoma
Briese and Evans, 1998 Briese et al., 1999)
10
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

11
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
12
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.
http//www.rirdc.gov.au/reports/Ras/01-27.pdf
13
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.
http//www.rirdc.gov.au/reports/Ras/01-27.pdf
Anderson 2004a
14
RUST LIFE CYCLES

• Rust needs to be autoecious (have all life
  stages on host plant)

• I
• aecia
• 
  macrocyclic
• 
  e.g. P.
  graminis
• 0
• pycnia
  demicyclic
• e.g. P. lagenophora
• microcyclic
• e.g. P.
  heterospora
• IV
  
  II
• basidia
  
  uredinia
• leptoforms
• e.g. P. malvacearum
• III
• telia

15
Rust on ST and CNG

• ST
• 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

Briese et al. (2001) Anderson (2004a)
16
Sphaerellopsis (Darluca) filum
http//www.niaes.affrc.go.jp/inventry/microorg/eng
/z65e-Sphaer-fil.html
17
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)

18
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
• Uredinia and telia in field (aecia on alternate
  host?)

Briese et al. (2001)
19
Puccinia nassellae - CNG
Anderson (2005a)
20
Puccinia nassellae - CNG
http//www.rirdc.gov.au/reports/Ras/01-27.pdf
Anderson (2005a)
21
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?)

Anderson (2004a)
22
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

Anderson (2004a)
23
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

Anderson (2005a) Anderson (2005c)
24
Puccinia graminella - CNG
Anderson (2005a)
25
Puccinia graminella - CNG
Aecia on Chilean needle grass at site NN 8
Germinating teliospores with basidiospores
Anderson (2005c)
26
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?

Anderson (2004) Anderson (2005)
27
Uromyces pencanus - CNG
Uredinia on inoculated plant
Uredinia on inoculated plant (site NN3)
Anderson (2005a)
28
Uromyces pencanus - CNG
Appresorium over stoma
Germinating urediniospore on leaf
Anderson (2005c)
29
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

Anderson (2005a)
30
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
• Much obvious damage in field
• plants currently being tested
• Uromyces pencanus
• Autoecious? (no teliospore infection yet)
• Much obvious damage in field
• urediospores infect but host-strain specificity

31
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)

32
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

Briese et al. (2001)
33
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

Anderson (2004a) Anderson (2005b)
34
Ustilago sp.

• Ustilago sp. on one population of ST
• Totally prevented seed production
• Important effect on plant populations?

Briese et al. (2001)
35
Ustilago sp. - ST

• Might not be U. hypodytes
• Glasshouse experiments
• Inoculated germinating seeds
• No infected plant produced seed (controls did)
• Little fusion of hyphae in germinating spores

Briese et al. (2001)
36
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

Briese et al. (2001)
37
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)

38
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)

39
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.
40
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.
41
Effects on seedlings of ST
Germination (n20) of Nassella trichotoma seeds
inoculated with fungi at 3x106 CFU. Data are
mean?SE.
Treatment No. germinated/20
None (control) 173a
Dinemasporium 0b
Ascochyta 53b
Fusarium 30.3b
Unidentified fungus 50.3b
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).
42
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.
43
Effectiveness of endemic fungi on ST
Species No. of plants infected/no. treated No. of plants infected/no. treated No. of plants infected/no. treated No. of plants infected/no. treated
Dinemasporium Fusarium Arthrinium Control
Nassella trichotoma (V8) 3/3 3/3 3/3 0/3
N. trichotoma (V10) 3/3 3/3 3/3 0/3

• 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.
44
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
• none died
• 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.
45
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.
46
Seed burial trial - ST
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.
47
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
Casonato, S.G. (2003). An investigation into the
biology and natural enemies of serrated tussock
in Australia. Ph.D. thesis, RMIT.
48
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 data5.08)

• 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.
49
Search for more biological control organisms for
ST

• looked in areas with established serrated tussock
• Bacchus Marsh
• Class 5 infestation

50
Property near Bacchus Marsh
Class 5 tussock
51
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

52
Property near Bacchus Marsh
Strange green patches seen in Class 5 fields
53
Cause of green patches

• Mushroom mycelium
• From fairy rings
• Useful in biological control in future?

54
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

55
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.