14 Protection Quarterly Vol.15(1) 2000 Final host specificity testing of the two isolates (FR3, IT2) was completed by May Biological control of pycnocephalus and C. 1993 and, following permission from the tenuiflorus using the fungus Puccinia cardui- Australian Quarantine Inspection Service and the then Australian Nature Conserva- pycnocephali tion Agency (now Environment Aus- tralia), the isolates were released in the J.J. BurdonAB, P.H. ThrallA, R.H. GrovesAB and P. ChaboudezC field across southern from Octo- A Centre for Plant Biodiversity Research, B CRC for Weed Management ber 1993 onwards. Systems, CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, The release program Australia. C At all release sites a mixed inoculum of le Fontenelle bât. G2, 663 rue Pré aux Clercs, Montpellier 34000, France. both isolates of P. cardui-pycnocephali was released by spraying a suspension of 300 mg of urediospores in 15 L of local tap Summary identified more than 20 insect species water onto 10 m2 patches of either or both Following extensive host-specificity test- feeding on the Carduus species but indi- C. tenuiflorus and C. pycnocephalus. A ing in southern France and in quarantine vidually none appeared to limit the size of mixed inoculum was used to prevent facilities in Canberra, two isolates of the host populations in the field (Sheppard et problems associated with distinguishing rust fungus Puccinia cardui-pycno- al. 1991). In contrast, extensive field obser- between these two closely related thistle cephali were released in southern Aus- vations of natural populations of Carduus taxa by untrained personnel. Plots were tralia in October 1993 as biological con- in southern France during the period inoculated in the late afternoon, covered trol agents for the slender thistles 1988-1991 and subsequent experiments with plastic sheeting overnight to main- Carduus pycnocephalus and C. tenui- indicated that the rust fungus Puccinia tain high humidity, and then uncovered florus. Initially, establishment was poor cardui-pycnocephali had a significant effect the following morning. Disease symp- due to prolonged dry conditions. Subse- on seed production (Chaboudez et al. toms became visible 10–15 days later de- quent releases in 1994–1995 became well 1993, P. Chaboudez unpublished data). pending on prevailing temperatures. established and consistently cause no- Once P. cardui-pycnocephali was identi- Initially, limited field releases were ticeable disease particularly in western fied as a potential biological control agent, made in New South Wales, Victoria and Victoria and Tasmania. In 1998, compara- two further complications came into play. Tasmania in late spring 1993 (October– tive assessments of fungicide-treated First, P. cardui-pycnocephali was known to November) with broad scale releases be- (disease absent) and untreated (disease have occurred in Australia for at least 50 ing undertaken throughout southern Aus- present) plots in both these areas showed years prior to this control program (Costin tralia during 1994. All these early releases reductions in the size of individual C. 1954). This presented questions regarding suffered from the prolonged drought that tenuiflorus and the number of in- the value of importing further strains of an prevailed across much of southern Aus- florescences as a consequence of infec- agent that, to date, had had no noticeable tralia during 1993–1994. While the patho- tion by P. cardui-pycnocephalus. While effect on thistle growth. Second, there was gen became established at several sites the impact of the pathogen alone was in- some suggestion that different isolates of during the winter/spring period, it failed sufficient to reduce thistle densities per- P. cardui-pycnocephali would show differ- to persist over summer probably because manently, it will have a role as an addi- ent degrees of aggressiveness on the two of the extremely dry conditions and con- tional tool in the integrated control of target species. These concerns were ad- sequent very high grazing pressures. these thistles. dressed during initial trials in France (con- Once the drought broke, further releases ducted at the CSIRO Biological Control were made through a network of collabo- Introduction Facility in Montpellier) by comparing the rators in many of the same areas in au- Carduus pycnocephalus and C. tenuiflorus virulence and aggressiveness of several tumn 1995. A strategy of release of P. are two closely related thistle species oc- isolates of P. cardui-pycnocephali collected cardui-pycnocephali at multiple sites rather curring naturally in Mediterranean to in Mediterranean Europe with a ‘control’ than detailed monitoring of just a few sites north-western Europe. Introduced to Aus- isolate collected in Australia. Two Medi- was adopted to maximize spread. A total tralia, probably as a contaminant of feed, terranean isolates (IT2 and FR3) were sub- of 478 ‘starter’ samples of both isolates of these thistles have become widely distrib- stantially more aggressive towards C. P. cardui-pycnocephali were distributed be- uted across southern Australia from pycnocephalus or C. tenuiflorus than the tween October 1993 and June 1995 (Figure Queensland to Western Australia and Tas- Australian isolate. Although both isolates 1). These post-drought releases were mania (Parsons and Cuthbertson 1992). In attacked both Carduus species, isolate IT2 much more successful, with reports of es- favourable sites, they may form dense induced a slightly more susceptible reac- tablishment and persistence coming from stands, leading to reduced growth of more tion on C. pycnocephalus than isolate FR3, collaborators in South Australia, Victoria, desirable pasture plants. Losses to Aus- which was similarly more successful on C. Tasmania and New South Wales. tralian industry resulting from reduced tenuiflorus (Chaboudez et al. 1993). Since 1995, anecdotal reports from stock carrying capacities and vegetable These two isolates of P. cardui- property owners and State government contamination of wool were estimated to pycnocephali were then subjected to ex- weed inspectors have suggested that P. exceed $5 million per annum in 1992. haustive host specificity testing on a range cardui-pycnocephali was generating suffi- As part of a broader strategy to control of plant species with a particular empha- cient disease pressure to cause reductions all weedy thistle species introduced to sis on members of the . With in the size and seed production of affected Australia, C. pycnocephalus and C. tenui- the exception of seedlings of Cynara slender thistle stands. This paper reports florus were identified as candidates for a scolymus (globe artichoke) where pustules an objective assessment of the perform- biological control program. The first step developed on cotyledons and the first true ance of P. cardui-pycnocephali as a biologi- in mounting the control program was to leaves, P. cardui-pycnocephali only attacked cal control agent against one (C. tenui- conduct a series of extensive surveys for plants of C. pycnocephalus and C. florus) of its two thistle hosts. natural enemies associated with the spe- tenuiflorus (Chaboudez et al. 1993, P. cies in their home ranges. Those surveys Chaboudez unpublished data). Plant Protection Quarterly Vol.15(1) 2000 15 significance of treatment effects (fungicide treated vs. control) on per cent disease, fe- cundity (as measured by the number of in- florescences) and final dry weight were based on Type III sums of squares. Plot density was used as a covariate. Regres- sion analysis was used to examine the re- lationship between disease severity and dry weight of infected plants.

Results and discussion There were substantial differences be- tween the Tasmanian and Victorian sites in terms of plot densities: mean densities were 27.7 and 52.0 m-2 for the two sites re- spectively. As a consequence of their higher density, individual plant dry weights were much lower in both fungi- cide treated and control plots at the Victo- rian site (Figure 2c). With respect to dis- ease levels, there was little difference in 60 the control plots, although fungicide- treated plots at the Victorian site were al- 124 most entirely disease-free. A low level of disease was present in many of the treated 210 plots at the Tasmanian site. There ap- peared to be little difference in number of 74 inflorescences per plant between the two sites. 10 As expected, there was a significant ef- fect of treatment (control vs. fungicide- Figure 1. General areas and number of releases of ‘starter’ samples of treated) on mean levels of disease for both inoculum of two isolates of P. cardui-pycnocephali from October 1993 to the Tasmanian (F=138.2, P<0.001) and Vic- June 1995. torian sites (F=265.1, P<0.001) (Figure 2a). Disease levels in control plots were rela- Materials and methods plants) were harvested approximately tively low being about 3–4%. However, Twenty 1 m2 plots were established in two weeks later in early December. Al- inspection of dried and withered leaves August 1998 in a large stand of C. though not all plots could be harvested, at still attached to plants indicated that dis- tenuiflorus in an improved pasture in both sites equal numbers of treated and ease levels had been higher in the weeks northern Tasmania (147°15'E, 41°04'E). A control plots were assessed. Within each preceding harvest. These differences were month later a second site was set up in a plot, the above-ground biomass of each reflected in significant effects of the fungi- similar manner in south-western Victoria plant was separately harvested and cide treatment on both the number of in- (141°26'E, 37°27'S). At both sites C. bagged. At that time, a visual estimate of florescences (Figure 2b) and plant dry tenuiflorus was the dominant thistle disease severity (per cent disease cover of weights (Figure 2c) at both sites. Mean present; although C. pycnocephalus was green leaves and stems) was also made plant dry weights for infected plants were present in the stands no individuals oc- and recorded. 28 and 22% lower than the fungicide- curred within the sample plots. Both sites Plants were returned to the laboratory, treated plants at the Tasmanian and Victo- were near locations at which both isolates dried for one week at 80°C and then rian sites respectively. However, in both of P. cardui-pycnocephali had been released weighed. For a subset of 50 plants from cases high variance in the data resulted in within the previous two years. At the time both sites, the total number of flowers as low R2 values. of plot establishment, thistles were at the well as the number of flower clusters (in- For the Tasmanian site, data pooled for rosette stage, and disease caused by P. florescences) were recorded. Due to the diseased and healthy plants showed a cardui-pycnocephali was apparent in both time-consuming nature of counting indi- strong positive correlation between the stands. At each site, plots were randomly vidual flowers and the high correlation number of inflorescences and dry weight assigned to two treatments: between the number of flowers and (r=0.92, P=0.0001), but no other significant i. regular treatment with fungicide, and number of inflorescences (r=0.81, P= relationships. Interestingly, when only in- ii. untreated controls in which disease 0.0001; r=0.90, P=0.0001 for Tasmania and fected plants were considered, significant was allowed to increase freely. Victoria respectively), only inflorescence negative correlations appeared between The fungicide-treated plots were sprayed data were recorded for the remaining per cent disease and both dry weight to run-off at 2–3 week intervals with plants. and number of inflorescences (r=-0.17, Baycor® [Bayer] (8.5 mL per 5 L water). Statistical analyses were carried out us- P=0.006; r=-0.16, P=0.007 respectively). This treatment substantially controlled the ing SAS statistical software (SAS Institute Figure 3 shows the relationship between disease such that at harvest time lesions 1985), and were done separately for the per cent infection and dry weight. caused by P. cardui-pycnocephali were ei- two sites. Pearson product-moment corre- Similarly, the combined data for the ther absent or at a trace level only. lations between per cent disease, total dry Victorian site showed a significant posi- All the plants in a total of 16 plots (443 weight and the number of inflorescences tive correlation between dry weight and plants) were harvested at the Tasmanian were calculated. Analyses of variance and the number of inflorescences (r=0.90, site in late November 1998, while at the regression analyses used the General P=0.0001). However, there was a signifi- Victorian site all the plants in 14 plots (728 Linear Models procedure, where the cant positive correlation between per cent 16 Plant Protection Quarterly Vol.15(1) 2000 disease and dry weight for the Victorian A 8 plots (r=0.08, P=0.03), suggesting that larger plants are more likely to acquire in- 7 fection. When only infected plants were considered for the Victorian site, this posi- 6 tive correlation became even stronger (r=0.21, P=0.0001) and a positive correla- 5 tion between per cent disease and number of inflorescences also occurred (r=0.18,

4 1234567890 P=0.0008). 12345678901 1234567890 12345678901 1234567890 The release of the two aggressive 12345678901 1234567890 3 12345678901 1234567890 strains of P. cardui-pycnocephali for biologi- 12345678901 1234567890 12345678901 1234567890 cal control of the thistles C. pycnocephalus 12345678901 1234567890

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Mean per cent infection C. tenuiflorus 2 12345678901 1234567890 and has resulted in success-

12345678901 1234567890

12345678901 1234567890 ful establishment of the pathogen at a

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1 12345678901 1234567890 range of sites across southern Australia.

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12345678901 1234567890 However, unlike the release of P. chon- 0 12345678901 1234567890 drillina to control Chondrilla juncea which Tasmania Victoria was accompanied by rapid spread and massive epidemic build-up of the rust over a very short period of time (Cullen et B al. 1973), and a subsequent decline in host 10 plant numbers (Burdon et al. 1981), the re- 9 leased strains of P. cardui-pycnocephali do not appear likely to cause similar massive 8 reductions in plant numbers. 7 Clearly under favourable conditions in the field, P. cardui-pycnocephali can reduce 6 the size and flower production of C. tenui- 5 florus. However, the data from this study 12345678901 in Tasmania and Victoria and casual ob- 12345678901 4 12345678901 servations indicate that for most years 12345678901 12345678901 such reductions alone would be insuffi- 3 12345678901 1234567890 12345678901 1234567890 cient to permanently reduce thistle densi- 12345678901 1234567890 12345678901 1234567890 ties. Rather, the role of P. cardui-pycno- 2 12345678901 1234567890

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Mean inflorescences per plant cephali 12345678901 1234567890 will be as an additional tool in the

1 12345678901 1234567890 12345678901 1234567890 integrated control of these thistles. Indeed,

12345678901 1234567890 0 Bendall (1973) has shown that in well- managed pastures, competition from pas- Tasmania Victoria ture grasses on its own may limit the weediness of C. pycnocephalus and C. tenui- florus. In such situations, the impact of P. C cardui-pycnocephali will provide an addi- 8 tional negative impact, further depressing the plant’s competitive ability. 7

6 Acknowledgments The authors are grateful to: 5 i. AWRPO for funding the testing and initial release program; 4 ii. the CRC for Weed Management Sys- tems for providing the financial sup- 3 port needed to establish the field plots; 12345678901 iii. Sandy Leighton (Tasmania) and Brad 12345678901

12345678901 Mean dry weight (g) Roberts and Nick Harvey (Victoria) 2 12345678901 12345678901 who established and sprayed the plots; 12345678901 12345678901 iv. the Tamar Valley Weeds Group (Tas- 1 12345678901 1234567890

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12345678901 1234567890 mania) for their support; and

12345678901 1234567890 0 v. especially to Ian Sauer for his enthusias- tic interest and generosity in making his Tasmania Victoria property ‘Farley’ available for this work.

Figure 2. The effect of the absence (fungicide-treated plots; filled bars) or References Bendall, G.M. (1973). Control of slender presence (control plots; cross-hatched bars) of the rust fungus P. cardui- thistle Carduus pycnocephalus L. and C. pycnocephali on: (a) the mean percent infection; (b) the mean number of tenuiflorus Curt. (Compositae) in pas- inflorescences per plant; and (c) mean dry weight of C. tenuiflorus plants ture by grazing management. Austral- growing at locations in northern Tasmania and western Victoria in 1998. ian Journal of Agricultural Research 24, The bars indicate standard deviations. 831-7. Plant Protection Quarterly Vol.15(1) 2000 17 300

250

200

150

100 Plant dry weight (g)

50

0 0246810121416 Per cent infection (severity)

Figure 3. The relationship between mean plant weight and percent infection by P. cardui-pycnocephali on C. tenuiflorus plants growing in northern Tasmania.

Burdon, J.J., Groves, R.H. and Cullen, J.N. Costin, A.B. (1954). ‘A study of the ecosys- (1981). The impact of biological control tems of the Monaro region of New on the distribution and abundance of South Wales’. (Soil Conservation Serv- Chondrilla juncea in south-eastern Aus- ice of New South Wales, NSW Govern- tralia. Journal of Applied Ecology 18, ment Printer, Sydney). 957-66. Cullen, J.M., Kable, P.F. and Catt, M. Chaboudez, P., Burdon, J.J. and Groves, (1973). Epidemic spread of a rust im- R.H. (1993). Application for field re- ported for biological control. Nature lease of the slender thistle rust fungus 244, 462-4. (Puccinia cardui-pycnocephali) – a classi- Parsons, W.T. and Cutherbertson, E.G. cal biological control agent for the nox- (1992). ‘Noxious weeds of Australia’. ious weeds C. pycnocephalus and C. (Inkata Press, Melbourne). tenuiflorus. CSIRO Division of Plant In- SAS Institute (1985). ‘SAS user’s guide. dustry Report for the Australian Quar- Version 5 Edition’. (SAS Institute Inc., antine Inspection Service and Environ- Cary, NC). ment Australia. Sheppard, A.W., Aeschlimann, J.-P., Chaboudez, P., Groves, R.H. and Burdon, Sagliocco, J.-L. and Vitou, J. (1991). J.J. (1993). Puccinia cardui-pycnocephali, a Natural enemies and population stabil- potential agent for the biological con- ity of the winter-annual Carduus trol of slender thistles in Australia. Pro- pycnocephalus L. in Mediterranean Eu- ceedings 10th Australian/14th Asian- rope. Acta Oecologica 12, 707-26. Pacific Weed Conference, Brisbane.