166 Protection Quarterly Vol.21(4) 2006 Further development of these previ- ously identifi ed fungi requires their patho- Biological control potential of endemic Australian genicity to be tested on actively growing fungal pathogens of Nassella trichotoma (serrated and their host specifi city to be test- ed against endemic Australian grasses and ) important pasture species. The aim of this study was to investigate the effect of two S.G. CasonatoA,B,D, A.C. LawrieA and D.A. McLarenB,C of the fungi previously isolated (Hussaini A Biotechnology and Environmental Biology, School of Applied Sciences, et al. 1998, Hussaini et al. 2000) and a third RMIT University, Bundoora West Campus, PO Box 71, Bundoora, newly isolated fungus, on fi ve month old Victoria 3083, Australia. serrated tussock and grass species impor- B tant within Australia. This would indicate Department of Primary Industries Frankston, PO Box 48, Frankston, if these fungi could potentially be used as Victoria 3199, Australia. biological control agents against serrated C CRC for Australian Weed Management. tussock in the fi eld. D Current address: HortResearch, Private Bag 92 169, Mt. Albert, Auckland, New Zealand. Materials and methods Target plants A trial was undertaken on 134 plants, in- cluding 19 species of Austrostipa, 12 other Summary grass species of importance to Australia Nassella trichotoma is a serious weed control than traditional methods where and two provenances of serrated tussock, in Australia. Three fungi, Arthrinium, natural enemies such as insects and/or both collected from Victoria (Table 1). The Dinemasporium and Fusarium, were fungi are used as control agents against emphasis in the trial was on the diversity identifi ed as infecting serrated tussock the host weed. Typically three strategies of grasses tested, and replication of the plants in the fi eld and were subsequent- use or apply plant pathogens in biologi- plants in the trial was limited by growth ly assessed for their ability to provide cal control: i) classical biological control of the plants from the original germination biological control in an inundative trial. often referred to as an inoculative release; (Table 1). Therefore, not all 31 plant species Host specifi city and pathogenicity was (ii) inundative biological control, which were inoculated with all three fungal path- tested on 5–6 month-old serrated tussock uses bioherbicides; and (iii) augmentative ogens, nor were all treatments fully rep- and 30 other grass species in a pot trial biological control, which is a supplemen- licated. All plants used were of a similar in a temperature-controlled glasshouse. tative approach. age and actively growing at the time of in- The fungi were not host-specifi c, as 2–18 A program is currently investigating oculation; N. trichotoma plants were about other grass species were infected nor did the possibility of implementing a classi- fi ve months old and other grass species, they kill serrated tussock plants. Howev- cal biological control program for serrated some of which had thickened tillers indi- er, six plants of the four Austrostipa spe- tussock using fungal pathogen sources in cating fl owering initiation, being about cies died after inoculation with Fusarium Argentina (Anderson et al. 2002). Classi- six months old. Some of the plants from oxysporum. Further research is needed cal biological control involves the transfer the endemic species were twice as tall as to assess the potential of the fungi as of fungal pathogens from the host plant those of the serrated tussock. Plants were biological control agents in inundative in its native range to the area where it is grown in 200 mm pots at the Department applications; this research has indicated problematic. of Primary Industries (DPI), Frankston that they have limited potential. In Australia fi ve fungi have previously Campus, in a glasshouse with a control- been observed infecting serrated tussock led temperature of 15–25°C and natural Introduction in Victoria in 1996–1997 (Hussaini et al. daylight hours during December. Nassella trichotoma (Nees) Arech., com- 1998, Hussaini et al. 2000); these were spe- monly referred to as serrated tussock, is cies of Ascochyta, Dinemasporium, Pleospo- Inoculum a major and problematic noxious weed in ra, Zinzipegasa and Fusarium, with the fi rst Plants were inoculated with fungal iso- Australia (Campbell 1998). It is predomi- four fungi observed in leaf spots and the lates of Fusarium sp. Link. and Dinemaspo- nantly located in New South Wales, the last in crown rot. Ascochyta, Fusarium and rium sp. Léveillé, that had been identifi ed Australian Capital Territory and Victoria, Dinemasporium reduced seed germination previously as infecting serrated tussock in with small infestations in Tasmania. With- in serrated tussock by 7–100% (Hussaini Victoria and subsequently cultured from in Victoria, serrated tussock infests an es- et al. 2000), suggesting their possible use the plants (Hussaini et al. 1998, Hussaini et timated 105 000 hectares with infestations in inundative or augmentative biological al. 2000). The Fusarium sp. was identifi ed costing up to $123 million to grazing in- control. The inundative strategy generally as Fusarium oxysporum Schltdl. An isolate dustries over a 30-year period (Morfe et al. uses indigenous fungi and involves the of Arthrinium sp. G.Kunze ex. E.M.Fries., 2002). Serrated tussock is mainly control- application of large quantities of inoculum observed on seeds of serrated tussock led by traditional means, such as manual (bioherbicides or mycoherbicides) gener- in the fi eld, was also used. Cultures of weed removal and herbicides. Herbicides ally annually, into specifi c weed-infested F. oxysporum, Dinemasporium sp. and Ar- have been heavily relied upon with much areas. This results in the localized control thrinium sp. were grown on 90 mm Petri research on the most appropriate herbicide of a weed infestation without the devel- dishes containing one-fi fth strength each and form of application (Campbell and opment of more widespread disease epi- of Potato Dextrose Agar (PDA; Oxoid, Bas- Annand 1962, Campbell 1987, Campbell demics (TeBeest 1996). The augmentative ingstoke, England) and Malt Extract Agar 1996, Campbell and Nicol 2001). Despite approach uses supplemental additions of (MEA; Oxoid, Basingstoke, England). this, some areas are unsuitable for routine the fungal biological control agents to ar- Fungi grown on both media were includ- herbicide use and it is predicted that ser- eas where the microorganism is already ed in the fungal suspension. Plates were rated tussock will continue to spread into present (Cook et al. 1996). The inoculum is grown at 25°C and 25 µE m-2 s-1 provided uninfested areas. neither mass produced nor applied as an by 36 W Osram ‘Warm White’ lights in a Biological control is potentially a more inundative dose over a large proportion of 12 h photoperiod. After 21 d growth, fungal environmentally sensitive means of weed the weed population (Charudattan 1991). plates were fl ooded with approximately Plant Protection Quarterly Vol.21(4) 2006 167 Table 1. Fungal infection of Nassella trichotoma, endemic Australian grasses and grasses of importance in Australia inoculated with fungi isolated from N. trichotoma: Dinemasporium sp., Fusarium oxysporum and Arthrinium sp. Infection noted in parenthesis; if no infection was evident, no value is given. ? possibly infected by F. oxysporum, with basal infection noted (other Fusarium spp. may have caused infection of the leaves). Species Authority Number of plants treated (number infected) Dinemasporium Fusarium Arthrinium Control Aristida sp. L. 2 2 (1) ? 0 0 Austrodanthonia duttoniana (Cashmore) H.P.Linder 1 1 1 2 Au. geniculata (J.M.Black) H.P.Linder 2 2 (2) ? 2 1 Dead = 2 Au. setacea (R.Br.) H.P.Linder 2 2 2 1 Austrostipa aristiglumis (F.Muell.) S.W.L.Jacobs & J.Everett 3 3 (3) ? 3 3 Dead = 1 A. breviglumis (J.M.Black) S.W.L.Jacobs & J.Everett 1 1 (1) ? 1 1 A. blackii (C.E.Hubb.) S.W.L.Jacobs & 1 1 (1) ? 1 1 J.Everett Dead = 1 A. curticoma (Vickery) S.W.L.Jacobs & J.Everett 1 1 (1) ? 1 2 A. drummondii (Steud.) S.W.L.Jacobs & J.Everett 1 0 0 0 A. elegantissima (Labill.) S.W.L.Jacobs & J.Everett 3 3 (2) ? 3 3 A. exilis (Vickery) S.W.L.Jacobs & J.Everett 2 (1) 2 (2) ? 2 0 A. fl avescens (Labill.) S.W.L.Jacobs & J.Everett 1 1 (1) ? 1 3 A. gibbosa (Vickery) S.W.L.Jacobs & J.Everett 1 0 0 0 A. mollis (R.Br.) S.W.L.Jacobs & J.Everett 3 (1) 3 (2) ? 3 2 A. mundula (J.M.Black) S.W.L.Jacobs & J.Everett 2 2 (2) ? 2 1 A. nullanulla (S.W.L.Jacobs & J.Everett) 1 1 (1) ? 1 1 S.W.L.Jacobs & J.Everett Dead =1 A. platycheata (Hughes) S.W.L.Jacobs & J.Everett 1 1 (1) ? 1 1 A. pubinodis (Trin. & Rupr.) S.W.L.Jacobs & 2 2 (2) ? 2 1 J.Everett Dead = 1 A. rudis (Spreng.) S.W.L.Jacobs & J.Everett 3 3 (2) ? 3 3 A. scabra (Lindl.) S.W.L.Jacobs & J.Everett 2 2 (2) ? 2 2 Dead = 1 A. setacea (R.Br.) S.W.L.Jacobs & J.Everett 1 1 (1) ? 0 0 A. stipoides (Hook. f.) S.W.L.Jacobs & J.Everett 2 2 (2) ? 2 2 A. trichophylla (Bentham) S.W.L.Jacobs & J.Everett 1 0 0 0 Dichanthium sericeum (R.Br.) A.Camus 1 1 1 0 crinita (L.f) Hook.f. 2 2 2 (1) 1 Diplachne fusca (L.) Beauv. 1 1 1 1 Elymus scabrus (R.Br.) A.Love 3 3 3 3 Enneapogon sp. Desv. ex. Beauv. 2 2 2 1 Enteropogon sp. Nees. 2 2 (2) ? 2 0 Nassella trichotoma (V8) (Nees) Arech 3 (3) 3 (3) ? 3 (3) 3 N. trichotoma (V10) (Nees) Arech 3 (3) 3 (3) ? 3 (3) 3 poiformis (Labill.) Druce 3 3 3 (1) 3

10 mL of 0.5% Tween 20 (v/v) (Labchem, thoroughly washed with undiluted com- Milli-Q water only. Plants were covered Auburn, Australia) in Milli-Q water and mercial bleach (active ingredient 12% with a clear freezer bag (Multiplex, Aus- were scraped with a sterile glass rod. NaOCl) with two drops (approximately tralia) or heavier clear polyethylene bags 4 µL) Tween 20 and rinsed four times (unknown manufacturer). Bags were se- Inoculation of target plants with sterile water before use. A sterile cured with a rubber band placed at the top For each fungus, the spore suspension container was used to pour the solution of the plastic pot. Plants were thus main- was made up to 1 × 106 spores mL-1 in onto the plants or the plant foliage was tained in approximately 100% relative hu- 0.5% Tween 20 in Milli-Q water. Suspen- submerged in the inoculum. Control midity for 48 hours before the bags were sions were placed in a 10 L plastic bucket, plants were dipped in 0.5% Tween 20 in removed. After inoculation, plants were 168 Plant Protection Quarterly Vol.21(4) 2006 returned to the same glasshouse facilities infected and both Austrodanthonia genticu- rots in many plant species, including the as before and randomly placed on a bench lata plants died (Table 1). Plants infected weed leafy spurge (Caesar 1996, Caesar et with capillary matting watering system. with F. oxysporum had visible rotting at al. 1998, Caesar et al. 1999). However, inoc- Water dripped onto the capillary matting the base, where white hyphal masses, ulation with F. oxysporum was associated via a wick system attached to a trough, so some with a salmon-pink tinge, were ob- in this study with the death of a number that a cohesive surface was kept between served. Many of the leaves of these plants of other grass species. As the fungus could the pot and the capillary matting. Plants were chlorotic. Due to the hyphal masses not be re-isolated and re-identifi ed, Koch’s were not rotated after their initial place- not sporulating, the fungus could not be postulates were not confi rmed and the ment. The symptoms and plants affected identifi ed. Plants that were infected after deaths could not be attributed conclusive- were recorded weekly. At the time of fi nal inoculation with the spore suspension ap- ly to F. oxysporum. Secondary fungal in- assessment, two months after inoculation, peared, on inspection, to have a smaller fections such as Penicillium and Alternaria plant material was removed and inspect- root biomass than plants uninfected or the may have contributed to plant death. The ed for disease symptoms with a dissect- non-inoculated control plants, although high humidity and limited air movement ing microscope. Associated fungi were this was not measured. Serrated tussock in the glasshouse probably heightened the examined with a compound microscope appeared to be least affected by F. oxyspo- infection by these fungi, which are com- and identifi ed using standard mycological rum. Infected plants also had secondary mon and widespread. Plants that died keys. infection, with serrated tussock having the were from the genera Austrostipa and Aus- least. Secondary infection by fungi such trodanthonia, which predominantly require Results as Alternaria sp. and Penicillium sp. was well drained soils and open plains (Aus- All three fungal species, Arthrinium sp., confi rmed microscopically on some ser- tralian Plants Society Maroondah 2001). Dinemasporium sp. and F. oxysporum, in- rated tussock plants, but was greatest on Excessive moisture possibly did not allow fected N. trichotoma plants (Table 1). Of the six plants of other species that died. On for optimal plant growth, hence making 30 grass species inoculated, two were in- N. trichotoma and six plants of other grass these plants vulnerable to fungal attack fected with Arthrinium sp., two with Din- species, F. roseum was present on necrotic in conditions that were optimal for fungal emasporium sp. and 18 with F. oxysporum areas of leaves. Control plants did not de- growth. (Table 1). Little infection occurred to N. velop this secondary infection. Further work could be undertaken on F. trichotoma after inoculation with the fungi oxysporum to determine if a forma specialis in this trial with none of the plants dying Discussion for serrated tussock exists, since this fun- as a result of the inoculations. Infections to The three fungi used in this trial, Arthrin- gus has been reported in the past to exhibit the other grass species varied from none to ium sp., Dinemsporium sp. and F. oxyspo- pronounced host specifi city (Caesar et al. slight in plants inoculated with Arthrinium rum were neither host-specifi c nor highly 1999). F. oxysporum could exhibit greater sp. and Dinemasporium sp. Of the plants virulent to serrated tussock, as other grass virulence on N. trichotoma by manipulat- inoculated with F. oxysporum, basal rot- species became infected but no serrated ing the infection window and the patho- ting and death occurred in six plants of tussock plant died. Therefore, they have gen population (Charudattan and Dinoor four other grass species. None of the un- only limited potential as inundative bio- 2000). Further investigation into the use of treated control plants had visible infection logical control agents. this fungus could only be warranted for from any of the fungi used as inoculum, No plant death occurred in any plants consideration as an inundative application although one plant of A. nullanulla, died inoculated with Arthrinium species. It ap- on dense stands of serrated tussock. without visible foliar infection (Table 1). peared growth was not hindered in plants Serious consideration would need to be Arthrinium sp. infected all N. trichotoma inoculated with Arthrinium sp., although undertaken before initiating an inundative plants, with the development of dark- this was not measured. Arthrinium species biological control program with any of the brown to black fruiting bodies on the sur- have a saprophytic nature and are found fungi investigated. Further work needs to face of the foliage. The fruiting body was on numerous other plant species (Barnett be initiated to ensure these fungi were suf- often associated with a small necrotic ring. and Hunter 1998). There are no known bi- fi ciently pathogenic for mycoherbicide de- Dichelachne crinita and Poa poiformis were ological control programs that utilize Ar- velopment. If control of serrated tussock infected with Arthrinium (Table 1) but no thrinium species and a signifi cant amount was sought using any of these fungi, only death occurred in any inoculated plant. of further work would be required before areas where there was blanket coverage of Infection was confi rmed by the observa- this fungus could be considered for use in the weed and no surrounding vegetation tion of fruiting bodies; however there ap- an inundative biological control program. of grass species of importance, particular- peared to be no internal penetration of the Dinemasporium sp. was largely found ly Australian endemic grass species, could leaves on microscopic investigation. on senescent leaves and may merely fol- be considered. Dinemasporium sp. infected all N. tri- low, rather than cause, tip death in ser- chotoma plants, with only two out of 28 rated tussock. In the conidial state, Dine- Acknowledgments other genera and species infected, one masporium is found all year round on dead Julio Bonilla (technical assistant) kindly plant each of A. exilis and one A. mollis stems, sheaths and leaves of many grasses germinated all seeds, except serrated tus- (Table 1). Fruiting bodies developed on including Agrostis, Bromus, Festuca, Phala- sock, used in the trial. Seona Casonato was the plants; however, no necrotic areas de- ris and Poa (Ellis and Ellis 1985). As this supported by a partial scholarship from veloped in association with these and no fungus has previously shown promise as a CRC Weed Management Systems. Thanks plants died. The greatest development of biological control agent in serrated tussock to the staff at DPI Frankston and for the infection occurred on N. trichotoma plants, seed germination (Hussaini et al. 2000), use of the glasshouse facilities. Thanks to in which leaf blades died from the tip further research is required to investigate the two referees, Mike Manning and Kym when fruiting bodies were present on the its life cycle and potential as an inundative Butler for comments on drafts. same blade. agent for younger serrated tussock. F. oxysporum infected N. trichotoma and The plants inoculated and subsequent- References other grass genera. Twenty six out of 29 ly infected with F. oxysporum were visibly Anderson, F., Pettit, W., Briese, D.T. and plants from 16 Austrostipa spp. became in- rotting at the base, with many leaves be- McLaren, D.A. (2002). Biological con- fected with F. oxysporum, with four species ing chlorotic; these symptoms agree with trol of serrated tussock and Chilean having a plant die. Five out of 21 plants those in the literature, with F. oxysporum needlegrass. Plant Protection Quarterly from 11 species of other grasses were penetrating into the roots and causing root 17, 104-11. 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