WILL AUSTRALIAN ENDEMIC PATHOGENS WEAKEN THE MIGHT OF GIANT RAT’S TAIL (GRT) GRASS?

Joseph S. Vitelli1, David G. Holdom1, Roger G. Shivas1, Claire Lock1, Yu Pei Tan1, Kaylene Bransgrove1, Annerose Chamberlain1, Natasha Riding1,James Hosking1 and Brett Cawthray2

1Biosecurity Queensland, DAF, Ecosciences Precinct, Brisbane 2Gladstone Regional Council, Miriam Vale

May 2019

NSW BIOTASKFORCE GRT / Identification

• Genus contains ~ 200 species in tropical and subtropical areas of the world, including Africa, Asia, Australasia, North and South America • In Australia, 18 species are endemic (native) and further 6 species introduced (naturalised) • Only 4 native species (Sporobolus actinocladus, S. caroli, S. mitchellii and S. virginicus) are regarded as favourable fodder species due to their high protein-content • Three of the native species, S. disjunctus, S. latzii and S. partimpatens are considered rare, and • S. pamelae is listed as endangered in Schedule 2 of the Queensland Nature Conservation Act 1992 Peterson, P.M., Romaschenko, K., Arrieta, Y. H., and Saarela, J.M. (2014). A molecular phylogeny and new subgeneric classification of Sporobolus (: : Sporobolinae). Taxon, 63: 1212–1243 3 Sporobolus distribution in Australia

Source: Australia Virtual Herbarium 2019 Clade A. Sporobolus indicus complex (23-26 species)

Introduced Natives Others S. africanus (Southern and eastern Africa ) *S. minor (Southern America) S. atrovirens (Mexico) S. molleri (Africa) S. berteroanus, S. natalensis (Africa) S. blakei (Australia) S. myrianthus (Africa) S. creber (Australia) S. pectinellus (Africa) **S. elongatus (Australia) S. pseudairoides, (Southern America) S. farinosus (Guam) S. pyramidalis (Africa/Brazil) S. fertilis (Asia) *S. quadratus (Africa) S. festivus (Africa) S. scabriflorus *S. fourcadii (South Africa) S. sessilis (Australia) S. infirmus (Africa) S. stapfianus (Africa) S. jacquemontii (America) S. tenuissimus (Mexico and South America) S. laxus (Australia) S. trichodes (Mexico) Sporobolus indicus complex distribution 10 Species belonging to Sporobolus indicus complex

Source: Australia Virtual Herbarium 2019 Plasticity seen within GRT (S. natalensis)

7 Molecular phylogeny of Australian Sporobolus species

Utilising molecular tools to better target weedy Sporobolus classical biological control agents and effectively study the genetic diversity of Sporobolus • Conduct DNA studies against all Sporobolus grasses occurring in Australia to determine their relatedness to native species in Australia and to species in the native range 102 7

Sporobolus natalensis Sporobolus jacquemontii

Sporobolus actinocladus

Sporobolus mitchelli 9 Endemic pathogen surveys

10 Endemic pathogen surveys

11 12 Sterilisation of lesions

Pathogen isolation

1. Surface sterilise visibly infected GRT leaves with ethanol.

2. Excise tissue adjacent to lesions and place sections on an agar plate with antibiotics.

3. Subculture emergent fungi to create a pure culture.

4. Send a sample to Brisbane’s Pathology Herbarium (BRIP) for identification. 13 Which pathogen is causing disease symptoms?

Up to 20 foliar pathogens found on an infected plant

14 Promising endemic pathogen Host species BRIP No. Pathogen S. fertilis 66084 a Alternaria arborescens S. natalensis 66616, 68299, 69018 1Colletotrichum spp. Sporobolus sp. 68238/9, 68820 1Colletotrichum spp. S. jacquemontii 66086 a, 66087 a, 66085 a Curvularia ravenelii S. natalensis 66088 a Curvularia ravenelii S. natalensis 69020 Curvularia sp. S. natalensis 66081 a Exserohilum rostratum S. natalensis 65635 a 1Fusarium sp. S. natalensis 66083 a Fusarium proliferatum S. natalensis 68300 Gen. nov. S. natalensis 68298 1Microdochium spp. S. natalensis 65649, 67439a 1,2Microdochium sp. nov. S. natalensis 66617 1,2Neopestalotiopsis sp. nov. S. elongatis 68237 1Neopestalotiopsis sp. S. natalensis Nigrospora spp. S. natalensis 66619 Paraphaeosphaeria michotii S. natalensis 66615 1,2Pestalotiopsis sp. nov. S. natalensis 65632 a, 63688 a, b, c 1Phoma sp. S. natalensis 66618 1Septoria sp. S. natalensis 65638 a 1Stagonospora sp. S. natalensis 65466, 66039, 66324, 66325 Ustilago sporoboli-indici 1Species within this genus are known to be pathogenic grass fungi worldwide 15 Koch’s Postulate methodology

Inoculation & re-isolation

1. Harvest spores from agar plate using a Tween 20 solution.

2. Use a hemocytometer to measure spore concentration and adjust to 106 spores per mL of Tween 20.

3. Use a pipetter to apply 10uL of inoculum to each GRT seedling, focusing on the leaf axils (growth points).

4. Place the seedlings in a plastic cage under high humidity (28°C, 60% RH) for five days.

5. After 4 weeks, use the isolation methodology to extract and identify fungi from within the leaf.

16 BRIP 65649 BRIP 65649

Microdochium sp BRIP 65649': A colony on ½PDA after 2 weeks, B colony on ½PDA after 1 week, C conidiomata on ½PDA, D conidial masses, E conidiogenous cells, H conidia. Scale bars: 1 cm (A, B); 200 μm (C); all others – 10 μm.

Microdochium dawsoniorum BRIP 65649

Microdochium lycopodinum CBS 125585 98 96 Microdochium phragmitis CBS 285.71 Microdochium fisheri CBS 242.91 S. creber S. natalensis Microdochium nivale CBS 116205 62 Microdochium neoqueenslandicum CBS 108926

37 Microdochium novae-zelandiae CPC 29376 100 Microdochium chrysanthemoides LC5363 82 Microdochium colombiense CBS 624.94 59 63 Microdochium musae CBS 143499 Microdochium seminicola KAS3576 63 Microdochium citrinidiscum CBS 109067 Microdochium tainanense CBS 269.76 81 Microdochium trichocladiopsis CBS 623.77 Microdochium dawsoniorum BRIP 65649 100 Microdochium dawsoniorum BRIP 67439a Idriella lunata CBS 204.56 0.04 BRIP 66615

Pestalotiopsis sp. BRIP 66615’: A colony on ½PDA after 2 weeks, B S. creber S. natalensis colony on ½PDA after 1 week, C-D conidiomata on ½PDA, E-G conidiogenous cells, H conidia. Scale bars: 1 cm (A, B); 200 μm (C); 100 μm (D); all others – 10 μm.

18 . BRIP 66617

S. creber S. natalensis

Neopestalotiopsis sp. BRIP 66617: A colony on ½PDA after 2 weeks, B colony on ½PDA after 1 week, C-D conidiomata on ½PDA, E-G conidiogenous cells, H conidia. Scale bars: 1 cm (A, B); 100 μm (C); 200 μm (D); all others – 10 μm. 19 Lesion on Sporobolus natalensis leaf sheath 15 weeks post-inoculation with Neopestalotiopsis sp. BRIP 66617 20 Dead S. natalensis seedling one month after ‘Microdochium sp. BRIP 65649' inoculation (right), 'Microdochium sp. BRIP 65649' growth 10 days after re-isolation (left). Scale bars: 1 cm

21 Dead S. natalensis seedling one month after 'Pestalotiopsis sp. BRIP 66615' inoculation (right), 'Pestalotiopsis sp. BRIP 66615' growth 10 days after re-isolation (left). Scale bars: 1 cm

22 Dead S. natalensis seedling one month after 'Neopestalotiopsis sp. BRIP 66617' inoculation (right), 'Neopestalotiopsis sp. BRIP 66617' growth 10 days after re-isolation (left). Scale bars: 1cm

23 Sporobolus species germinant mortality associated with isolate treatment

Lock, C (2018). Investigation of fungal pathogens for the biological control of giant rat’s tail grass (Sporobolus natalensis) in Australia. Honours Research Thesis. Bachelor of Environmental Management The University of Queensland, Brisbane. 24 Phoma sp. (preference for mature ) Pathogen - black stem fungus from dying GRT grass – Fungus collected from GRT plants at Taunton – Identified genus level as Phoma, New recorded species, Based on ITS sequences – Phoma a ubiquitous (present everywhere), cosmopolitan fungus commonly found in soils – Pathogen causes leaf spot, foliar collapse and wilting of the entire plant – Wilted leaves and blackening shoots 1st observed at plant base, with symptoms progressing upward – Phoma known to overwinter in diseased material – Burning paddocks reduces the presences of Phoma GRT Endemic pathogen survey commenced February 2017 GRT paddock with minimal seed heads was found to contain a leaf smut

26 “Unthrifty” GRT plant with leaf smut

27 Leaf smut around Taunton – characterised with black lines on the leaf blades called sori

Sori

28 Leaf smut more prominent on young growth – from heavily grazed GRT plants or slashed plants

29 Burning encouraged new growth

Unburnt control

Leaf smut re- establishing on GRT plants 8 weeks post fire from a nearby paddock infested with leaf smut

30 Leaf smut (Ustilago sporoboli-indici)

Ruptured epidermis, releasing a powdery mass of blackish-brown, spores that are stuck together

Germinating spores in agar plate

Sporobolus stems (circled) with Leaf smut has been found on 17 formation of teliospores (black) properties across Taunton, Conondale, Gin Gin and Bundaberg. 31 GRT paddock with minimal seed heads GRT seed viability following leaf smut (Ustilago sporoboli-indici) infection (in progress) Immature flower heads infected with leaf smut have minimal-viable seed

33 GRT seed production following leaf smut infection

Inflorescence damage Inflorescence Length (cm) Seed production/inflor 4 (severely deformed) 3.3 <1 3 (stem inflorescence) 12.2 12 2 (“healthy”) 56 896 1 (control) 44.5 870

34 Summary • This is no silver bullet – but maybe the glimmer of hope • An integrated control program using herbicides, pasture competition, fertilising, pasture spelling will be required • Effective South African biocontrol agents may be found but in the meantime we have at least 1 leaf smut (Ustilalgo sporoboli-indici) and 40 promising pathogens in Australia that appear damaging • Koch’s Postulate has determined that 3 pathogens are pathogenic against GRT – more KP’s to do followed by pathogenicity testing • Mass producing the agent will hopefully be cheap and easy • Landholders noticing die-back of Sporobolus species are encouraged to contact Biosecurity Queensland to help identify potential pathogens

35 Thank you

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