WILL AUSTRALIAN ENDEMIC PATHOGENS WEAKEN

THE MIGHT OF GIANT RAT’S TAIL (GRT) GRASS?

NSW BIOTASKFORCE

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, Brisbane2Gladstone Regional Council, Miriam Vale

May 2019

• Genus Sporobolus 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

GRT Taxonomy / Identification

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Peterson, P.M., Romaschenko, K., Arrieta, Y. H., and Saarela, J.M. (2014). A molecular phylogeny and new subgeneric classification of Sporobolus(Poaceae: Chloridoideae: Sporobolinae). Taxon, 63: 1212–1243

Sporobolus distribution in Australia

Source: Australia Virtual Herbarium 2019

Clade A. Sporobolus indicus complex (23-26 species)

*S. minor (Southern America)S. molleri (Africa)S. natalensis (Africa)S. myrianthus (Africa)S. pectinellus (Africa)S. pseudairoides, (Southern America)S. pyramidalis (Africa/Brazil)*S. quadratus (Africa)S. scabriflorusS. sessilis (Australia)S. stapfianus (Africa)S. tenuissimus (Mexico and South America)S. trichodes (Mexico)

Introduced Natives OthersS. africanus (Southern and eastern Africa )S. atrovirens (Mexico)S. berteroanus, S. blakei (Australia)S. creber (Australia)**S. elongatus (Australia)S. farinosus (Guam)S. fertilis (Asia)S. festivus (Africa)*S. fourcadii (South Africa)S. infirmus (Africa) S. jacquemontii (America)S. laxus (Australia)

Sporobolus indicus complex distribution 10 Species belonging to Sporobolus indicus complex

Source: Australia Virtual Herbarium 2019

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Plasticity seen within GRT (S. natalensis)

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

Molecular phylogeny of Australian Sporobolus species

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Sporobolus natalensis

Sporobolus jacquemontii

Sporobolus actinocladus

Sporobolus mitchelli

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Endemic pathogen surveys

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Endemic pathogen surveys

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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 Plant Pathology Herbarium (BRIP) for identification.

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Up to 20 foliar pathogens found on an infected plant

Which pathogen is causing disease symptoms?

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Host species BRIP No. PathogenS. fertilis 66084 a Alternaria arborescensS. natalensis 66616, 68299, 69018 1Colletotrichum spp. Sporobolus sp. 68238/9, 68820 1Colletotrichum spp. S. jacquemontii 66086 a, 66087 a, 66085 a Curvularia raveneliiS. natalensis 66088 a Curvularia raveneliiS. natalensis 69020 Curvularia sp.S. natalensis 66081 a Exserohilum rostratumS. natalensis 65635 a 1Fusarium sp.S. natalensis 66083 a Fusarium proliferatumS. 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 michotiiS. 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

Promising endemic pathogen

1Species within this genus are known to be pathogenic grass fungi worldwide

Host species BRIP No. PathogenS. fertilis 66084 a Alternaria arborescensS. natalensis 66616, 68299, 69018 1Colletotrichum spp. Sporobolus sp. 68238/9, 68820 1Colletotrichum spp. S. jacquemontii 66086 a, 66087 a, 66085 a Curvularia raveneliiS. natalensis 66088 a Curvularia raveneliiS. natalensis 69020 Curvularia sp.S. natalensis 66081 a Exserohilum rostratumS. natalensis 65635 a 1Fusarium sp.S. natalensis 66083 a Fusarium proliferatumS. 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 michotiiS. 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

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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.

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Microdochium lycopodinum CBS 125585

Microdochium phragmitis CBS 285.71

Microdochium fisheri CBS 242.91

Microdochium nivale CBS 116205

Microdochium neoqueenslandicum CBS 108926

Microdochium novae-zelandiae CPC 29376

Microdochium chrysanthemoides LC5363

Microdochium colombiense CBS 624.94

Microdochium musae CBS 143499

Microdochium seminicola KAS3576

Microdochium citrinidiscum CBS 109067

Microdochium tainanense CBS 269.76

Microdochium trichocladiopsis CBS 623.77

Microdochium dawsoniorum BRIP 65649

Microdochium dawsoniorum BRIP 67439a

Idriella lunata CBS 204.56 0.04

Microdochium dawsoniorum BRIP 65649

BRIP 65649

S. creber S. natalensis

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.

BRIP 65649

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S. creber S. natalensisPestalotiopsis sp. BRIP 66615’: 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); 200 μm (C); 100 μm (D); all others – 10 μm.

BRIP 66615

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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.

. BRIP 66617

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Lesion on Sporobolus natalensis leaf sheath 15 weeks post-inoculation with Neopestalotiopsis

sp. BRIP 66617

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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

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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

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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

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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.

– 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

Phoma sp. (preference for mature plants)

Pathogen - black stem fungus from dying GRT grass

GRT Endemic pathogen survey commenced February 2017 GRT paddock with minimal seed heads was found to contain a leaf smut

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“Unthrifty” GRT plant with leaf smut

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

Sori

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

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

Burning encouraged new growth

Unburnt control

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Leaf smut (Ustilago sporoboli-indici)

Germinating spores in agar plate

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

Sporobolus stems (circled) with

formation of teliospores (black)

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Leaf smut has been found on 17 properties across Taunton, Conondale, Gin Gin and Bundaberg.

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

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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

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• 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

Summary

Thank you

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