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Go to Table of Contents DETECTION OF A POTENTIAL PATHOGEN, CAUSING AZORELLA DIE-BACK ON MAQUARIE ISLAND, USING NEXT-GENERATION SEQUENCING M. Glen A , T. Rudman B , Y.Z. Qing B , H. Achurch B , M. Dieckmann B and C.L. Mohammed A , A Tasmanian Institute of Agriculture. University of Tasmania, Private Bag 98, Hobart, Tas, 7001 Email: Morag.Glen@utas.edu.au B Department of Primary Industries, Parks, Water and the Environment. GPO Box 44, Hobart, Tas, 7001 ABSTRACT. Next-generation sequence technologies were used to characterise fungal bacterial and oomycete communities associated with healthy and die-back affected plants of Azorella maquariensis to determine whether a potential pathogen is associated with the die-back. A species of Rosellinia was the most frequently detected fungal species overall and was strongly associated with roots and rhizosphere soil of die-back affected plants. While Koch’s postulates must be fulfilled to demonstrate that this pathogen is a primary cause of the die-back on Macquarie Island, this finding provides strong evidence to support this hypothesis. Further attempts to isolate the pathogen responsible for Azorella dieback will include techniques or selective media suitable for this genus. INTRODUCTION Azorella macquariensis, a keystone species of Macquarie Island's feldmark habitat, has been adversely affected by dieback in recent years. The remote location and extreme climate of Maquarie Island increases the difficulty of isolating a potential pathogen. Attempts to isolate a pathogen associated with the dieback have failed, and it has been hypothesized that the dieback is a response to changes in climate. Recent advances in DNA sequencing technologies have increased the capacity to characterise fungal and microbial communities. Next-generation sequencing technologies were exploited to characterise fungal, oomycete and bacterial communities associated with healthy plants and those affected by dieback in an attempt to determine whether a potential pathogen was associated with dieback-affected plants. MATERIALS AND METHODS Sampling: 36 healthy and 36 die-back affected A. macquariensis were sampled from 20 sites across Macquarie Island, two to three replicate plants at each location. Rhizosphere soil from each plant was also sampled. DNA extraction, PCR and Sequencing: DNA was extracted from the roots, adventitious roots and leaves using previously described methods (Glen et al 2002). DNA was also extracted from rhizosphere soil using a MoBio Powersoil DNA extraction kit. Target gene regions (fungal rDNA ITS and beta-tubulin, oomycete rDNA ITS2 and bacterial 16S rDNA) were amplified using primers with different markers for healthy and diseased samples and for the different substrates. PCR products were pooled and sequenced on a Roche FLX 454 sequencer by Research and Testing Laboratories, Texas. Sequences were grouped into OTUs using CD-hit (Huang et al., 2010). Statistical analyses: PERMANOVA, canonical analysis of principal co-ordinates (CAP) and non-metric multidimensional scaling (MDS) analyses of microbial communities were carried out using PRIMER6 (Clarke and Gorley, 2006). Statistical analyses were based on percentage composition of OTUs in each sample. RESULTS Amplification was successful from all primer/sample combinations. A total of 87,065 sequences were obtained, though three samples (two from the β-tubulin data-set and one from the Oomycete data-set) produced no or very few sequences. Xylariaceae, also associated with roots and soil of diseased plants. More precise identification was not possible due to the lack of reference sequences with high sequence similarity. These two OTUs may represent the same species, but confirmation of this is dependent on obtaining an isolate and comparing both sequences. While precise species identification was not possible based on the sequence data, it is closely related to R. quercina and R. desmazierii. CAP analyses based on Euclidean distance and Bray-Curtis similarity gave concordant results, with roots having the most distinctive fungal communities, and roots from healthy plants quite distinct from those of diseased plants. The Rosellinia sp. was a major contributor to differentiation of the fungal community from diseased roots. An oomycete species, unable to be identified more accurately, was also associated with diseased roots, but was detected at much lower abundance. DISCUSSION From the CAP analysis of the fungal datasets, one fungal species is strongly associated with roots of diseased plants and is also present in the soil around these roots. The genus Rosellina includes the well-known and wide-spread plant pathogen R. necatrix, that is a serious pathogen of several fruit and nut tree species. R. desmazieresii is considered more of a saprophyte that can become parasitic. No species of Rosellinia has been isolated from the Azorella maquariensis plants so no pathogenicity tests can be conducted until an isolate is obtained, but this species is considered a likely cause of the dieback. No other candidate fungus, oomycete or bacterial species is so strongly associated with diseased roots. Further isolation attempts, particularly targeting Rosellinia spp., are a high priority for future work. As the analysis described here was based on pooled data, it is not known whether the Rosellinia sp. was present in all diseased root samples. The next steps will therefore include the design of specific primers and qPCR of individual root and soil DNA samples to ascertain that the Rosellinia sp. is consistently present. Samples from Marion Island, where a similar dieback is affecting another Azorella species, will also be tested to determine whether the same species is also present there. REFERENCES Clarke, K.R., Gorley, R.N., 2006. PRIMER v6: User Manual/Tutorial. PRIMER-E, Plymouth. The most abundant OTU in the fungal rDNA ITS dataset Huang, Y., Niu, B., Gao, Y., Fu, L. and Li, W. (2010) CD-HIT was a species of Rosellinia (Xylariaceae), that was strongly Suite: a web server for clustering and comparing associated with roots and soil from diseased plants. The most abundant OTU in the β-tubulin dataset was a species of biological sequences. Bioinformatics 26; 680. 7th Australasian Soilborne Diseases Symposium 17
Go to Table of Contents RHIZOCTONIA SOLANI AG8 INOCULUM LEVELS IN AUSTRALIAN SOILS ARE INFLUENCED BY CROP ROTATION AND SUMMER RAINFALL V.V.S.R. Gupta A , A. McKay C , S. Diallo A , D. Smith C , A. Cook D , J. Kirkegaard B , K. Ophel-Keller C , W. Davoren A , R. Llewellyn A and D.K. Roget E A CSIRO Ecosystem Sciences, Urrbrae, 5064 SA, B CSIRO Plant Industry, Canberra ACT 2601 C SARDI, Urrbrae, 5064, SA, D SARDI, Minnipa, SA; E formerly CSIRO Email: Gupta.Vadakattu@csiro.au ABSTRACT Rhizoctonia solani Kühn AG-8 causes seedling diseases in a wide range of cereal, legume and oilseed crop plants. The expression of Rhizoctonia disease in any specific field is a result of management and environmental factors that influence the level of pathogen inoculum, inherent suppressive activity, N availability and crop/root vigour. Changes in the pathogen inoculum DNA level both in-crop and off-season were measured in field experiments in SA and NSW. Inoculum levels generally increased within cereal crops whereas non-cereal rotation options either reduced or caused no change. Inoculum levels were consistently lowest after canola or mustard in all sites and experiments. The effect of rotation on Rhizoctonia inoculum levels lasted for one crop season. Multiple rainfall events during summer can reduce inoculum levels from high to low disease risk. The identification that brassica oilseeds can provide an effective control of Rhizoctonia provides growers with the first documented rotational option for Rhizoctonia disease control. INTRODUCTION Rhizoctonia bare patch caused by Rhizoctonia solani Kühn AG-8 is a seedling disease of a wide variety of crops which decreases root length resulting in reduced plant growth and yield losses. Previous research has given us a variety of management options that may reduce disease incidence in direct drill systems and some weed management options to remove inoculum sources. However, Rhizoctonia bare patch disease is still causing significant losses to production in cereals, in particular in the southern Australian dry land crops, and has increased in recent dry seasons. R. solani fungus grows on soil organic matter and produces a hyphal network in the surface soil (1). It has been the previous inability to define and link the various edaphic, plant and environmental factors that has limited the predictability and management of this disease. Improved capabilities including DNA (inoculum and communities) and biochemical (catabolic diversity) techniques will now allow us better measure the changes in pathogen and soil microbial communities and link them to disease incidence. We investigated the effect of management and environmental factors on inoculum levels under different rotation and tillage systems. MATERIALS AND METHODS Surface soil (0-10cm) samples were collected from selected crop rotation and tillage treatments in multi-year field experiments (2008-2011) at Waikerie (Alfisol) and Streaky Bay (Calcarosol) and Karoonda (Calcarosol) in SA and Galong (Red Brown Earth) in NSW. Samples were collected at monthly intervals in crop and after crop harvest. Soils were analysed for R. solani AG8 DNA concentration (PredictaB ® , SARDI, RDTS), microbial activity, dissolved organic C and mineral N levels. RESULTS and DISCUSSION R. solani DNA levels were dynamic both within the crop season and during summer (Table 1). Results from more than 15 field experiments over 3 crop seasons showed a significant build-up of the inoculum in cereal crops (wheat, barley and cereal rye), whereas, in the non-cereal crops inoculum levels were either reduced or no change was observed (Figure 1). R. solani DNA concentrations generally decreased during summer in all the treatments and at all four sites. In the absence of host plants, summer rainfall events of >25mm in a week substantially reduce the level of inoculum, mostly due to microbial competition, whereas inoculum levels can increase during dry periods lasting 4 or more weeks. Soil microbial activity at sowing had a strong influence on the level of disease incidence (data not shown). R. solani AG8 (pg DNA/g soil) 1600 1400 1200 1000 800 600 400 200 0 Continuous Wheat NT Continuous Wheat Cultiv Whet after fallow NT Wheat after Canola NT Wheat after Pasture NT Canola NT Pasture NT Fallow NT May July September October December Figure 1. Effect of crop type and fallowing on the buildup of R. solani AG8 DNA in a field experiment at Streaky Bay in SA. Dashed line represent inoculum level considered high disease risk. Table 1. Effect of rotation crops, fallowing and summer rainfall on the concentration of R. solani AG8 DNA (pg / g soil) in soils. Crop / Treatment # sites (soils) No. of seasons Inoculum level at harvest compared to sowing Inoculum decline with summer rainfall Wheat 4 (3) 3 Increased (***) Sig Barley 2 2 Increased (***) Sig Cereal Rye 1 / 3 2 Increased (***) Mod Canola 4 (3) 3 Decreased (***) Sig Mustard 1 (2) 1 Decreased (***) Sig Peas 1 (2) 1 Decreased (*) Highly sig Lupins 1 (3) 2 Increased (**) Highly sig Decreased/no Medic pasture 4 (3) 3 change (*)& Sig (variable) Decreased/no Fallow 2 3 change (*)& Sig (variable) NB: *=10-fold; &=affected by the presence of grasses. ACKNOWLEDGEMENTS Financial support was provided by Grains RDC and host institutions of researchers. REFERENCES 1. Neate, S. M. (1987). Plant debris in soil as a source of inoculum of Rhizoctonia in wheat, Transactions of the British Mycological Society 88: 157-162. 2. Gupta, V.V.S.R. et al. (2011). Principles and management of soil biological factors for sustainable rainfed farming systems. In ‘Rainfed farming systems’, eds. P Tow et al. (Springer Science and Business Media) pp. 149-184. 7th Australasian Soilborne Diseases Symposium 18
Page 2 and 3: The following organisations sponsor
Page 4 and 5: Welcome to the Seventh Australasian
Page 6 and 7: 7 th Australasian Soilborne Disease
Page 8 and 9: THURSDAY 20 SEPTEMBER 08:30 Chair -
Page 10 and 11: TABLE OF CONTENTS KEYNOTE SPEAKERS
Page 12 and 13: ANTI-FUNGAL METABOLITES PRODUCED BY
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Page 16 and 17: Go to Table of Contents BENEFICIAL
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Page 20 and 21: Go to Table of Contents THE ROLE OF
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Page 58 and 59: Go to Table of Contents RHIZOCTONIA
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Page 62 and 63: Go to Table of Contents SEED POTATO
Page 64 and 65: Go to Table of Contents APPLICATION
Page 66 and 67: Go to Table of Contents EVALUATION
Page 68 and 69: Go to Table of Contents MANAGEMENT
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Go to Table of Contents WHAT IS THE
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Go to Table of Contents THE PATHOGE
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Go to Table of Contents ASSESSMENT
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Go to Table of Contents BREAK CROPS
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Go to Table of Contents Miyan, S. 2
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