Effective, consistent, and rapid non-contact application methods for seedling basal stem infection by Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum (Lib.) de Bary, an economically devastating soil-borne fungal pathogen known to cause disease across a wide range of plants, produces long-term inoculum called sclerotia that can either germinate carpogenically by ascospores infecting above-ground plant parts or myceliogenically to infect stem base and roots. Typically for research purposes S. sclerotiorum diseases are initiated by direct contact methods, using S. sclerotiorum mycelium agar plugs wrapped around the stem or sclerotia placed directly beneath root mass. However, reproducible non-contact methods leading to basal stem infection are not currently available. Therefore, the objective of this study was to develop effective non-contact protocols that consistently generate basal plant stem infection from S. sclerotiorum in the soil. Using three host plant species (canola, lupin, and lettuce) we determined two methods that reliably produced basal stem infection. The first method, where mycelial agar plugs were positioned just below the soil surface at a distance of 5 mm from each seedling, led to 100% infection in all plants. The second method used pathogen-infested soil by mixing the soil with dry inoculum in the form of a powder prepared from mycelium-colonized organic substrates. Four substrates consistently produced 100% seedling infection at four days after inoculation (DAI); wheat bran, wheat grain, red rice, and hulled millet. In contrast, chia, canary, sesame, and ryegrass seed substrates resulted in less than 50% seedling infection at 10 DAI and infection levels did not progress further. The two soil inoculation methods outlined in this study will enhance future research on the progression of S. sclerotiorum diseases, with the potential to screen disease-resistant host genotypes to basal S. sclerotiorum infection, and in particular to test the effectiveness of soil applications of fungicides or biocontrol agents against S. sclerotiorum basal infection.

Impact of Preconditioning Temperature and Duration Period on Carpogenic Germination of Diverse Sclerotinia sclerotiorum Populations in Southwestern Australia.

The soilborne pathogen Sclerotinia sclerotiorum (Lib.) de Bary is the causal agent of Sclerotinia stem rot, a severe disease of broad-leaf crops including canola/rapeseed (Brassica napus) that can result in significant yield losses. Sclerotia, the hard melanized resting structure of the pathogen, requires preconditioning before carpogenic germination can occur. We investigated the effect of preconditioning temperature (4, 20, 35, 50°C, and field conditions) and duration (0, 30, 60, 120, 179, 240, and 301 days) on germination of S. sclerotiorum sclerotia collected from five canola fields in the southwestern Australia grain belt. The ecological diversity of each population was characterized using mycelial compatibility group (MCG) typing. No response was observed for isolates conditioned at 4°C at any time period, indicating that chilling is not a preconditioning requirement for these isolates. Sclerotia required preconditioning for a minimum of 60 days before any significant increase in germination occurred, with no further increases in germination recorded in response to longer conditioning after 60 days. The highest germination was observed in sclerotia conditioned at 50°C. The MCG results indicated significant diversity within and between populations, suggesting local adaptation to different environments as well as ensuring the ability to respond to seasonal variation between years.

Crop-Zone Weed Mycobiomes of the South-Western Australian Grain Belt.

In the absence of a primary crop host, secondary plant hosts may act as a reservoir for fungal plant pathogens of agricultural crops. Secondary hosts may potentially harbor heteroecious biotrophs (e.g., the stripe rust fungus Puccinia striiformis) or other pathogens with broad host ranges. Agricultural grain production tends toward monoculture or a limited number of crop hosts over large regions, and local weeds are a major source of potential secondary hosts. In this study, the fungal phyllospheres of 12 weed species common in the agricultural regions of Western Australia (WA) were compared through high-throughput DNA sequencing. Amplicons of D2 and ITS were sequenced on an Illumina MiSeq system using previously published primers and BLAST outputs analyzed using MEGAN. A heatmap of cumulative presence-absence for fungal taxa was generated, and variance patterns were investigated using principal components analysis (PCA) and canonical correspondence analysis (CCA). We observed the presence of several major international crop pathogens, including basidiomycete rusts of the Puccinia spp., and ascomycete phytopathogens of the Leptosphaeria and Pyrenophora genera. Unrelated to crop production, several endemic pathogen species including those infecting Eucalyptus trees were also observed, which was consistent with local native flora. We also observed that differences in latitude or climate zones appeared to influence the geographic distributions of plant pathogenic species more than the presence of compatible host species, with the exception of Brassicaceae host family. There was an increased proportion of necrotrophic Ascomycete species in warmer and drier regions of central WA, compared to an increased proportion of biotrophic Basidiomycete species in cooler and wetter regions in southern WA.

Carpogenic Germinability of Diverse Sclerotinia sclerotiorum Populations Within the Southwestern Australian Grain Belt.

Sclerotinia stem rot, caused by the necrotrophic plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary, is a major disease of canola and pulses in Australia. Current disease management relies greatly on cultural and chemical means of control. Timing of fungicide applications remains a challenge, because efficacy is dependent on accurate prediction of ascospore release and presence on the plant. The aims of this study were to determine the optimal temperature for carpogenic germination of S. sclerotiorum populations sampled from canola and lupin fields in southwestern Australia and characterize diversity using mycelial compatibility groupings (MCGs). Sclerotia were collected from four diseased canola and one diseased lupin field from across southwestern Australia. Forty sclerotia from each population were incubated at four alternating temperatures of 30/15, 20/15, 20/4, and 15/4°C (12-h/12-h light/dark cycle) and assessed every 2 to 3 days for a 180-day period. MCG groupings for populations were characterized using 12 reference isolates. Results indicated the time to initial carpogenic germination decreased as diurnal temperature fluctuations decreased, with a fluctuation of 5°C (20/15°C) having the most rapid initial germination followed by 11°C (15/4°C) followed by 16°C (20/4°C). Optimal germination temperature for all five populations was 20/15°C; however, population responses to other diurnal temperature regimes varied considerably. No germination was observed at 30/15°C. MCG results indicate extensive diversity within and between populations, with at least 40% of sclerotia within each population unable to be characterized. We suggest that this diversity has enabled S. sclerotiorum populations to adapt to varying environmental conditions within southwestern Australia.

Potential distribution of the Australian native Chloris truncata based on modelling both the successful and failed global introductions.

Our aim was to model the current and future potential global distribution of Chloris truncata (windmill grass) based on the plant's biology, soil requirements and colonisation success. The growth response of C. truncata to constant temperatures and soil moisture levels were measured and estimated respectively, to develop parameters for a CLIMEX bioclimatic model of potential distribution. The native distribution in eastern Australia and naturalised distribution in Western Australia was also used to inform the model. Associations with soil types were assessed within the suitable bioclimatic region in Australia. The global projection of the model was tested against the distribution of soil types and the known successful and failed global introductions. The verified model was then projected to future conditions due to climate change. Optimal temperature for plant development was 28°C and the plant required 970 degree-days above a threshold of 10°C. Early collection records indicate that the species is native to Queensland, New South Wales and Victoria. The plant has been introduced elsewhere in Australia and throughout the world as a wool contaminant and as a potential pasture species, but some of the recorded establishments have failed to persist. The CLIMEX model projected to the world reflected effectively both the successful and failed distributions. The inclusion of soil associations improved the explanation of the observed distribution in Australia, but did not improve the ability to determine the potential distribution elsewhere, due to lack of similarity of soil types between continents. The addition of a climate change projection showed decreased suitability for this species in Australia, but increased suitability for other parts of the world, including regions where the plant previously failed to establish.