Tan (syn. yellow) spot (TS) is an economically important disease of wheat and causes significant crop losses worldwide [1]. The causal agent of TS is the ascomycete fungus Pyrenophora tritici-repentis (Ptr) which feeds and kills off the host tissue, thereby forming the characteristic necrotic and/or chlorotic spot-like lesions [2]. Yield losses can peak as high as 50 % on susceptible varieties and results in global industry impacts with annual losses estimated to exceed US$150 million dollars in Australia alone and TS ranking consistently in the top three most damaging fungal wheat pathogens in the USA [3–5]. Moreover, it has been reported that Ptr has also been isolated from other cereal crops such as barley and other grasses [6, 7]. This emphasizes the importance of identifying and understanding the pathogenicity mechanisms between Ptr and its hosts as to reduce the damage caused by this economically important plant pathogen.
Ptr secretes necrotrophic effectors (NE) during infection to kill the host tissue [8]. The necrosis and/or chlorosis will only manifest itself if the host carries the corresponding susceptibility gene, in accordance to the ‘inverse’ gene-for-gene pathosystem model [9]. Thus far three NEs have been characterized in Ptr, namely ToxA, ToxB and ToxC [6, 10]. Whereas ToxA and ToxB are small secreted proteins, ToxC is a metabolic effector and each NE induces specific phenotypic disease symptoms [10–12]. ToxA induces dark necrotic lesions occasionally associated with a chlorotic halo, whilst ToxB induces chlorosis as does ToxC, with the latter able to spread the chlorosis through the leaf [13, 14]. These disease symptoms will only effectuate on those wheat lines carrying the dominant susceptibility genes which are Tsn1, Tsc2 and Tsc1 that interact with ToxA, ToxB and ToxC, respectively [15]. Through the identification of three differential wheat lines, each carrying one of these susceptibility genes, it was now possible to globally classify and distinguish eight races of Ptr [16]. The classification of Ptr into race 1 to 8 is based on the Ptr NE profile and their disease scoring in these three differential wheat lines (Glenlea, 6B662, 6B365) [1]. The molecular knowledge gained within the Ptr-wheat pathosystem and the establishment of a global race structure now allows for the screening of host genetic resistance and the development of more effective and sustainable control measures [17].
Besides the progress made in untangling the complex NE regulation [18, 19], valuable genomic resources have been generated of multiple Ptr isolates across different races. For example, a high quality Ptr reference genome of the Australian race 1 isolate M4 (ToxA, ToxC) was assembled and annotated using PacBio sequencing technology [20]. In addition, seven pathogenic Ptr isolates, including the race 5 DW5 isolate (ToxB) from the USA, were sequenced using Illumina paired-end reads [14]. Along with the ability to genetically transform this fungal pathogen [12, 18], these attributes make Ptr an ideal species for virulence studies.
Inoculation experiments with asexual spores (or conidia) form a cornerstone in the research field of many fungal-plant pathosystems. This includes the need to carry out disease resistance screening [17, 21], comparative phenotypic assays [22], isolate maintanence, quantitative trait locus analyses of biparental populations and genome wide association studies of diversity panels [23, 24]. Already in 1977, the effects of substrate, temperature and photoperiod on sporulation of Ptr were studied [25]. Odvody and Boosalis (1981) reported a sporulation technique which involved carrying out an initial culture on potato-dextrose agar, transferring mycelial plugs onto V8 agar followed by induction of conidiophore production under a light source emitting wavelengths of less than 350nm, crucial for subsequent conidia production [26]. This method was further simplified by Raymond et al. (1985) by including both a PDA and V8 sector in the same petri dish to help induce conidiophore production [27]. It was not until Lamari and Bernier (1989) formulated their inoculum production method in 1989 which combined these culture media to a single V8-PDA agar, that this would become a commonly used method to collect Ptr conidia [28]. The Lamari/Bernier method is a modification of Raymonds et al. with the main adaptations being the use of a single medium (V8-PDA), growing the mycelial cultures in continuous darkness as well as further growing the transferred single plugs per petri dish in the dark. Conidiophore formation was induced by flooding the grown colony with water and flattening of the mycelia followed by a 24 light incubation. A subsequent dark incubation at 15°C induced conidia formation. However, this approach remained time-consuming and labour-intensive to harvest sufficient conidia numbers for applications such as infection assays [21]. To overcome this limitation, Dinglasan et al. (2016) raised conidia by overlaying infected wheat stubbles on a TS-susceptible wheat variety followed by overhead watering to generate a substantial amount of conidia (2x105 conidia in 20 ml of spore suspension) for infection studies. Despite the high inoculum number, the production of conidia using live plants from stubble-borne infection is time intensive and non-axenic, thus run the risk of containing co-infecting fungal pathogens of wheat [29].
In this study, we set out to develop an optimized sporulation protocol using in vitro culturing of Ptr that is simple, time-efficient and can consistently generate high spore inoculum numbers, free of mycelial/microbial contaminations. To efficiently harvest the conidia raised through this optimised approach, we devised a simple harvesting strategy by taking advantage of the hydrophobic property of fungal conidia. By adopting these modified approaches, we were able to devise a simple protocol that repeatedly generates three orders of magnitude more conidia per vegetative petri dish (3x106) compared to the Lamari and Bernier-based method [28]. Moreover, no mycelial contamination could be observed in the conidial extract. Here, we describe the modifications and optimisations that led to our inoculum production method and evaluate this optimized protocol on a range of Ptr isolates. We also tested this improved method on taxonomically related fungal pathogens of barley that causes net form diseases of barley, Pyrenophora teres f. teres and f. maculata, and similarly observed significant improvements in conidia production and purity.