The Graminicolous Downy Mildews

Taxonomy and identification
The downy mildews are a group of obligate fungal pathogens of vascular plants which belong in the kingdom Straminipila, phylum Heteroknota, class Peronosporomycetes, order Peronosporales, family Peronosporaceae.

Members of the Kingdom Straminipila possess, or have evolved from organisms that did possess, biflagellate zoospores which have an anterior flagellum bearing 2 rows of tripartite, tubular hairs (a straminipilous flagellum). Within the Straminipila, most modern taxonomists agree that although the species belonging in the Class Peronosporomycetes (traditionally referred to as Oomycetes) have many of the morphological and morphological features of the ‘true fungi’ which comprise the Kingdom Mycota, they are phylogenetically separate from that Kingdom.

The Order Peronosporales is separated from other Orders within the Peronosporomycetes by centripetal oosporogenesis (protoplasmic organisation subsequent to meiosis occurs at or near the centre of the oogonium) and a persistent periplasm (the outer protoplasm of the oogonium) which ultimately develops into the exospore wall (exosporium) of the oospore, and by well differentiated conidiophores or sporangiophores. In turn, the Family Peronosporaceae is characterised by fungi that infect only grass hosts (family Poaceae), produce large lobate haustoria (a specialised hyphal branch which absorbs food from host cells), and have unbranched, dichotamously or pseudo-dichotamously branched conidiophores or sporangiophores, which bear conidia or sporangia borne on pedicels.

There are over 700 described (not necessarily valid) species of downy mildew fungi throughout the world in 20 described genera (excluding Phytophthora), with most species being confined to particular host genera or families. Eight genera, Baobabopsis, Eraphthora, Graminivora, Peronosclerospora, Poakatesthia, Viennotia, Sclerophthora, and Sclerospora have been described on wild and crop grasses (F. Poaceae). Throughout the world many downy mildews cause economic losses of ornamental plants, horticultural crops, and broadacre grain, oilseed and pulse crops that require a combination of practices including plant resistance and fungicide applications for effective management.

Over 50 downy mildew species in 11 genera have been described in Australia, with 75% of species belonging in the genus Peronospora. Only 4 genera of grass-infecting downy mildew genera have been identified in Australia, namely Baobabopsis, Eraphthora, Peronosclerospora and Sclerophthora. Although some species of Peronosclerospora (eg., P. maydis, P. philippinensis and P.sorghi) and Sclerospora (S. graminicola) cause significant losses in maize, sorghum, pearl millet and other grass crops overseas, none of these species have been found in Australia.

Traditionally, the delimitation of downy mildew genera has been based primarily on the morphology of the asexual state (mainly the shape and branching of conidiophores or sporangiophores, the morphology of the ultimate spore-bearing cells, and shape and dimensions of the asexual spores) and secondly on that of the sexual state (mainly the dimensions and ornamentation of oogonia and the dimensions of oospores). Of the Australian grass downy mildew genera the asexual spore-bearing structures of Baobabopsis and Eraphthora are short and unbranched, but they differ in that those of Baobabopsis are club-shaped-cylindrical with conidia borne on ampulliform projections, while those of Eraphthora are obconical with sporangia borne on short pedicels. The sporangiophores of Sclerophthora are also short, but sympodially branched and bear 4-5 sporangia at the tips of branches. On the other hand, the conidiophores of Peronosclerospora species are long (>150 µm long), dichotomously branched and bear many conidia at the tips of the ultimate branches. The asexual spores of Eraphthora and Sclerophthora germinate by the release of motile zoospores (sporangia) whilst those of Peronosclerospora germinate by the production of 1 or occasionally 2 germ tubes (conidia). Spore germination in Baobabopsis has not been observed. Peronosclerospora (conidium-producing) was separated from Sclerospora (sporangium-producing) (Shaw 1978) on the basis of the mode of asexual spore germination, although the morphology of the spore-bearing structures is similar.

Within genera, species can be delimited by a combination of (i) the shape  of the conidia or sporangia (ii) the ornamentation of the oogonia (if any), (iii) the diameter of the oospores and (iv) for some species by the host. Some morphological characters, especially the dimensions of conidiophores, sporangiophores and asexual spores are unreliable for species identification because they are greatly influenced by the environment. The dimensions of oogonia are also unreliable because over time the oogonial wall slowly erodes and becomes thinner with longer exposure to the weather.

The integrated use of traditional and new morphological characters (eg., haustorium morphology) and molecular techniques has led to a revolution in the taxonomy of the DM fungi, with many new genera and species being described. Since 2000, 10 new downy mildew genera, including Baobabopsis, Eraphthora, Graminovora, Poakatesthia, and Viennota which are found exclusively on grasses have been described, based primarily on molecular data.

Symptoms and signs
On grasses, the first symptoms of infection are yellow spots, flecks or streaks which appear between the parallel veins of young leaves. At night a white downy growth, consisting of conidiophores or sporangiophores bearing the asexual spores, develops on these chlorotic areas. This evanescent white down is best seen in the early morning prior to sunrise .and is more commonly found on the lower leaf surface. Often the first infected leaves on an infected plant or tiller are only partly chlorotic, but on later leaves the entire blade becomes chlorotic. Leaves systemically infected with a downy mildew fungus tend to be wider than healthy leaves and are often held in an upright manner. Oogonia ultimately develop in the discoloured areas between the veins, and as they grow they split the leaf in some pathogen x host combinations, causing it to fray. Frayed leaves held high above the ground are a common symptom of downy mildew infection on wild, perennial grasses during the winter months in Australia (eg., Baobabopsis donbarrettii on Perotis rara, B. enneapogonis on Enneapogon spp., Eraphthora butleri on Eragrostis sp., Peronosclerospora noblei on Sorghum leiocladum, P. australiensis on Sorghum plumosum and Sorghum timorense and P. sargae on Sorghum timorense). However, there are no reports of frayed leaves on grasses infected by Sclerophthora macrospora. The oogonia of some species, eg., P. maydis have not been found.

Downy mildew infection can also cause other symptoms. Grasses infected by species of Peronosclerospora, Sclerospora and Sclerophthora may display the following - stunting (eg., Sclerophthora macrospora, Peronosclerospora sacchari), excessive tillering (eg., P. sorghi, Sclerophthora macrospora), elongation of culms (eg., P. sacchari), increase in node number and  upright infected leaves (P. eriochloae, P. noblei, P. sacchari), phyllody of the tassels and ears of maize and other grasses(P. australiensis, P. sacchari, Sclerophthora macrospora, Sclerospora graminicola), twisting of the upper leaves on culms (P. noblei, P. sacchari), and sterility. Investigations by Ryley (1985, 2001, 2002) and Ryley and Langdon (2001) revealed that most tillers of Sorghum leiocladum infected by P. noblei and of Eriochloa pseudoacrotricha infected by P. eriochloae are vegetative. Any inflorescences which develop on these DM-infected tillers are always small, underdeveloped and do not produce viable caryopses.

The downy mildew fungi survive in a variety of ways (i) fungal strands (hypha) in seeds (some species of Peronosclerospora, Sclerospora and Sclerophthora) (ii) hypha in vegetative plant parts (species of Peronosclerospora), (iii) oospores in infected plant residues and soil (species of Sclerophthora, Sclerospora, Peronosclerospora), and (iv) on/in alternative hosts  (species of Sclerophthora and Peronosclerospora).
Hypha in seeds and vegetative plant parts (eg., P. sorghi in sorghum seeds, P. maydis in maize seeds and P. sacchari in sugarcane planting setts) grow into the seedling or young plants and may spread throughout the plant, resulting in a systemic infection. Hyphae of some downy mildew species have been demonstrated to perennate in the tiller bases of perennial, wild grasses, including P. noblei on Sorghum leiocladum and P. eriochloae on Eriohloa pseudoacrotricha (Ryley 1985, 2001, 2002; Ryley and Langdon 2001).

Thick-walled oogonia which survive in the soil or in infected plant residues germinate, usually in response to exudates from the roots of a nearby host, by producing a germ tube (species of Peronosclerospora) or a sporangium (species of Sclerophthora). Germ tubes which grow from the germinated oospores of Peronosclerospora species grow towards the roots, directly penetrate the roots and invade the rest of the plant. Motile spores (zoospores) of Sclerophthora species released from the sporangia swim through soil water towards the host’s roots where they encyst. Then a germ tube penetrates the root surface and hyphae invade the roots. The subsequent invasion of the roots and shoots of the developing seedling usually results in a systemic infection.

Grass plants can also be infected after sporangia or conidia which develop on plants of another grass are transported by wind and deposited on aboveground parts. There they germinate rapidly in free water from dew or rain, with conidia producing short germ tubes and sporangia producing zoospores. The germ tubes from conidia or from zoospores grow through the openings of leaf stomata, and hyphae invade the sub-stomatal cavities then grow throughout the leaf and often into other plant parts.  Systemic infection can occur if hyphae grow into the apical meristem of the stem, otherwise the hyphae remain localised.

Overseas, all of the important downy mildew pathogens of grass crops, including P. maydis, P. philippinensis, P. sacchari, P. sorghi, Sclerophthora macrospora and Sclerospora graminicola have a wide host range, some including wild grasses. Sorghum halepense, a widespread weed throughout the world including Australia, is a host of P. sacchari and P. sorghi. In Australia, Sclerophthora macrospora has a wide host range including grasses growing in the wild, but no wild grass has been positively confirmed as an alternative host of P. sacchari in Australia. Sorghum timorense and Eriochloa pseudoacrotricha are believed to be the significant alternative hostsassociated with occasional outbreaks of P. australiensis and P. eriochloae on maize crops in northern Australia and southern Queensland respectively.

Asexual sporulation occurs at night when there is a layer of water on the leaf surfaces and temperatures are cooler. For example, Bonde et al., (1992) reported that the optimum temperature range for asexual sporulation of P. sorghi and P. phillipinensis is 18-23°C after 5-6 hours of dew. Other researchers report different optimum ranges, such as 24-26°C for P. sorghi and 21-26°C for P. phillipinensis (Smith and Renfro, 1999). The optimum temperature range for infection of leaves by conidia is similar to that for sporulation; for example (Smith and Renfro, 1999) reported a range of 21-25°C for P. sorghi and 19-20°Cfor P. phillipinensis. The sporangiophores and conidiophores grow from hyphae in the substomatal cavities, through the stomatal openings into the boundary layer above the leaf surfaces and other invaded plant parts and develop rapidly. Mature conidia and sporangia are released by pressure when rain drops hit the leaves or soon after rain ceases when the relative humidity is still relatively high. Ingold (1971) reported that at the flat point of attachment of the conidia of P. phillipinensis and the tips of the subtending pedicels both rapidly round off and the conidia are projected for a millimetre or so. There is a noted periodicity in spore release, which for most species occurs during the early morning hours before sunrise and only very rarely during the night. Spores are transported in air currents and under some circumstances may travel for many hundreds of kilometres. The sporangia and/or zoospores of some genera (eg., Sclerophthora) are spread in water. The conidiophores of all the graminicolous DMs except Graminivora, Poakatesthia and Viennotia are evanescent and shrivel up rapidly after the release of conidia/sporangia.

The production of the sexual state (oogonia and oospores) in leaves and other plant parts begins after asexual sporulation has ceased and as the plant parts begin to senescence. Similar to the asexual state, oospores develop only in those parts of the grass plants that have previously been colonised by hyphae (including roots, inflorescences, flower parts, and seeds for somedowny mildew species). Oogonia and oospores remain in the residues of plant part in which they are formed until those parts disintegrate. Then they are released into the soil where they can remain dormant for many years. The sexual spores can be spread in infected plant residues, seeds (eg., P. sorghi and P. maydis), vegetative organs and  infested soil by water, wind or mechanical means or directly by the wind (Peronosclerospora sorghi oospores in frayed leaves of Sorghum bicolor).

Downy mildew fungi have a unique place in the history of Plant Pathology. The first recognised and widely used fungicide in history, Bordeaux mixture, was developed by the French scientist Professor Pierre-Marie-Alexis Millardet after he made observations that grape vines sprayed with mixture of copper sulphate and lime or verdigres to discourage pilfering remained greener for longer than unsprayed vines. During his studies he found that sprays of Bordeaux mixture provided excellent control of grape downy mildew (caused by Plasmopara viticola) (Large, 2003). Fungicides still play an important role in the management of downy mildew diseases on horticultural and broadacre field crops and ornamentals, in addition to other management practices such as plant resistance, clean planting material, crop rotation and hygiene.

The first documented record of a graminicolous downy mildew in Australia was that of Sclerospora macrospora (= Sclerophthora macrospora) on leaves of Alopecurus (Cooke 1892), with the oldest herbarium record being that of the same fungus collected on oats (Avena sativa) in 1924. In 1929 the American William Henry Weston Jr (1890-1978) described Sclerospora noblei from specimens of oogonia attached to frayed leaves of the perennial, indigenous grass Sorghum plumosum (sic, now Sorghum leiocladum) collected near Glen Innes, New South Wales and sent to him by Robert Jackson Noble (1894-1981). Later, Weston (1942) described the asexual state, again from material sent to him by Noble. He noted that the conidia of S. noblei lacked the thickened, modified papillae for dehiscence of zoospores and that the spores germinated by germ tubes, considering that these features precluded any alliance with the sporangium-producing S. graminicola, the type species of that genus. On the basis of Weston’s observations, Shaw (1980) transferred S. noblei to Peronosclerospora.

Malcolm John Ryley’s (1953-) studies (Ryley, 1985, 2001, 2002; Ryley and Langdon 2001) on P. noblei on Sorghum leiocladum in southern Queensland confirmed its identity and also revealed critical aspects of its biology. He found that hyphae of P. noblei perennate in tiller bases of the grass during the winter months and invade some of the tiller buds in early spring when wild sorghum becomes active in response to rain events. Tiller buds that were not invaded developed into healthy, flowering tillers whilst tiller buds invaded by hyphae of P. noblei develop into systemically infected, vegetative tillers bearing wide, chlorotic leaves held in a bunched, upright manner. Downy mildew-infected tillers of S. leiocladum are thicker and have more nodes than healthy, flowering tillers. The hyphae invade the growing point of tillers and grow into the leaf primordia, and in the leaf sheaths and blades as they develop. The invaded parts of leaf blades become chlorotic with asexual sporulation occurring at night and only from this discoloured leaf tissue. After asexual sporulation ceases in chlorotic tissue, oogonia develop between the parallel vascular bundles within leaf blades, ultimately causing the blades to fray.

During a study of downy mildew fungi on perennial grasses between 1978 and 1981, Ryley and his PhD supervisor Raymond Forbes Newton Langdon (1917-2015) found a downy mildew on early spring grass (Eriochloa pseudoacrotricha) at several locations in southern Queensland. As the fungus differed in morphology (primarily the dimensions and shape of the conidia and oogonia) from all described species of Peronosclerospora, Ryley and Langdon (2001) named the fungus Peronosclerospora eriochloae. Recent molecular studies have confirmed that P. eriochloae is a valid species. Although Kubicek and Kenneth (1984) briefly described a new downy mildew fungus (Peronosclerospora globosa) that they had found on several Eriochloa species in Texas, USA, they never validly published the name according to rules of the International Code of Botanical Nomenclature. The progression of symptoms and activity of hyphae of P. eriochloae in plants of E. pseudoacrotricha is almost identical to that described above for P. noblei on Sorghum leiocladum (Ryley and Langdon 2001). Other downy mildew species which infect perennial grasses may also perennate and grow in a similar manner to that of P. eriochloae and P. noblei. Seedlings of E. psuedoacrotricha infected with P. eriochloae were commonly found growing in the wild; healthy seedlings of the grass were successfully inoculated with conidia of the pathogen and grew into systemically-infected plants.

Ryley and Langdon (2001) also provided descriptions of, and comments on, Queensland specimens of downy mildew fungi on an Eragrostis species and on Triodia pungens. The papillate oogonia of the fungus on Eragrostis were very similar to those described for the only other known downy mildew species on Eragrostis at that time, Basidiophora butleri. Telle and Thines (2012) re-examined this fungus and based on morphology and molecular features showed that this species differed significantly from the type species B. entospora, erecting the genus Eraphthora to accommodate the species butleri. Three downy mildew specimens on Eragrostis cilianensis from New South Wales that were included in Telle and Thine’s molecular study were confirmed as Eraphthora butleri. Examination of two specimens of the Triodia downy mildew fungus showed that the morphology of its sexual state was very similar that of P. noblei, but the authors did not speculate on its identity.

The genus Baobabopsis was described by Thines et al. (2015) based on molecular evidence and on the morphology of its asexual and sexual states, namely broad, club-shaped – cylindrical conidiophores with a cluster of terminal ampulliform projections bearing conidia, and oogoonia with a verrucose, warted outer wall (exosporium). Both Baobabopsis species described to date were found on indigenous grasses in northern Australia – B. donbarrettii on the annual or short-lived perennial Perotis rara and B. enneapogonis on perennial Enneapogon species Together with the Peronosclerospora species australiensis, eriochloae, noblei and sargae, the two Baobabopsis species are apparently restricted to Australia.
Downy mildew fungi have caused losses on grasses grown for crops in Australia. Although Sclerophthora macrospora has been recorded on crop grasses including barley, maize, oats, sorghum and wheat, losses have been negligible. Affected plants are usually found in low-lying, wetter areas of paddocks or in crops that have received high rainfall. Symptoms include excessive tillering, rolling and twisting of the upper leaves and phyllody in inflorescences, the latter effect often termed “crazy top”.

In his article on downy mildew disease of sugarcane and other grasses in Australia, Leece (1941) attributed an outbreak of a new disease on sugarcane growing in the Herbert River district of northern Queensland in 1901 to the downy mildew pathogen Sclerospora sacchari (now Peronosclerospora sacchari). He suspected that it had been introduced into Australia from cane consignments imported from New Guinea. In addition to providing a detailed account of the symptoms caused by P. sacchari on sugarcane, Leece reported that plants of maize and teosinte (Euchlaena mexicana) grown very near sugarcane plants infected with P. sacchari exhibited symptoms and signs of DM infection (leaf chlorosis and development of the asexual state). As a result of replacing susceptible sugarcane varieties with resistance ones and using disease-free planting material, P. sacchari was considered to have been eradicated from Australia in 1972 when the last experimental plots downy mildew-infected sugarcane plants were destroyed, although the last infected crop was recorded in 1959 (Plant Health Australia 2009).

Ramsey and Jones (1988) documented several detections of downy mildew on maize and sweet corn across northern Western Australia, the Northern Territory and northern Queensland prior to 1988, which at the time were variously attributed to either Peronosclerospora maydis or P. sorghi, two of the most serious downy mildew pathogens of maize and grain sorghum (Sorghum bicolor). During an outbreak of downy mildew on maize and sweet corn in 1985 which was attributed to P. maydis, Ramsey and Jones found the same downy mildew fungus on nearby plants of Sorghum plumosum, a wild perennial grass. The identifications of these downy mildew fungi on maize and sweet corn were based on the characteristics of the asexual states, which are known to be highly variable. Peronosclerospora australiensis and Peronosclerospora sargae were described in 2012 (Shivas et al., 2012) from specimens on wild, indigenous species of Sorghum collected during several surveys of tropical regions of northern Australia. Although the asexual state was not found for either species, molecular studies conducted on the oogonia from frayed leaves of their grass hosts revealed that they differed from all other known Peronosclerospora species. Molecular phylogenetic analysis has shown that two specimens of maize from the Northern Territory were part of a clade which included the type specimen of P. australiensis (Telle et al., 2010). There have been several subsequent outbreaks of downy mildew on maize in northern Australia, all shown to be caused by P. australiensis. The current evidence suggests that most, if not all, of these previous outbreaks of downy mildew on Zea mays in northern Australia were in fact due to P. australiensis which survived from season to season on the wild, indigenous Sorghum species.

In 1995, a limited outbreak of downy mildew on one maize line amongst many others in a breeder’s nursery in southern Queensland was attributed to an unknown species of Peronosclerospora, possibly P. maydis. Telle et al., (2010) used DNA sequence analysis to show that the causal fungus was actually P. eriochloae, which had not previously been recorded on maize. One of its hosts, Eriochloa psuedoacrotricha, is known from the immediate vicinity of that downy mildew outbreak, so it is highly probable that conidia of P. eriochloae that had developed on nearby plants of E. pseudoacrotricha infected the highly susceptible maize breeding line during a period of conducive weather.