An unexpected diversity of powdery mildew species infecting the Fabaceae in Australia

Abstract

The Fabaceae family has been reported to host more than fifty species of powdery mildew worldwide. Despite being commonly found on fabaceous hosts throughout Australia, the accurate identification of many powdery mildew species remains uncertain. The objective of this study was to identify powdery mildew species that naturally occur on fabaceous hosts in Australia and provide insight into those native and weedy species that may host crop pathogens and contribute to disease in cropping systems. The ribosomal DNA internal transcribed spacer (ITS) sequences and morphology of 34 fresh and 40 herbarium powdery mildew specimens infecting diverse Fabaceae species in Australia were characterised in this study. Altogether, a total of eleven powdery mildew species were identified from 51 Fabaceae species. Podosphaera xanthii was the most common powdery mildew in this study and was detected on 18 host species across ten genera. Ten species of Erysiphe were confirmed on 37 host species covering 17 host genera, with E. diffusa and E. cf. trifoliorum the most prevalent. This work provides the most comprehensive catalogue of powdery mildew species infecting legume hosts throughout Australia.

Introduction

The Fabaceae, commonly known as legumes, are one of the largest and economically important plant families worldwide, comprised of more than 640 genera and 18,000 species [1]. Legumes are widely distributed across every continent except Antarctica, occurring in almost every habitat type [1], and are known to improve soil fertility through their symbiosis with nitrogen-fixing bacteria [2]. Grain legumes, known as pulses, and forage legumes provide an important food source to humans and animals worldwide [2]. Several pulses are economically important broad-acre, horticultural or pasture crops in Australia, including common bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), faba bean (Vicia faba L.), vetch (Vicia spp.), mungbean (Vigna radiata [L.] Wilczek), black gram (V. mungo [L.] Hepper), cowpea (V. unguiculata [L.] Walp.), adzuki bean (V. angularis [Willd.]), soybean (Glycine max [L.] Merr.), lupin (Lupinus angustifolius [L.]), pigeonpea (Cajanus cajan [L.]), and various medics (Medicago spp.). The inclusion of these legume crops in the agricultural system provides multiple benefits to growers when grown in rotation with cereals, such as minimizing the build-up of cereal pathogen populations, improving soil fertility and biodiversity, and providing an additional source of income [3]. Several introduced Fabaceae have become significant weeds throughout Australia [4]. One example is Macroptilium lathyroides, commonly known as phasey bean, which was originally introduced as a pasture legume but now commonly grows throughout urban and regional habitats [5].

The land flora of Australia is unique as it includes approximately 11,119 native species that have evolved largely in isolation during the past 100 million years, following the separation of the Australian continent from other lands [6–7]. More than twice the number of native plant species were introduced to Australia as crops, pasture species, ornamentals, and also inadvertently as weeds, since the beginning of European colonization of the continent in the late 1700s [6–7]. During the past 5–10,000 years, and possibly also earlier, new plant species may have entered Australia naturally, as well, through land bridges that were formed between the continent and some of the neighbouring lands due to low sea levels during ice ages [8]. Some legume species are considered as Australian natives while others, including all commercially grown pulses, have been introduced since the first European settlers arrived in Australia [6].

The full life cycle of most powdery mildews includes an asexual (anamorph) and a sexual (teleomorph) stage [9]. During the asexual life cycle, airborne conidia are produced in large numbers to allow the pathogen to spread across wider regions and infect the green tissues, and sometimes also other aerial tissues of their living host plants if available in the environment [10]. Many species are also capable of sexual reproduction, whereby hyphae of opposite mating types fuse, and produce the sexual fruiting bodies (the sexual morphs) known as chasmothecia. These contain ascospores and may survive for long periods of time without a living host [10–11]. Many powdery mildew species do not develop chasmothecia in tropical and subtropical regions where they can produce conidia on their hosts throughout the year [11].

The Fabaceae family has been reported to host more than fifty species of powdery mildew (Erysiphaceae, Helotiales) worldwide [9,12–15]. Powdery mildews have commonly been found on fabaceous hosts throughout Australia; however, the accurate identification of many historically collected specimens remains uncertain. To complicate the accurate identification of these species further, the sexual morphs of powdery mildews infecting species of the Fabaceae have never been reported in Australia [15–17]. Distinguishing species of powdery mildew belonging to the same genus, based solely on morphological characteristics of the asexual morph can be difficult [16]. Sequencing the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) is considered a reliable method for distinguishing species of powdery mildew [16]. To date, eight powdery mildew species, belonging to two genera, Podosphaera and Erysiphe, have been identified on fabaceous hosts in Australia based on morphology and ITS sequences (Table 1) [15–23]. In an earlier study by Cunnington et al. [15], restriction fragment length polymorphisms (RFLPs) and sequencing of the ITS region had also revealed the presence of one more taxon, identified as Oidium hardenbergiae on Hardenbergia spp., in Australia (Table 1).

[Figure omitted. See PDF.]

Powdery mildews cause economically important diseases in several legume crops in Australia. This disease is reported to reduce grain yields by 20% in soybean [24] and up to 40% in mungbean [25–26]. A recent study by Kelly et al. [17] revealed that Glycine tabacina, a legume species native to Australia, is a host of the soybean pathogen, Erysiphe diffusa, and may be a source of inoculum to soybean crops growing nearby, as well as a means of survival on a perennial host between soybean cropping seasons. This case study has indicated that other members of the Fabaceae family may also act as alternate hosts for powdery mildew pathogens infecting crops grown in Australia.

Another recent study has revealed that powdery mildew epidemics on mungbean and black gram are caused by two species in Australia, not just P. xanthii as previously indicated [20]. Erysiphe vignae, the other causal agent of mungbean and black gram powdery mildew, may have been present in Australian paddocks long before it was discovered by Kelly et al. [20]. Little is known about E. vignae in general, including its possible alternate hosts. This knowledge gap triggered the current study that aimed at identifying powdery mildew species that naturally occur on fabaceous hosts in Australia based on nrDNA ITS sequences and/or morphological characteristics. The finding of alternate hosts of E. vignae and other crop pathogens may provide insight into the origin of diverse pathogens in cropping systems. This work provides the most comprehensive catalogue of powdery mildew species infecting legume hosts throughout Australia.

Materials and methods

Collection of powdery mildew specimens

Leaves of wild and cultivated legumes with naturally occurring powdery mildew infections were collected during ad hoc surveys throughout Queensland between 2020 and 2024. Specimens were also collected from other states in Australia, when possible. Most collections occurred at sites near cropping paddocks, where powdery mildew pathogens are most likely to occur. No materials were collected from natural settings that require a collection permit. Powdery mildew-infected tissues were microscopically examined and processed for DNA extraction as described below, prior to being dried and pressed as herbarium specimens and deposited at the Queensland Plant Pathology Herbarium (BRIP). Historically collected powdery mildew specimens from fabaceous hosts were also obtained from the Queensland Plant Pathology Herbarium and examined using light microscopy and ITS sequencing in order to identify the species based on the most recent taxonomic methods.

Microscopic examination

Microscopic examination was performed using a Nikon Eclipse Ni-U (Tokyo, Japan) microscope with bright field and differential interference contrast (DIC) optics and photographs were taken with an Olympus DP23-CU 6.4MP (Tokyo, Japan) microscope camera. For fresh samples, actively growing mycelia, conidia and conidiophores were removed with cellotape and mounted on a microscope slide containing a droplet of lactic acid. Powdery mildew mycelia from infected herbarium specimens were rehydrated by boiling pieces of infected plant tissues in lactic acid on a microscope slide, as described by Shin and La [27]. During microscopy, the following characteristics were examined: shape and size of conidia (n = 25); presence or absence of fibrosin bodies in fresh conidia; nature of conidiogenesis; morphology of the conidiophore; shape of hyphal appressoria; and when observed, position of conidial germ tubes and shape of germ tube apices.

DNA extraction, PCR amplification and sequencing

Powdery mildew mycelia were removed from fresh and herbarium infected plant tissues using 1–1.5 cm² pieces of cellotape, then total genomic DNA was extracted using an Extract-N-Amp Plant PCR kit (Sigma-Aldrich, St. Louis, MO) as per the manufacturer’s instructions. Two DNA samples were prepared from each specimen. The nested polymerase chain reaction (PCR) method developed by Cunnington et al. [19], using primers PMITS1 and PMITS2 first, and ITS1-F and ITS4 primers in the second reaction, was modified to amplify the ITS region from the DNA samples of this study as described by Kiss et al. [16]. PCR products were purified and each strand was sequenced by Macrogen Inc. (Seoul, Korea) using primers ITS1-F and ITS4, respectively.

Phylogenetic analyses

The ITS sequences of all specimens were edited using Geneious Prime 2024.0.5 (Dotmatics). Chromatograms were visually inspected for potential sequencing errors, trimmed and then the forward and reverse sequences were assembled to produce consensus sequences. These were used as queries in BLASTn searches in standard databases in NCBI GenBank and also in BLASTn searches that were limited to sequences from type materials. The consensus sequences produced in this study were deposited in GenBank (Table 2). Alignments of ITS sequences belonging to the Podosphaera and Erysiphe genera were constructed separately using MAFFT v. 7.388 [28], visually inspected for potential misalignments, then trimmed to the length of the shortest sequence. An Erysiphe ITS data set was constructed, consisting of 48 sequences from this study, 17 previously published sequences from Fabaceae hosts in Australia, and four sequences of representative specimens obtained from GenBank (Table 2). Erysiphe glycines MUMH 1462 (AB078807) was used as the outgroup in this alignment based on Takamatsu et al. [29]. This resulted in an alignment with a total length of 557 characters, including 417 identical and 140 variable sites.

[Figure omitted. See PDF.]

A separate data set was constructed for specimens belonging to the Podosphaera genera. This dataset consisted of 26 sequences obtained in this study, two sequences from powdery mildew on Vigna spp. [20], and eight sequences of representative specimens obtained from GenBank (Table 2). Cystotheca wrightii MUMH 137 (AB000932) was used as the outgroup based on Takamatsu et al. [30]. This resulted in an alignment with a total length of 488 characters, including 363 identical and 125 variable sites.

Phylogenetic analyses were conducted separately on Erysiphe and Podosphaera ITS datasets using Bayesian Inference and Maximum Likelihood approaches. The Akaike Information Criterion was estimated for Bayesian Inference using MrModeltest v. 2.3. [31] and PAUP v. 4.0 [32] and used to determine the best-fit nucleotide substitution model for each alignment. Two Markov Chain Monte Carlo chains were run using MrBayes v. 3.2.4 [33], where one tree per 100 generations was saved, and the runs were ended when the standard deviation of split frequencies was below 0.01. The 50% majority rule consensus tree was estimated after a 25% burn-in of the saved trees. RaxML v. 8.2.11 [34] was used with the GTRGAMMA model of nucleotide substitution and 1,000 bootstrap replicates for the Maximum Likelihood analysis.

Results

Fabaceous hosts of powdery mildews in Australia

A total of 34 fresh powdery mildew specimens were collected in this study from 21 fabaceous host species across Australia (Table 2). An additional 40 herbarium specimens, from 27 host species, dating back to 1928, were also examined. Of all the hosts, the highest number of powdery mildew specimens were collected from the genus Vigna, with 17 specimens collected on six Vigna species (Table 2). The next largest group were powdery mildews on Glycine and Vicia species, with nine specimens of each. When combined with recently published specimens with available ITS sequences, this study included 93 specimens on the Fabaceae, including powdery mildews from 17 native Australian host species and 34 hosts that were introduced to the continent from overseas (Table 2). All specimens were collected from Queensland (QLD) except for seven from Victoria (VIC); two each from Western Australia (WA), New South Wales (NSW), and South Australia (SA); and one from the Northern Territory (NT) (Table 2).

Identification of powdery mildews on fabaceous hosts

Light microscopy observations of freshly collected powdery mildews and specimens rehydrated from herbarium materials morphologically identified all these pathogens as either Erysiphe or Podosphaera species. All specimens identified as Erysiphe had conidiophores that produced conidia singly; at least some of the hyphal appressoria were lobed; conidia did not contain fibrosin bodies; and when present, the position of the germ tubes that emerged from conidia was terminal or sub-terminal with simple or lobed ends (Figs 1-3).

[Figure omitted. See PDF.]

(A) Symptoms on leaves of Cullen australasicum BRIP 76638, (B) Cajanus cajan BRIP 76646, (C) Glycine max BRIP 76637, and (D) Glycine tabacina BRIP 76160. (E) Germinated conidium mounted in lactic acid. (F - G) Conidiophores mounted in lactic acid. Bar = 10 µm.

In contrast, powdery mildews identified as Podosphaera always produced conidia on their conidiophores in chains; their hyphae had simple appressoria; fibrosin bodies were present in fresh conidia; germ tubes stemmed from the middle of conidia and terminated in simple apices (Fig 4).

[Figure omitted. See PDF.]

(A) Symptoms on leaves of Vicia sativa BRIP 76629, (B) Vicia villosa BRIP 76641, and (C) Vicia faba BRIP 74256. (D - E) Conidiophores mounted in lactic acid. (F) Germinated conidium mounted in lactic acid. Bar = 10 µm.

BLASTn searches of the consensus ITS sequences determined in the fresh and herbarium specimens confirmed the morphological identifications at genus level. The sexual morphs known as chasmothecia were not found in any fresh or herbarium specimens examined in this study.

Phylogenetic analyses

ITS sequences were determined for all 74 fresh and herbarium specimens examined in this study. The newly determined Erysiphe sequences were analysed together with 17 Erysiphe sequences published earlier from Australian fabaceous hosts [16–21,23,35] and four reference sequences [13,29,30,36], while the new Podosphaera sequences were included in a separate phylogenetic analysis that contained two previously published Podosphaera sequences from the Fabaceae in Australia [20] with eight reference sequences [16,19]. Altogether, 65 specimens were identified as Erysiphe and 28 as Podosphaera species on the Fabaceae in Australia (Table 3).

[Figure omitted. See PDF.]

The analysis of the Erysiphe specimens revealed the occurrence of a total of ten species on 37 fabaceous hosts in Australia (Table 3 and Fig 5) while the other phylogeny indicated that all Podosphaera specimens from 18 hosts in the Fabaceae belong to a single species, P. xanthii (Table 3 and Fig 6). Altogether, eleven powdery mildew species were identified in Australia from more than 50 host species belonging to 24 genera of the Fabaceae (Table 2).

[Figure omitted. See PDF.]

Symptoms on (A) Vigna radiata BRIP 71005 infected with Erysiphe vignae, (B) Acacia glaucoptera BRIP 76682 infected with Erysiphe sp., (C) Gastrolobium celsianum BRIP 76684 infected with Erysiphe guarinonii, (D) Acacia sp. BRIP 76686 infected with Erysiphe quercicola, (E) Acacia sophorae BRIP 71600 infected with Erysiphe quercicola, and (F) Cullen tenax BRIP 76633 infected with Erysiphe medicaginis. Conidiophores of Erysiphe medicaginis (G), Erysiphe quercicola (H), and Erysiphe vignae (I) mounted in lactic acid. (J) Germinated and non-germinated conidium of Erysiphe vignae mounted in lactic acid. (K) Lobed, hyphal appressorium of Erysiphe vignae. Bar = 10 µm.

[Figure omitted. See PDF.]

(A) Symptoms on leaves of Macroptilium lathyroides BRIP 76642, (B) Vigna lanceolata BRIP 76624, (C) Vigna radiata BRIP 71599, and (D) Vigna angularis BRIP 76647. (E and I) Germinated conidia mounted in lactic acid. (F - G) Conidiophores mounted in lactic acid. (H) Conidia mounted in lactic acid. Arrows point to fibrosin bodies. Bar = 10 µm.

[Figure omitted. See PDF.]

The specimens collected from fabaceous hosts are in bold. Previously published sequences from Australia are followed by ‘*’. All other specimens were obtained from GenBank. The tree is rooted to the ITS sequence of Erysiphe glycines MUMH 1462. Maximum Likelihood bootstrap values >80% and Bayesian Posterior Probability values >0.80 are shown above or below the branches. Thickened branches represent Maximum Likelihood bootstrap values of 100% and Bayesian Posterior Probability of 1.00. The scale bar represents nucleotide substitutions per site.

[Figure omitted. See PDF.]

The specimens collected from fabaceous hosts are in bold. Previously published sequences from Australia are followed by ‘*’All other sequences were obtained from GenBank. The tree is rooted to the ITS sequence of Cystotheca wrightii MUMH 137. Maximum Likelihood bootstrap values >80% and Bayesian Posterior Probability values >0.80 are shown above the branches. Thickened branches represent Maximum Likelihood bootstrap values of 100% and Bayesian Posterior Probability of 1.00. The scale bar represents nucleotide substitutions per site.

Specimens belonging to the genus Erysiphe

Based on ITS sequences and microscopy, a total of 65 Fabaceae specimens examined in this study were naturally infected by powdery mildews belonging to the Erysiphe genus (Table 2; Table 3). In total, this study reports 28 new host records for Erysiphe species in Australia (Table 2). Ten species of Erysiphe were confirmed in this study, naturally occurring on 37 Fabaceae host species covering 17 host genera (Tables 2 and 3). Of the ten Erysiphe species, E. diffusa and E. cf. trifoliorum, were the most common, found on eleven and ten hosts, respectively (Table 3). Several other Erysiphe species were detected in this study on fewer hosts, such as E. pisi which was only found on a single host. Each of these species caused typical powdery mildew symptoms on the leaves, and sometimes phyllodes, of their hosts that included native Australian plants (Figs 1-3).

Erysiphe diffusa was found on eleven hosts, including three species of Glycine (Table 2 and Fig 1). All samples of E. diffusa had similar morphology and identical ITS sequences, except specimen BRIP 68827 from Glycine clandestina, which differed from all other Australian specimens examined in this work by one nucleotide [16]. The ITS sequences of E. diffusa determined in this study were identical to several E. diffusa ITS sequences available in GenBank, including AB078800, reported as E. diffusa from soybean in Japan [29]. Erysiphe diffusa was detected on eleven Fabaceae species in this study, including several important agricultural crops, such as soybean, pigeonpea, and common bean (Table 2). Several Australian native Fabaceae hosts were naturally infected with E. diffusa, including Cullen australasicum, G. tabacina, and G. clandestina.

Erysiphe cf. trifoliorum was confirmed on ten Fabaceae host species, covering seven genera, in this study (Tables 3 and Fig 2). Erysiphe cf. trifoliorum was found on several important agricultural crops, including several species of Vicia, pea (Pisum sativum), and common bean (Table 2). Fifteen E. cf. trifoliorum specimens had identical ITS sequences, while BRIP 2236, BRIP 76640 and BRIP 76641 differed by up to two nucleotides. ITS sequences that are identical to those determined in fifteen specimens in this work are available in GenBank, including MN216308 from Trifolium hybridum in Korea, reported as E. trifoliorum, and FJ378877 from Medicago sp. in the USA, and reported as E. trifolii. The taxonomy of E. cf. trifoliorum remains unresolved [12,14,16].

Erysiphe medicaginis was recently described from Australia [18] and detected on six Fabaceae species in this study (Table 2 and Fig 3). Medicago species, which are grown as pasture crops and are also common weeds throughout Australia, were the most common hosts. Two Australian native species, Cullen tenax and Hardenbergia comptoniana, were also infected with E. medicaginis. Erysiphe medicaginis was detected in Queensland, New South Wales, South Australia, and Western Australia (Table 2). ITS sequences of E. medicaginis determined in this study were identical to several ITS sequences in GenBank, including LC009919 from Sophora flavescens in Japan, another Fabaceae host, and reported as a Pseudoidium species [37].

Erysiphe quercicola was confirmed on four Fabaceae species, all belonging to the Acacia genus (Table 2 and Fig 3). All E. quercicola ITS sequences in this study were identical to several ITS sequences on GenBank, such as OM033344 reported as E. quercicola from Mangifera indica in Taiwan [38]. Two powdery mildews on Cassia fistula and another one on Arachis pintoi were identified as E. aquilegiae. The ITS sequences on the two C. fistula specimens were identical and differed by one nucleotide from the A. pintoi specimen. Specimens identified as E. neolycopersici were detected on two Fabaceae species and grouped together within the E. aquilegiae clade (Table 2 and Fig 5). ITS sequences of the E. neolycopersici specimens were identical and differed by one or two nucleotides to E. aquilegiae. Erysiphe guarinonii was found on two Fabaceae hosts, both native to Australia, Hardenbergia violacea and Gastrolobium celsianum. Erysiphe pisi was confirmed on pea and was the only pathogen detected on a single host in this study. One Erysiphe specimen, BRIP 76682, collected from Acacia glaucoptera in Western Australia differed by one nucleotide from AB292705, the ITS sequence of a powdery mildew identified earlier as E. alphitoides on Quercus sp. in Australia [35].

Erysiphe vignae described recently from mungbean and black gram [20], was detected on a new host in Australia in this study. The ITS sequence and morphology of the powdery mildew fungus on perennial horse gram (Macrotyloma axillare) confirmed its identity as E. vignae (Table 2 and Fig 5). A recent BLASTn search revealed several ITS sequences from overseas that are identical to E. vignae, including powdery mildew samples from Phaseolus vulgaris in Spain, deposited in GenBank as Erysiphe sp. (KU320678) and China, as E. vignae (MW579545); P. acutifolius in Puerto Rico, as E. diffusa (PP938951); P. coccineus in Mexico, as Erysiphe sp. (OQ448664); V. unguiculata in Brazil, as E. diffusa (KY515231) and China, as E. vignae (ON073844); G. max in China, as E. diffusa (MG171170); Erythrina indica in Brazil, as Erysiphe sp. (MF326644); and Strophostyles pauciflora in USA, as E. vignae (PP681083).

Of all the Fabaceae hosts included in this study, those within the Australian native Acacia genus were infected with the most diverse powdery mildew species. Four different species of Erysiphe were detected on the different Acacia host species, including E. quercicola, E. diffusa, E. cf. trifoliorum, and an Erysiphe sp. with an ITS sequence that most closely matched E. alphitoides (Table 2). More than one species of powdery mildew was recorded from many of the established crop hosts, including common bean, pea, soybean, mungbean, and black gram. Often these crops were hosts of both P. xanthii and one or more Erysiphe species.

Specimens belonging to the genus Podosphaera

Twenty-eight Fabaceae specimens were confirmed to be infected with Podosphaera xanthii (Tables 2 and 3). Podosphaera xanthii was the most common powdery mildew that infected Fabaceae hosts in this study, consisting of 18 host species across ten genera (Table 3). This study reports 15 new host records for P. xanthii in Australia (Table 2). Podosphaera xanthii was most prevalent on the Vigna genus and was found on seven different species, including the Australian natives, wild mungbean (V. radiata ssp. sublobata) and maloga bean (V. lanceolota) (Table 2 and Fig 4). All but two P. xanthii sequences determined in this study were identical and grouped together within a single clade with a Bayesian posterior probability of 1.0 and a Maximum Likelihood bootstrap value of 91% (Fig 6). The ITS sequence of the P. xanthii specimen BRIP 71439 from V. unguiculata differed by one nucleotide, and BRIP 8404 from P. vulgaris by five nucleotides from the other P. xanthii sequences; however, these two specimens remained grouped together within the P. xanthii clade (Fig 6).

Discussion

This study was triggered by a lack of knowledge on the host range of Erysiphe vignae, a species that was recently discovered on mungbean and black gram in Australia [20]. Morphological examination and ITS sequencing of 34 freshly collected field samples of powdery mildews infecting diverse Fabaceae species in Australia, and 40 Australian herbarium specimens collected from 1928, detected E. vignae on M. axillare in Queensland. Macrotyloma axillare, commonly known as perennial horse gram, is a heat and drought tolerant legume originating from sub-Saharan Africa and cultivated for livestock forage in subtropical and tropical regions of Australia [39–40]. Like many other legumes originally introduced from overseas as pasture crops [4], M. axillare has become an environmental weed in Queensland. It has a negative impact on the regeneration of native species by climbing on woody plants in open forests and woodlands [5].

BLASTn searches in GenBank revealed several powdery mildews with ITS sequences identical to E. vignae, which were collected outside Australia from hosts other than mungbean, black gram and M. axillare. These records from Brazil, China, Mexico, Spain, Puerto Rico, and the USA indicate that E. vignae may have a global distribution on diverse legume species and could be present in Australia on, as yet, unknown hosts.

Outside of Australia, E. polygoni was repeatedly reported as the causal agent of powdery mildew on Vigna spp. and other fabaceous hosts [41]. This binomial often refers to all powdery mildews on Fabaceae [12]. Interestingly, powdery mildews with ITS sequences that are highly similar to diverse E. polygoni records in GenBank were not detected in this study.

Molecular identification of powdery mildew species was solely based on ITS sequence analyses in this work. ITS and other nrDNA sequences have long been used as reliable molecular tools for powdery mildew identifications and phylogenies [37,42,43] and a genome-scale phylogeny of the Erysiphaceae based on 751 single-copy orthologs extracted from 24 powdery mildew genomes supported the lineages that were previously established based on nrDNA analyses [11]. Recent advances in the identification and phylogeny of different groups of the Erysiphaceae included analyses of a number of secondary DNA species barcodes, including fragments of genes for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), glutamine synthetase synthase (GS), β-tubulin (TUB2), actin (ACT), and mini-chromosome maintenance protein 7 (MCM7) [36,44–47]. These multi-locus analyses focusing on species-level identifications highlighted that some of the secondary barcode sequences confirmed even single nucleotide differences in the ITS region of diverse specimens [36,45–47]. Therefore, ITS sequences were considered as sufficient to achieve the aims of the present study and even single nucleotide differences in this locus were noted during analyses.

Prior to this study, only eight species of powdery mildew had been identified on fabaceous hosts in Australia based on morphological characteristics and ITS analyses [15–23]. This study revealed the presence of P. xanthii and ten species of Erysiphe on more than 50 fabaceous hosts in Australia, including 17 native host species. Overall, this study reports 43 new host records for powdery mildews infecting the Fabaceae in Australia.

Podosphaera xanthii was detected more on fabaceous hosts than any other powdery mildew in this study. It is regarded as a species complex, comprised of multiple races, with distinct or broad host ranges [10,48,49]. This study supported its presence on mungbean, cowpea, wild mungbean (V. radiata ssp. sublobata), and black gram [16,20] and identified an additional 14 fabaceous hosts of P. xanthii in Australia. Podosphaera xanthii is a major pathogen of mungbean in Australia, occurring in all regions of production each year [20]. Two Australian native Vigna species were identified as hosts of P. xanthii in this study, namely wild mungbean and maloga bean (V. lanceolota). Both species are known to grow in close proximity to mungbean paddocks in Australia; therefore, may contribute to the survival of the pathogen between mungbean cropping seasons.

Erysiphe diffusa had the broadest host range of all Erysiphe spp. and was detected on eleven fabaceous species in this study. Previously, it was recorded on soybean and two Australian natives, G. clandestina and G. tabacina in Australia [16,17,20,22]. Those three species were confirmed as hosts in this study, in addition to Acacia flavescens, Clitoria sp., pigeonpea, common bean, various Macrotyloma spp., and the Australian native, Cullen australasicum. Although pigeonpea is currently only a minor crop in Australia, it has been identified as having a high production potential in Queensland and its industry is growing [50]. As its production expands throughout Queensland, it is possible that E. diffusa could become a significant constraint to its production.

A recent study by Kelly et al. [17] confirmed that G. tabacina is an alternate host for E. diffusa infecting soybean. It is likely that the other hosts identified in this study also act as alternate hosts for this crop pathogen. Outside of Australia, E. glycines has also been reported as a pathogen of soybean [29], however this species was not detected in this study.

Erysiphe cf. trifoliorum was the third most common powdery mildew pathogen of fabaceous hosts in Australia. It was found on ten host species across seven genera. The taxonomy of E. cf. trifoliorum remains unresolved; Bradshaw et al. [12] proposed that the E. cf. trifoliorum complex includes at least four separate species that are widely reported on the Fabaceae. Cunnington et al. [15] also reported E. cf. trifoliorum as the most common species of powdery mildew on the Fabaceae in Australia.

This study has reported several new hosts of E. medicaginis in Australia. Erysiphe medicaginis was only recently described as a new species in Australia, occurring on M. polymorpha [18]. Six fabaceous species were newly identified as hosts of E. medicaginis in this study, mostly within the Medicago genera. Various Medicago species are grown as pasture crops throughout Australia and can be commonly found on roadsides as weeds. Two Australian native hosts, Cullen tenax and Hardenbergia comptoniana, were also infected with E. medicaginis. Milk vetch (Astragalus hamosus) was also found to host E. medicaginis.

Erysiphe guarinonii was identified on two fabaceous hosts, both native to Australia, Hardenbergia violacea and Gastrolobium celsianum. A comprehensive study by Bradshaw et al. [12] revealed that all hosts of E. guarinonii belong to the Fabaceae family. In our study, the ITS sequences of the powdery mildew on H. violacea (BRIP 76685) were identical to Oidium hardenbergiae in GenBank (AY450959). Cunnington et al. [15] had previously identified O. hardenbergiae on three Hardenbergia spp. in Australia, including H. comptoniana. In our study, we report E. medicaginis on H. comptoniana. Further specimens should be examined to determine whether H. comptoniana is a host of both species of powdery mildew.

Historically, E. pisi was considered to cause disease in most pulse crops and was made up of several formae speciales [3,51]. More recent studies suggest that E. pisi is likely confined to powdery mildew on Pisum [12]. In this work, E. pisi was only detected on pea, similar to a previous Australian study [15]. Erysiphe cf. trifoliorum was also detected on pea in this work and overseas [12,52].

Several crop pathogens were identified in this study on hosts that are not grown as commercial crops in Australia, indicating the opportunity for these to act as alternate hosts. Recently, ITS and MCM7 sequencing and cross-inoculations confirmed that G. tabacina, and Australian native legume is a host of E. diffusa pathogenic to soybean [17]. The current work identified M. axillare as an alternate host of E. vignae infecting mungbean and black gram based on ITS sequences and morphology; and also, a number of new hosts of the crop pathogens, P. xanthii and E. diffusa. Successful cross-inoculation tests would be needed to support these results. However, it is often difficult to carry out such tests that require fresh inoculum, ideally from all potential host plant species, and disease-free host plants grown in isolation before and after inoculations with the respective powdery mildews [17,20]. Cross-inoculation tests were not included in this work.

No chasmothecia were observed on any host tissues during this study. Few powdery mildew species have been reported to develop chasmothecia in tropical and subtropical regions around the world [11]. It is likely that the Australian climate, and in some species, a broad host range, allows the continual survival of the asexual stage of powdery mildew species on multiple hosts throughout the year.

Lists of plant pathogens present in Australia are essential for biosecurity awareness and risk assessment. This study provides the most comprehensive catalogue of powdery mildew species infecting fabaceous hosts in Australia, providing insights into alternate hosts of key crop pathogens and strengthening Australia’s plant biosecurity awareness. From a crop disease management perspective, the results highlight the importance of weed control in and around crop paddocks to reduce the sources of inoculum during crop production and limit the survival of powdery mildews on alternate hosts within and between cropping seasons.

Acknowledgments

The authors would like to greatly acknowledge the support of the Australian Department of Agriculture, Fisheries and Forestry Brisbane Science and Surveillance team members, Jennifer Morrison, Dr Louisa Parkinson, and Jamie Summerhayes for the collection and identification of Erysiphe vignae on Macrotyloma axillare; Dr Trevor Volp and Adam Quade (Queensland Department of Primary Industries) for the collection of two specimens; and Drs Anke Martin (University of Southern Queensland), Andrew Taylor (Government of Western Australia, Department of Primary Industries and Regional Development) and Eden Tongson (The University of Melbourne) for the collection of one specimen. This research was supported by the University of Southern Queensland (UniSQ), and the Queensland Department of Primary Industries (DPI).

References

1. 1. Harris SA. Tropical forests - woody legumes (excluding Acacias). In: Burley J, editor. Encyclopedia of Forest Sciences. Oxford, UK: Elsevier Ltd; 2004. p. 1793–97.

2. 2. Smýkal P, Coyne C, Ambrose M, Maxted N, Schaefer H, Blair M. Legume crops phylogeny and genetic diversity for science and breeding. Crit Rev Plant Sci. 2015;34:43–104.

* View Article

* Google Scholar

3. 3. Sulima AS, Zhukov VA. War and Peas: Molecular Bases of Resistance to Powdery Mildew in Pea (Pisum sativum L.) and Other Legumes. Plants (Basel). 2022;11(3):339. pmid:35161319

* View Article

* PubMed/NCBI

* Google Scholar

4. 4. Lonsdale W. Inviting trouble: Introduced pasture species in northern Australia. Aust J Ecol. 1994;19:345–54.

* View Article

* Google Scholar

5. 5. Brisbane City Council (internet). Australia: Weed Identification Tool. [cited 14 Sep 2024]. Available from: https://weeds.brisbane.qld.gov.au/weeds//.

6. 6. Randall RP. The introduced flora of Australia and its weed status. Adelaide: CRC for weed management; 2007.

7. 7. Fensham RJ, Laffineur B. Defining the native and naturalized flora for the Australian continent. Aust J Bot. 2019;67:55–69.

* View Article

* Google Scholar

8. 8. Kirkpatrick JB. A continent transformed: human impact on the natural vegetation of Australia. Oxford: Oxford University Press; 1999.

9. 9. Braun U, Cook RTA. Taxonomic Manual of the Erysiphales (Powdery Mildews). The Netherlands: CBS-KNAW Fungal Biodiversity Centre; 2012.

10. 10. Pérez-García A, Romero D, Fernández-Ortuño D, López-Ruiz F, De Vicente A, Torés JA. The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits. Mol Plant Pathol. 2009;10(2):153–60. pmid:19236565

* View Article

* PubMed/NCBI

* Google Scholar

11. 11. Vaghefi N, Kusch S, Németh MZ, Seress D, Braun U, Takamatsu S, et al. Beyond nuclear ribosomal DNA sequences: evolution, taxonomy, and closest known saprobic relatives of powdery mildew fungi (Erysiphaceae) inferred from their first comprehensive genome-scale phylogenetic analyses. Front Microbiol. 2022;13:903024. pmid:35756050

* View Article

* PubMed/NCBI

* Google Scholar

12. 12. Bradshaw M, Braun U, Götz M, Jurick Ιι W. Phylogeny and taxonomy of powdery mildew caused by Erysiphe species on Lupinus hosts. Mycologia. 2022;114(1):76–88. pmid:34851235

* View Article

* PubMed/NCBI

* Google Scholar

13. 13. Bradshaw MJ, Boufford D, Braun U, Moparthi S, Jellings K, Maust A, et al. An in-depth evaluation of powdery mildew hosts reveals one of the world’s most common and widespread groups of fungal plant pathogens. Plant Dis. 2024;108(3):576–81. pmid:37755416

* View Article

* PubMed/NCBI

* Google Scholar

14. 14. Darsaraei H, Khodaparast SA, Asgari B, Götz M, Takamatsu S, Braun U. Erysiphe spp. on Fabaceae from Iran: A new insights into some complex species. Mycol Progress. 2024;23(1).

* View Article

* Google Scholar

15. 15. Cunnington JH, Lawrie AC, Pascoe IG. Molecular determination of anamorphic powdery mildew fungi on the Fabaceae in Australia. Austral Plant Pathol. 2004;33(2):281.

* View Article

* Google Scholar

16. 16. Kiss L, Vaghefi N, Bransgrove K, Dearnaley JDW, Takamatsu S, Tan YP, et al. Australia: a continent without native powdery mildews? the first comprehensive catalog indicates recent introductions and multiple host range expansion events, and leads to the re-discovery of Salmonomyces as a new lineage of the Erysiphales. Front Microbiol. 2020;11:1571. pmid:32765452

* View Article

* PubMed/NCBI

* Google Scholar

17. 17. Kelly LA, Ahmad A, Dahanayaka BA, Dearnaley JDW, Vaghefi N, Kiss L. Glycine tabacina, native to Australia, is an alternate host of Erysiphe diffusa causing powdery mildew on soybean. Plant Pathology. 2024;73(9):2528–36.

* View Article

* Google Scholar

18. 18. Crous P, Wingfield M, Chooi Y, Gilchrist C, Lacey E, Pitt J. Fungal planet description sheets: 1042-1111. Persoonia. 2020;44:301–459.

* View Article

* Google Scholar

19. 19. Cunnington JH, Takamatsu S, Lawrie AC, Pascoe IG. Molecular identification of anamorphic powdery mildews (Erysiphales). Austral Plant Pathol. 2003;32(3):421.

* View Article

* Google Scholar

20. 20. Kelly LA, Vaghefi N, Bransgrove K, Fechner NA, Stuart K, Pandey AK, et al. One crop disease, how many pathogens? Podosphaera xanthii and Erysiphe vignae sp. nov. identified as the two species that cause powdery mildew of Mungbean (Vigna radiata) and Black Gram (V. mungo) in Australia. Phytopathology. 2021;111(7):1193–206. pmid:33487024

* View Article

* PubMed/NCBI

* Google Scholar

21. 21. Limkaisang S, Takamatsu S, Cunnington JH, Wui LK, Salleh B, Sato Y, et al. Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew. Mycoscience. 2006;47(6):327–35.

* View Article

* Google Scholar

22. 22. McTaggart AR, Ryley MJ, Shivas RG. First report of the powdery mildew Erysiphe diffusa on soybean in Australia. Australasian Plant Dis Notes. 2012;7(1):127–9.

* View Article

* Google Scholar

23. 23. Young A, Kiss L. First report of powdery mildew of coastal wattle (Acacia sophorae) caused by Erysiphe quercicola. Australasian Plant Dis Notes. 2021;16(1).

* View Article

* Google Scholar

24. 24. Dunn M, Gaynor L. Impact and control of powdery mildew on irrigated soybean varieties grown in Southeast Australia. Agronomy. 2020;10(4):514.

* View Article

* Google Scholar

25. 25. Thompson S. Mungbeans vs fungus: two sprays for optimum control. GRDC GroundCover. 2016.

* View Article

* Google Scholar

26. 26. Weir D, Kelly L, Sparks A. The impact of different management strategies on the control of powdery mildew in mungbeans – southern downs. Australia: Queensland Department of Agriculture and Fisheries; 2017.

27. 27. Shin HD, La YJ. Morphology of edge lines of chained immature conidia on conidiophores in powdery mildew fungi and their taxonomic significance. Mycotaxon. 1993;46:445–51.

* View Article

* Google Scholar

28. 28. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. pmid:23329690

* View Article

* PubMed/NCBI

* Google Scholar

29. 29. Takamatsu S, Taguchi Y, Shin H-D, Paksiri U, Limkaisang S, Thi Binh N, et al. Two Erysiphe species associated with recent outbreak of soybean powdery mildew: results of molecular phylogenetic analysis based on nuclear rDNA sequences. Mycoscience. 2002;43(4):333–41.

* View Article

* Google Scholar

30. 30. Takamatsu S, Hirata T, Sato Y. A parasitic transition from trees to herbs occurred at least twice in tribe Cystotheceae (erysiphaceae): evident from nuclear ribosomal DNA. Mycol Res. 2000;104:1304–11.

* View Article

* Google Scholar

31. 31. Nylander J. Mrmodeltest version 2. Sweden: Evolutionary Biology Centre, Uppsala University; 2004.

32. 32. Swofford D. Phylogenetic analysis using parsimony (*and other methods). Massachusetts: Sinauer Associates; 2002.

33. 33. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4. pmid:12912839

* View Article

* PubMed/NCBI

* Google Scholar

34. 34. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3. pmid:24451623

* View Article

* PubMed/NCBI

* Google Scholar

35. 35. Takamatsu S, Braun U, Limkaisang S, Kom-Un S, Sato Y, Cunnington JH. Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato. Mycol Res. 2007;111(Pt 7):809–26. pmid:17681225

* View Article

* PubMed/NCBI

* Google Scholar

36. 36. Shirouzu T, Takamatsu S, Hashimoto A, Meeboon J, Ohkuma M. Phylogenetic overview of Erysiphaceae based on nrDNA and MCM7 sequences. Mycoscience. 2020;61(5):249–58.

* View Article

* Google Scholar

37. 37. Takamatsu S, Ito Arakawa H, Shiroya Y, Kiss L, Heluta V. First comprehensive phylogenetic analysis of the genus Erysiphe (Erysiphales, Erysiphaceae) I. The Microsphaera lineage. Mycologia. 2015;107(3):475–89. pmid:25724999

* View Article

* PubMed/NCBI

* Google Scholar

38. 38. Hsiao H-Y, Ariyawansa HA, Hsu C-C, Wang C-J, Shen Y-M. New records of powdery mildews from Taiwan: Erysiphe ipomoeae comb. nov., E. aff. betae on buckwheat, and E. neolycopersici comb. nov. on Cardiospermum halicacabum. Diversity. 2022;14(3):204.

* View Article

* Google Scholar

39. 39. Blumenthal MJ, Staples IB. Origin, evaluation and use of Macrotyloma as forage—a review. Trop Grasslands. 1993;27:16–29.

* View Article

* Google Scholar

40. 40. Fisher D, Reynolds I, Chapman M. The perennial horse gram (Macrotyloma axillare) genome, phylogeny, and selection across the Fabaceae. In: Chapman M, editor. Underutilised crop genomes. Cham: Springer; 2022. p. 255–79.

41. 41. Reddy KS. Identification and inheritance of a new gene for powdery mildew resistance in mungbean (Vigna radiata L. Wilczek). Plant Breeding. 2009;128(5):521–3.

* View Article

* Google Scholar

42. 42. Takamatsu S. Origin and evolution of the powdery mildews (Ascomycota, Erysiphales). Mycoscience. 2013;54(1):75–86.

* View Article

* Google Scholar

43. 43. Takamatsu S. Molecular phylogeny reveals phenotypic evolution of powdery mildews (Erysiphales, Ascomycota). J Gen Plant Pathol. 2013;79(4):218–26.

* View Article

* Google Scholar

44. 44. Ellingham O, David J, Culham A. Enhancing identification accuracy for powdery mildews using previously underexploited DNA loci. Mycologia. 2019;111(5):798–812. pmid:31449476

* View Article

* PubMed/NCBI

* Google Scholar

45. 45. Qiu P-L, Liu S-Y, Bradshaw M, Rooney-Latham S, Takamatsu S, Bulgakov TS, et al. Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus. BMC Microbiol. 2020;20(1):51. pmid:32138640

* View Article

* PubMed/NCBI

* Google Scholar

46. 46. Bradshaw MJ, Guan GX, Nokes L, Braun U, Liu SY, Pfister DH. Secondary DNA barcodes (cam, gapdh, gs, and rpb2) to characterize species complexes and strengthen the powdery mildew phylogeny. Front Ecol Evol. 2022;10:918908.

* View Article

* Google Scholar

47. 47. Bradshaw M, Braun U, Takamatsu S, Németh MZ, Seress D, Pfister DH. The Erysiphe alphitoides complex (powdery mildews)–unravelling the phylogeny and taxonomy of an intricate assemblage of species. New Zealand J. Bot. 2023:1–17.

* View Article

* Google Scholar

48. 48. Braun U, Shishkoff N, Takamatsu S. Phylogeny of Podosphaera sect. Sphaerotheca subsect. Magnicellulatae (Sphaerotheca fuliginea auct. s. lat.) inferred from rDNA ITS sequences - a taxonomic interpretation. Schlechtendalia. 2001;7:45–52.

* View Article

* Google Scholar

49. 49. Hirata T, Cunnington J, Paksiri U, Limkaisang S, Shishkoff N, Grigaliunaite B. Evolutionary analysis of subsection Magnicellulatae of Podosphaera section Sphaerotheca (Erysiphales) based on the rDNA internal transcribed spacer sequences with special reference to host plants. Can J Bot. 2000;78:1521–30.

* View Article

* Google Scholar

50. 50. C.N.Rachaputi R, Motuma Bedane G, James Broad I, Sepp Deifel K. Genotype, Row Spacing and Environment Interaction for Productivity and Grain Quality of Pigeonpea (Cajanus cajan) in sub-tropical Australia. Biosci, Biotech Res Asia. 2018;15(1):27–38.

* View Article

* Google Scholar

51. 51. Falloon R, Viljanen-Rollinson S. Powdery mildew. In: Kraft J, Pfleger F, editors. Compendium of pea diseases and pests. St. Paul, MN, USA: American Phytopathological Society; 2001. p. 28–9.

52. 52. Attanayake RN, Glawe DA, McPhee KE, Dugan FM, Chen W. Erysiphe trifolii– a newly recognized powdery mildew pathogen of pea. Plant Pathology. 2010;59(4):712–20.

* View Article

* Google Scholar

Citation: Kelly LA, Dahanayaka BA, Vaghefi N, Ahmad A, Kiss L (2025) An unexpected diversity of powdery mildew species infecting the Fabaceae in Australia. PLoS One 20(5): e0323505. https://doi.org/10.1371/journal.pone.0323505

About the Authors:

Lisa A. Kelly

Roles: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing

E-mail: [email protected]

Affiliations: Centre for Crop Health, University of Southern Queensland, Toowoomba, Australia, Department of Primary Industries, Queensland Government, Toowoomba, Australia

ORICD: https://orcid.org/0000-0002-2310-8597

Buddhika A. Dahanayaka

Roles: Investigation

Affiliation: Centre for Crop Health, University of Southern Queensland, Toowoomba, Australia

Niloofar Vaghefi

Roles: Formal analysis, Methodology, Supervision, Writing – review & editing

Affiliations: Centre for Crop Health, University of Southern Queensland, Toowoomba, Australia, School of Agriculture, Food and Ecosystem Sciences, Faculty of Science, The University of Melbourne, Parkville, Australia

ORICD: https://orcid.org/0000-0003-0430-4856

Aftab Ahmad

Roles: Investigation

Affiliation: Centre for Crop Health, University of Southern Queensland, Toowoomba, Australia

Levente Kiss

Roles: Conceptualization, Data curation, Investigation, Methodology, Resources, Supervision, Writing – review & editing

Affiliation: Centre for Crop Health, University of Southern Queensland, Toowoomba, Australia

ORICD: https://orcid.org/0000-0002-4785-4308

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

[/RAW_REF_TEXT]

References

1. Harris SA. Tropical forests - woody legumes (excluding Acacias). In: Burley J, editor. Encyclopedia of Forest Sciences. Oxford, UK: Elsevier Ltd; 2004. p. 1793–97.

2. Smýkal P, Coyne C, Ambrose M, Maxted N, Schaefer H, Blair M. Legume crops phylogeny and genetic diversity for science and breeding. Crit Rev Plant Sci. 2015;34:43–104.

3. Sulima AS, Zhukov VA. War and Peas: Molecular Bases of Resistance to Powdery Mildew in Pea (Pisum sativum L.) and Other Legumes. Plants (Basel). 2022;11(3):339. pmid:35161319

4. Lonsdale W. Inviting trouble: Introduced pasture species in northern Australia. Aust J Ecol. 1994;19:345–54.

5. Brisbane City Council (internet). Australia: Weed Identification Tool. [cited 14 Sep 2024]. Available from: https://weeds.brisbane.qld.gov.au/weeds//.

6. Randall RP. The introduced flora of Australia and its weed status. Adelaide: CRC for weed management; 2007.

7. Fensham RJ, Laffineur B. Defining the native and naturalized flora for the Australian continent. Aust J Bot. 2019;67:55–69.

8. Kirkpatrick JB. A continent transformed: human impact on the natural vegetation of Australia. Oxford: Oxford University Press; 1999.

9. Braun U, Cook RTA. Taxonomic Manual of the Erysiphales (Powdery Mildews). The Netherlands: CBS-KNAW Fungal Biodiversity Centre; 2012.

10. Pérez-García A, Romero D, Fernández-Ortuño D, López-Ruiz F, De Vicente A, Torés JA. The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits. Mol Plant Pathol. 2009;10(2):153–60. pmid:19236565

11. Vaghefi N, Kusch S, Németh MZ, Seress D, Braun U, Takamatsu S, et al. Beyond nuclear ribosomal DNA sequences: evolution, taxonomy, and closest known saprobic relatives of powdery mildew fungi (Erysiphaceae) inferred from their first comprehensive genome-scale phylogenetic analyses. Front Microbiol. 2022;13:903024. pmid:35756050

12. Bradshaw M, Braun U, Götz M, Jurick Ιι W. Phylogeny and taxonomy of powdery mildew caused by Erysiphe species on Lupinus hosts. Mycologia. 2022;114(1):76–88. pmid:34851235

13. Bradshaw MJ, Boufford D, Braun U, Moparthi S, Jellings K, Maust A, et al. An in-depth evaluation of powdery mildew hosts reveals one of the world’s most common and widespread groups of fungal plant pathogens. Plant Dis. 2024;108(3):576–81. pmid:37755416

14. Darsaraei H, Khodaparast SA, Asgari B, Götz M, Takamatsu S, Braun U. Erysiphe spp. on Fabaceae from Iran: A new insights into some complex species. Mycol Progress. 2024;23(1).

15. Cunnington JH, Lawrie AC, Pascoe IG. Molecular determination of anamorphic powdery mildew fungi on the Fabaceae in Australia. Austral Plant Pathol. 2004;33(2):281.

16. Kiss L, Vaghefi N, Bransgrove K, Dearnaley JDW, Takamatsu S, Tan YP, et al. Australia: a continent without native powdery mildews? the first comprehensive catalog indicates recent introductions and multiple host range expansion events, and leads to the re-discovery of Salmonomyces as a new lineage of the Erysiphales. Front Microbiol. 2020;11:1571. pmid:32765452

17. Kelly LA, Ahmad A, Dahanayaka BA, Dearnaley JDW, Vaghefi N, Kiss L. Glycine tabacina, native to Australia, is an alternate host of Erysiphe diffusa causing powdery mildew on soybean. Plant Pathology. 2024;73(9):2528–36.

18. Crous P, Wingfield M, Chooi Y, Gilchrist C, Lacey E, Pitt J. Fungal planet description sheets: 1042-1111. Persoonia. 2020;44:301–459.

19. Cunnington JH, Takamatsu S, Lawrie AC, Pascoe IG. Molecular identification of anamorphic powdery mildews (Erysiphales). Austral Plant Pathol. 2003;32(3):421.

20. Kelly LA, Vaghefi N, Bransgrove K, Fechner NA, Stuart K, Pandey AK, et al. One crop disease, how many pathogens? Podosphaera xanthii and Erysiphe vignae sp. nov. identified as the two species that cause powdery mildew of Mungbean (Vigna radiata) and Black Gram (V. mungo) in Australia. Phytopathology. 2021;111(7):1193–206. pmid:33487024

21. Limkaisang S, Takamatsu S, Cunnington JH, Wui LK, Salleh B, Sato Y, et al. Molecular phylogenetic analyses reveal a close relationship between powdery mildew fungi on some tropical trees and Erysiphe alphitoides, an oak powdery mildew. Mycoscience. 2006;47(6):327–35.

22. McTaggart AR, Ryley MJ, Shivas RG. First report of the powdery mildew Erysiphe diffusa on soybean in Australia. Australasian Plant Dis Notes. 2012;7(1):127–9.

23. Young A, Kiss L. First report of powdery mildew of coastal wattle (Acacia sophorae) caused by Erysiphe quercicola. Australasian Plant Dis Notes. 2021;16(1).

24. Dunn M, Gaynor L. Impact and control of powdery mildew on irrigated soybean varieties grown in Southeast Australia. Agronomy. 2020;10(4):514.

25. Thompson S. Mungbeans vs fungus: two sprays for optimum control. GRDC GroundCover. 2016.

26. Weir D, Kelly L, Sparks A. The impact of different management strategies on the control of powdery mildew in mungbeans – southern downs. Australia: Queensland Department of Agriculture and Fisheries; 2017.

27. Shin HD, La YJ. Morphology of edge lines of chained immature conidia on conidiophores in powdery mildew fungi and their taxonomic significance. Mycotaxon. 1993;46:445–51.

28. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30(4):772–80. pmid:23329690

29. Takamatsu S, Taguchi Y, Shin H-D, Paksiri U, Limkaisang S, Thi Binh N, et al. Two Erysiphe species associated with recent outbreak of soybean powdery mildew: results of molecular phylogenetic analysis based on nuclear rDNA sequences. Mycoscience. 2002;43(4):333–41.

30. Takamatsu S, Hirata T, Sato Y. A parasitic transition from trees to herbs occurred at least twice in tribe Cystotheceae (erysiphaceae): evident from nuclear ribosomal DNA. Mycol Res. 2000;104:1304–11.

31. Nylander J. Mrmodeltest version 2. Sweden: Evolutionary Biology Centre, Uppsala University; 2004.

32. Swofford D. Phylogenetic analysis using parsimony (*and other methods). Massachusetts: Sinauer Associates; 2002.

33. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4. pmid:12912839

34. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3. pmid:24451623

35. Takamatsu S, Braun U, Limkaisang S, Kom-Un S, Sato Y, Cunnington JH. Phylogeny and taxonomy of the oak powdery mildew Erysiphe alphitoides sensu lato. Mycol Res. 2007;111(Pt 7):809–26. pmid:17681225

36. Shirouzu T, Takamatsu S, Hashimoto A, Meeboon J, Ohkuma M. Phylogenetic overview of Erysiphaceae based on nrDNA and MCM7 sequences. Mycoscience. 2020;61(5):249–58.

37. Takamatsu S, Ito Arakawa H, Shiroya Y, Kiss L, Heluta V. First comprehensive phylogenetic analysis of the genus Erysiphe (Erysiphales, Erysiphaceae) I. The Microsphaera lineage. Mycologia. 2015;107(3):475–89. pmid:25724999

38. Hsiao H-Y, Ariyawansa HA, Hsu C-C, Wang C-J, Shen Y-M. New records of powdery mildews from Taiwan: Erysiphe ipomoeae comb. nov., E. aff. betae on buckwheat, and E. neolycopersici comb. nov. on Cardiospermum halicacabum. Diversity. 2022;14(3):204.

39. Blumenthal MJ, Staples IB. Origin, evaluation and use of Macrotyloma as forage—a review. Trop Grasslands. 1993;27:16–29.

40. Fisher D, Reynolds I, Chapman M. The perennial horse gram (Macrotyloma axillare) genome, phylogeny, and selection across the Fabaceae. In: Chapman M, editor. Underutilised crop genomes. Cham: Springer; 2022. p. 255–79.

41. Reddy KS. Identification and inheritance of a new gene for powdery mildew resistance in mungbean (Vigna radiata L. Wilczek). Plant Breeding. 2009;128(5):521–3.

42. Takamatsu S. Origin and evolution of the powdery mildews (Ascomycota, Erysiphales). Mycoscience. 2013;54(1):75–86.

43. Takamatsu S. Molecular phylogeny reveals phenotypic evolution of powdery mildews (Erysiphales, Ascomycota). J Gen Plant Pathol. 2013;79(4):218–26.

44. Ellingham O, David J, Culham A. Enhancing identification accuracy for powdery mildews using previously underexploited DNA loci. Mycologia. 2019;111(5):798–812. pmid:31449476

45. Qiu P-L, Liu S-Y, Bradshaw M, Rooney-Latham S, Takamatsu S, Bulgakov TS, et al. Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus. BMC Microbiol. 2020;20(1):51. pmid:32138640

46. Bradshaw MJ, Guan GX, Nokes L, Braun U, Liu SY, Pfister DH. Secondary DNA barcodes (cam, gapdh, gs, and rpb2) to characterize species complexes and strengthen the powdery mildew phylogeny. Front Ecol Evol. 2022;10:918908.

47. Bradshaw M, Braun U, Takamatsu S, Németh MZ, Seress D, Pfister DH. The Erysiphe alphitoides complex (powdery mildews)–unravelling the phylogeny and taxonomy of an intricate assemblage of species. New Zealand J. Bot. 2023:1–17.

48. Braun U, Shishkoff N, Takamatsu S. Phylogeny of Podosphaera sect. Sphaerotheca subsect. Magnicellulatae (Sphaerotheca fuliginea auct. s. lat.) inferred from rDNA ITS sequences - a taxonomic interpretation. Schlechtendalia. 2001;7:45–52.

49. Hirata T, Cunnington J, Paksiri U, Limkaisang S, Shishkoff N, Grigaliunaite B. Evolutionary analysis of subsection Magnicellulatae of Podosphaera section Sphaerotheca (Erysiphales) based on the rDNA internal transcribed spacer sequences with special reference to host plants. Can J Bot. 2000;78:1521–30.

50. C.N.Rachaputi R, Motuma Bedane G, James Broad I, Sepp Deifel K. Genotype, Row Spacing and Environment Interaction for Productivity and Grain Quality of Pigeonpea (Cajanus cajan) in sub-tropical Australia. Biosci, Biotech Res Asia. 2018;15(1):27–38.

51. Falloon R, Viljanen-Rollinson S. Powdery mildew. In: Kraft J, Pfleger F, editors. Compendium of pea diseases and pests. St. Paul, MN, USA: American Phytopathological Society; 2001. p. 28–9.

52. Attanayake RN, Glawe DA, McPhee KE, Dugan FM, Chen W. Erysiphe trifolii– a newly recognized powdery mildew pathogen of pea. Plant Pathology. 2010;59(4):712–20.