Regional Comparisons of Sensitivities of Phytophthora citrophthora and P. syringae Causing Citrus Brown Rot in California to Four New and Two Older Fungicides.

Isolates of the citrus brown rot pathogens Phytophthora citrophthora and P. syringae from the Inland Empire (IE) and Ventura Co. (VE) regions of southern California were evaluated for their sensitivity to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, and the previously published baselines that were generated for Central Valley (CV) isolates of California were expanded. Fungicides were generally more toxic to CV isolates of both species for all four fungicides. Specific differences were found in the toxicity of ethaboxam to P. syringae where CV isolates on average were 6.8 or 8.2 times more sensitive than those from the VE or IE regions, respectively. Based on the grouping of isolates in an unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, as well as fastStructure analyses and plotting of principal component analyses (PCAs), differences in ethaboxam sensitivity could be related to differences in genetic background of the isolates. Isolates of P. citrophthora from the IE and VE had slightly reduced (i.e., 1.5×) sensitivity to mandipropamid as compared with isolates from the CV and were found on distinct branches in the UPGMA dendrogram. Differences in genetic background of less sensitive isolates within each species indicate that these two phenotypes emerged multiple times independently. IE and VE isolates of both species were sensitive to mefenoxam. Moderate resistance to potassium phosphite (EC50 values of 25 to 75 µg/ml) was present in IE and VE isolates of P. syringae, whereas some IE isolates of P. citrophthora were considered resistant with EC50 values of up to 113.69 µg/ml. Resistance to potassium phosphite did not relate to distinct genotypes.

Aphanomyces astaci in Mexico: A new haplotype from dwarf crayfish Cambarellus montezumae.

The crayfish plague is an emerging infectious disease caused by the pathogen Aphanomyces astaci (Oomycota), which is responsible for the decimation of Eurasian freshwater crayfish. This pathogen can coexist with the North American crayfish. These are chronic carriers of the disease as consequence of an immune response that can contain the growth of the pathogen without killing it. The origin of A. astaci locates in the southeastern United States and coincides with the origin of the family Cambaridae. This diverse family of decapods is distributed in North America from southern Canada to Honduras. However, only the native crayfish species from Canada and the USA have been examined for the presence of A. astaci. In this study, we describe for the first time the presence of A. astaci in Mexico in a population of the native species Cambarellus montezumae. By analyzing the small (rrnS) and large (rrnL) mitochondrial ribosomal regions, we showed the presence of two haplotypes of A. astaci within the same population (d1-haplotype and, a novel haplotype that was named, mex1-haplotype). The finding of A. astaci in Mexico confirms the occurrence of this pathogen within the range of the family Cambaridae. The individuals of C. montezumae appear to be chronic carriers of A. astaci, indicated by the lack of documented crayfish plague outbreaks in this population, similar to the pattern observed in other North American species. Thus, the results are of special concern to susceptible species of southern regions of America, i.e., Parastacidae. Therefore, this work emphasizes the need to better understand the distribution and genetic diversity of A. astaci within the distribution range of the natural carriers, i.e., North American species, especially the unexplored area of the family Cambaridae.

Microsatellite Markers from Peronospora tabacina, the Cause of Blue Mold of Tobacco, Reveal Species Origin, Population Structure, and High Gene Flow.

Peronospora tabacina is an obligate parasite that causes blue mold of tobacco. The pathogen reproduces primarily by sporangia, whereas the sexual oospores are rarely observed. A collection of 122 isolates of P. tabacina was genotyped using nine microsatellites to assess the population structure of individuals from subpopulations collected from central, southern, and western Europe; the Middle East; Central America; North America; and Australia. Genetic variations among the six subpopulations accounted for ∼8% of the total variation, including moderate levels of genetic differentiation, high gene flow among these subpopulations, and a positive correlation between geographic and genetic distance (r = 0.225; P < 0.001). Evidence of linkage disequilibrium (P < 0.001) showed that populations contained partially clonal subpopulations but that subpopulations from Australia and Mediterranean Europe did not. High genetic variation and population structure among samples could be explained by continuous gene flow across continents via infected transplant exchange and/or long-distance dispersal of sporangia via wind currents. This study analyzed the most numerous P. tabacina collection and allowed conclusions regarding the migration, mutation, and evolutionary history of this obligate biotrophic oomycete. The evidence pointed to the species origin in Australia and identified intracontinental and intercontinental migration patterns of this important pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Oxathiapiprolin Alone or Mixed with Metalaxyl Seed Treatment for Management of Soybean Seedling Diseases Caused by Species of Phytophthora, Phytopythium, and Pythium.

Species of Phytophthora, Phytopythium, and Pythium affect soybean seed and seedlings each year, primarily through reduced plant populations and yield. Oxathiapiprolin is effective at managing several foliar diseases caused by some oomycetes. The objectives of these studies were to evaluate oxathiapiprolin in a discriminatory dose assay in vitro; evaluate oxathiapiprolin as a soybean seed treatment on a moderately susceptible cultivar in 10 environments; compare the impact of seed treatment on plant populations and yields in environments with low and high precipitation; and compare a seed treatment mixture on cultivars with different levels of resistance in four environments. There was no reduction in growth in vitro among 13 species of Pythium at 0.1 µg ml-1. Soybean seed treated with the base fungicide plus oxathiapiprolin (12 and 24 µg a.i. seed-1) alone, oxathiapiprolin (12 µg a.i. seed-1) plus mefenoxam (6 µg a.i. seed-1), or oxathiapiprolin (24 µg a.i. seed-1) plus ethaboxam (12.1 µg a.i. seed-1) had greater yields in environments that received ≥50 mm of precipitation within 14 days after planting compared with those that received less. Early plant population and yield were significantly higher for seed treated with oxathiapiprolin (24 µg a.i. seed-1) + metalaxyl (13.2 µg a.i. seed-1) compared with nontreated for six of seven cultivars in at least one of four environments. Oxathiapiprolin combined with another Oomycota fungicide applied to seed has the potential to be used to protect soybean plant establishment and yield in regions prone to poor drainage after high levels of precipitation.

Resistance to Potassium Phosphite in Phytophthora Species Causing Citrus Brown Rot and Integrated Practices for Management of Resistant Isolates.

Phytophthora citrophthora, P. syringae, P. nicotianae, and less commonly P. hibernalis are causal agents of brown rot of citrus fruit in California. The chronic disease occurs during the winter season, requires annual management, and has limited California citrus exports because of quarantines in some markets. Potassium phosphite (KPO3) is registered as a pre- and postharvest fungicide in the United States to manage Phytophthora brown rot. We evaluated the in vitro toxicity of KPO3 to 65, 60, and 38 isolates of P. citrophthora, P. syringae, and P. nicotianae, respectively, that were obtained from major growing regions of California. Frequency distributions of effective concentrations to inhibit mycelial growth by 50% (EC50 values) were not normally distributed, with skewness values of 1.84, 1.60, and -0.51 for each species, respectively. Isolates considered sensitive (EC50 values <25 µg/ml), moderately resistant (EC50 values from 25 to 75 µg/ml), or resistant (EC50 values >75 µg/ml) were identified for each species. The majority of P. citrophthora (83.1%) and P. syringae (78.3%) isolates were sensitive, whereas most P. nicotianae isolates (86.8%) were moderately resistant or resistant. Resistance factors were calculated as 65, 19, and 10 for the three species, respectively. In preharvest field trials, KPO3 (2,280 g/ha) applications were not effective in reducing citrus brown rot incidence when orange fruit were inoculated with a resistant (EC50 = 161.9 µg/ml) isolate of P. citrophthora, demonstrating the potential for field resistance. Oxathiapiprolin (32.6 g/ha), however, was highly effective, indicating the absence of multidrug resistance. Postharvest treatments with KPO3 were only effective in reducing brown rot caused by the resistant isolate of P. citrophthora to a low incidence when high rates (8,000 µg/ml) were used in heated (54°C) applications. The sensitive and moderately resistant isolates were managed using rates of 4,000 µg/ml, but heated treatments at this rate were needed to reduce brown rot to commercially acceptable levels when decay was caused by a moderately resistant isolate.

In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles.

Pythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 µg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

Molecular and morphological characterization of three new species of Pythium from Iran: P. ershadii, P. pyrioosporum, and P. urmianum.

Three new species of Pythium: P. ershadii, P. pyrioosporum, and P. urmianum from soils of various regions in Iran are described and illustrated. These species are morphologically distinct from all other known species. Pythium ershadii is morphologically characterized by pyriform ornamented oogonia and rarely production of pyriform oospores. Pythium pyrioosporum differs from other species of the genus by the production of pyriform oospores and smooth walled oogonia, oospores with a tapering elongation toward a hypogynous antheridium and intercalary hypogynous antheridia. Pythium urmianum is distinguished by the presence of intercalary hypogynous antheridia, smooth walled oogonia formed laterally on hyphae or on short side branches and peanut-shaped oospores. Phylogenetic relationships of these new taxa with other Pythium species were investigated using internal transcribed spacers rDNA and partial coxI sequence data. The three species reside in clade E1 and are separated from closely related species.

Phytopythium: molecular phylogeny and systematics.

The genus Phytopythium (Peronosporales) has been described, but a complete circumscription has not yet been presented. In the present paper we provide molecular-based evidence that members of Pythium clade K as described by Lévesque & de Cock (2004) belong to Phytopythium. Maximum likelihood and Bayesian phylogenetic analysis of the nuclear ribosomal DNA (LSU and SSU) and mitochondrial DNA cytochrome oxidase subunit 1 (COI) as well as statistical analyses of pairwise distances strongly support the status of Phytopythium as a separate phylogenetic entity. Phytopythium is morphologically intermediate between the genera Phytophthora and Pythium. It is unique in having papillate, internally proliferating sporangia and cylindrical or lobate antheridia. The formal transfer of clade K species to Phytopythium and a comparison with morphologically similar species of the genera Pythium and Phytophthora is presented. A new species is described, Phytopythium mirpurense.

Equidae pythiosis in Brazil and the world: a systematic review of the last 63 years (1960-2023).

This systematic review compiles reports of clinical pythiosis in horses, mules and donkeys from 1960 to 2023 worldwide, focusing on Brazil. We searched databases and included 71 articles detailing clinical characteristics, geographic distribution, epidemiology, diagnostic methods, therapies, and outcomes. The results showed that publications on equine pythiosis have significantly increased since 2010. Brazil reported the highest incidence, comprising 55% of cases, predominantly in the southern, northeastern, and central-western regions during summer and autumn. Cutaneous pythiosis was the most prevalent form, generally presenting as single lesions in the appendicular region, and affected females more than males. Diagnosis typically involved histopathology, used alone or with other methods. Various treatments have been employed, with surgery, often combined with chemotherapy and immunotherapy, being the most common. Notably, 80.84% of treated animals recovered, highlighting the effectiveness of these therapies in enhancing survival rates. The limitations of the study included the lack of data in published case reports, which made it difficult to collect and calculate epidemiological data. Additionally, we recognize that pythiosis in Brazil is underreported, since this disease does not have mandatory notification and several cases are not registered and/or reported in the literature. Lastly, it is hypothesized that equid pythiosis may be more widespread than currently known, and its real occurrence in Brazil remains uncertain.

Novel nanotechnological approaches for managing Phytophthora diseases of plants.

Members of the Phytophthora genus are soil-dwelling pathogens responsible for diseases of several important plants. Among these, Phytophthora infestans causes late blight of potatoes, which was responsible for the Irish potato famine during the mid-19th century. Various strategies have been applied to control Phytophthora, including integrated management programs (IMPs) and quarantine, but without successful full management of the disease. Thus, there is a need to search for alternative tools. Here, we discuss the emerging role of nanomaterials in the detection and treatment of Phytophthora species, including slow delivery of agrochemicals (microbicides and pesticides). We propose integrating these tools into an IMP, which could lead to a reduction in pesticide use and provide more effective and sustainable control of Phytophthora pathogens.

Exposure of Culex quinquefasciatus to the oomycete Pythium insidiosum: A protocol for in vitro studies.

Pythium insidiosum causes pythiosis, an infection that affects different species of mammals, including humans, and inhabits marshy ecosystems of tropical, subtropical, and temperate regions worldwide. Therefore, this study proposes a protocol to expose Culex quinquefasciatus to P. insidiosum zoospores. Cx. quinquefasciatus immatures (eggs, larvae, and pupae) were exposed to zoospores (8x103 zoospores/mL) of the oomycete for 24 h. The exposure of Cx. quinquefasciatus to the zoospores from L1 to the emergence of adults was evaluated, and P. insidiosum detection was performed by microbiological culture, polymerase chain reaction, and histopathological analysis of stage 4 larvae. The protocol used to produce Cx. quinquefasciatus colonies and adapted for this study proved viable for research on the interaction between P. insidiosum and this Culicidae species. Moreover, P. insidiosum presence was evident in all larval stages of the mosquito, although the presence of the oomycete was not detected in the eggs, pupae, and adults. This study is a pioneer in the development of a protocol to evaluate Cx. quinquefasciatus exposure to P. insidiosum zoospores, and under experimental conditions, P. insidiosum can establish itself in Cx. quinquefasciatus larval stages. The developed protocol is expected to serve as a basis for developing studies to evaluate the interactions of P. insidiosum with these mosquitoes and shed more light on the participation of culicids in expanding the ecological niche of P. insidiosum.

Antifungal activity of a novel synthetic polymer M451 against phytopathogens.

Phytopathogenic fungi are the predominant causal agents of plant diseases. Available fungicides have substantial disadvantages, such as being insufficiently effective owing to intrinsic tolerance and the spread of antifungal resistance accumulating in plant tissues, posing a global threat to public health. Therefore, finding a new broad-spectrum fungicide is a challenge to protect plants. We studied the potency of a novel antimicrobial agent, M451, a 1,6-diaminohexane derivative, against different phytopathogenic fungi of the Ascomycota, Oomycota, and Basidiomycota phyla. M451 exhibited significant antifungal activity with EC50 values from 34-145 µg/mL. The minimal fungicidal concentration against Fusarium oxysporum ranged from 4 to 512 µg/mL depending on the exposure times of 5 min to 24 h. M451 has the highest activity and significantly lower exposure times compared to different polyene, azole, and phenylpyrrole antifungals. The conidial germination assay revealed that M451 induced 99 and 97.8% inhibition against F. oxysporum within 5 min of exposure to 5,000 and 500 µg/mL, respectively. Germ tube elongation, spore production, and spore germination were also significantly inhibited by M451 at concentrations of ≥50 µg/mL. Based on the broad spectrum of antifungal effects across different plant pathogens, M451 could be a new chemical fungicide for plant disease management.

Pythium insidiosum complex hides a cryptic novel species: Pythium periculosum.

Early phylogenetic analysis of Pythium insidiosum, the etiologic agent of pythiosis in mammals, showed the presence of a complex comprising three monophyletic clusters. Two included isolates recovered from cases of pythiosis in the Americas (Cluster I) and Asia (Cluster II), whereas the third cluster included four diverged isolates three from humans in Thailand and the USA, and one isolate from a USA spectacled bear (Cluster III). Thereafter, several phylogenetic analyses confirmed the presence of at least three monophyletic clusters, with most isolates placed in clusters I and II. Recent phylogenetic analyses using isolates from environmental sources and from human cases in India, Spain, Thailand, and dogs in the USA, however, showed the presence of two monophyletic groups each holding two sub-clusters. These studies revealed that P. insidiosum possesses different phylogenetic patterns to that described by early investigators. In this study, phylogenetic, population genetic and protein MALDI-TOF analyses of the P. insidiosum isolates in our culture collection, as well as those available in the database, showed members in the proposed cluster III and IV are phylogenetically different from that in clusters I and II. Our analyses of the complex showed a novel group holding two sub-clusters the USA (Cluster III) and the other from different world regions (Cluster IV). The data showed the original P. insidiosum cluster III is a cryptic novel species, now identified as P. periculosum. The finding of a novel species within P. insidiosum complex has direct implications in the epidemiology, diagnosis, and management of pythiosis in mammalian hosts.

Peronosporaceae species causing downy mildew diseases of Poaceae, including nomenclature revisions and diagnostic resources.

Downy mildew pathogens of graminicolous hosts (Poaceae) are members of eight morphologically and phylogenetically distinct genera in the Peronosporaceae (Oomycota, Peronosporales). Graminicolous downy mildews (GDMs) cause severe losses in crops such as maize, millets, sorghum, and sugarcane in many parts of the world, especially in tropical climates. In countries where the most destructive GDMs are not endemic, these organisms are often designated as high-risk foreign pathogens and subject to oversight and quarantine by regulatory officials. Thus, there is a need to reliably and accurately identify the causal organisms. This paper provides an overview of the Peronosporaceae species causing graminicolous downy mildew diseases, with a description of their impact on agriculture and the environment, along with brief summaries of the nomenclatural and taxonomic issues surrounding these taxa. Key diagnostic characters are summarized, including DNA sequence data for types and/or voucher specimens, morphological features, and new illustrations. New sequence data for cox2 and 28S rDNA markers are provided from the type specimens of three species, Peronosclerospora philippinensis, Sclerospora iseilematis, and Sclerospora northii. Thirty-nine species of graminicolous downy mildews are accepted, and seven previously invalidly published taxa are validated. Fifty-five specimens are formally designated as types, including lectotypification of 10 species, neotypification of three species, and holotype designation for Sclerophthora cryophila. Citation: Crouch JA, Davis WJ, Shishkoff N, Castroagudín VL, Martin F, Michelmore R, Thines M (2022). Peronosporaceae species causing downy mildew diseases of Poaceae, including nomenclature revisions and diagnostic resources. Fungal Systematics and Evolution 9: 43-86. doi: 10.3114/fuse.2022.09.05.

High-Temperature-Tolerant Fungus and Oomycetes in Korea, Including Saksenaea longicolla sp. nov.

Global temperatures are steadily increasing, leading to significant changes in microbial diversity and ecology. In the present study, we isolated high-temperature-growing fungi and fungi-like group (Oomycota) strains from freshwater environments of Korea and identified them based on cultural, morphological, and multilocus phylogenetic analyses. As a result, we introduce Saksenaea (Fungi) isolates as a new species, Saksenaea longicolla sp. nov. and record Phytophthora chlamydospora and P. lagoariana (Oomycota) new to Korea. In the growth experiments, they exhibited high-temperature tolerance, which can grow at 35-40 °C but become inactive at 4 °C and below. This study confirms the presence of high-temperature-tolerant fungi and oomycetes in Korea and suggests that the Korean climate conditions are changing in favor of these species. This indicates that climate warming is altering microbial distributions in freshwater environments.

Taxonomic and pathogenic characterization of Phytopythium species from West Azarbaijan, Iran, and description of two new species.

Phytopythium is a globally distributed genus, species of which occur in various ecological niches. Despite their importance as plant pathogens, information on species diversity and their distribution in West Azarbaijan Province of Iran is limited. This study aimed to investigate the taxonomy and plant pathogenicity of Phytopythium isolates recovered from soils in the province. A polyphasic taxonomy based on morphological, cultural, and multilocus sequence data revealed two new species, described here as P. babaiaharii and P. longitubum. In addition, a putatively new species and five known species, P. boreale, P. carbonicum, P. mercuriale, P. ostracodes, and P. vexans, were found in the studied region. Phytopythium ostracodes, P. mercuriale, and P. boreale were the three most frequent species isolated from soil, although P. mercuriale was only found in one field. Among the studied species, P. mercuriale, P. ostracodes, and P. vexans exhibited various levels of pathogenicity on sugar beet, sunflower, and tomato, and P. boreale was documented for first time as a plant pathogen. Phytopythium ostracodes was highly aggressive on sugar beet and sunflower but lowly aggressive on tomato, whereas P. vexans exhibited high aggressiveness toward the three crop plants. Both P. boreale and P. mercuriale were found to be lowly aggressive on the host plants. The results indicated that members of Phytopythium, particularly plant-pathogenic species, are common in arable soils of West Azarbaijan Province and can pose a threat to agricultural crops in the region.

The Omics Hunt for Novel Molecular Markers of Resistance to Phytophthora infestans.

Wild Solanum accessions are a treasured source of resistance against pathogens, including oomycete Phytophthora infestans, causing late blight disease. Here, Solanum pinnatisectum, Solanum tuberosum, and the somatic hybrid between these two lines were analyzed, representing resistant, susceptible, and moderately resistant genotypes, respectively. Proteome and metabolome analyses showed that the infection had the highest impact on leaves of the resistant plant and indicated, among others, an extensive remodeling of the leaf lipidome. The lipidome profiling confirmed an accumulation of glycerolipids, a depletion in the total pool of glycerophospholipids, and showed considerable differences between the lipidome composition of resistant and susceptible genotypes. The analysis of putative resistance markers pinpointed more than 100 molecules that positively correlated with resistance including phenolics and cysteamine, a compound with known antimicrobial activity. Putative resistance protein markers were targeted in an additional 12 genotypes with contrasting resistance to P. infestans. At least 27 proteins showed a negative correlation with the susceptibility including HSP70-2, endochitinase B, WPP domain-containing protein, and cyclase 3. In summary, these findings provide insights into molecular mechanisms of resistance against P. infestans and present novel targets for selective breeding.

Sirolpidium bryopsidis, a parasite of green algae, is probably conspecific with Pontisma lagenidioides, a parasite of red algae.

The genus Sirolpidium (Sirolpidiaceae) of the Oomycota includes several species of holocarpic obligate aquatic parasites. These organisms are widely occurring in marine and freshwater habitats, mostly infecting filamentous green algae. Presently, all species are only known from their morphology and descriptive life cycle traits. None of the seven species classified in Sirolpidium, including the type species, S. bryopsidis, has been rediscovered and studied for their molecular phylogeny, so far. Originally, the genus was established to accommodate all parasites of filamentous marine green algae. In the past few decades, however, Sirolpidium has undergone multiple taxonomic revisions and several species parasitic in other host groups were added to the genus. While the phylogeny of the marine rhodophyte- and phaeophyte-infecting genera Pontisma and Eurychasma, respectively, has only been resolved recently, the taxonomic placement of the chlorophyte-infecting genus Sirolpidium remained unresolved. In the present study, we report the phylogenetic placement of Sirolpidium bryopsidis infecting the filamentous marine green algae Capsosiphon fulvescens sampled from Skagaströnd in Northwest Iceland. Phylogenetic reconstructions revealed that S. bryopsidis is either conspecific or at least very closely related to the type species of Pontisma, Po. lagenidioides. Consequently, the type species of genus Sirolpidium, S. bryopsidis, is reclassified to Pontisma. Further infection trials are needed to determine if Po. bryopsidis and Po. lagenidioides are conspecific or closely related. In either case, the apparently recent host jump from red to green algae is remarkable, as it opens the possibility for radiation in a largely divergent eukaryotic lineage. Citation: Buaya AT, Scholz B, Thines M (2021). Sirolpidium bryopsidis, a parasite of green algae, is probably conspecific with Pontisma lagenidioides, a parasite of red algae. Fungal Systematics and Evolution 7: 223-231. doi: 10.3114/fuse.2021.07.11.

Protists in the Plant Microbiome: An Untapped Field of Research.

Protists are mostly unicellular eukaryotes. Some protists are beneficial for plants, while others live as endosymbionts and can cause severe plant diseases. More detailed studies on plant-protist interactions exist only for plant pathogens and parasites. A number of protists live as inconspicuous endophytes and cause no visible disease symptoms, while others appear closely associated with the rhizosphere or phyllosphere of plants, but we still have only a vague understanding on their identities and functions. Here, we provide a protocol on how to assess the plant-associated protist community via Illumina-sequencing of ribosomal marker-amplicons and describe how to assign taxonomic affiliation to the obtained sequences.