Received: 10 October 2022 Accepted: 5 December 2022

DOI: 10.1111/1462-2920.16303

MINI REVIEW

Plant associated protists—Untapped promising candidates

for agrifood tools

Bao-Anh Thi Nguyen 1 | Kenneth Dumack 2 | Pankaj Trivedi 3 |

Zahra Islam 1,4 | Hang-Wei Hu 1,4

1
School of Agriculture and Food, Faculty of
Veterinary and Agricultural Sciences, The Abstract
University of Melbourne, Parkville, Victoria, The importance of host-associated microorganisms and their biotic interac-
Australia
2
tions for plant health and performance has been increasingly acknowl-
Terrestrial Ecology, Institute of Zoology,
University of Cologne, Köln, Germany edged. Protists, main predators and regulators of bacteria and fungi, are
3
Microbiome Network and Department of abundant and ubiquitous eukaryotes in terrestrial ecosystems. Protists are
Agricultural Biology, Colorado State considered to benefit plant health and performance, but the community
University, Fort Collins, Colorado, USA structure and functions of plant-associated protists remain surprisingly
4
ARC Hub for Smart Fertilisers, The University underexplored. Harnessing plant-associated protists and other microbes
of Melbourne, Parkville, Victoria, Australia
can potentially enhance plant health and productivity and sustain healthy
Correspondence
food and agriculture systems. In this review, we summarize the knowledge
Hang-Wei Hu, School of Agriculture and Food, of multifunctionality of protists and their interactions with other microbes in
Faculty of Veterinary and Agricultural plant hosts, and propose a future framework to study plant-associated pro-
Sciences, The University of Melbourne,
Parkville, Victoria 3010, Australia.
tists and utilize protists as agrifood tools for benefiting agricultural
Email: hang-wei.hu@unimelb.edu.au production.

Funding information
Australian Research Council, Grant/Award
Number: IH200100023

INTRODUCTION perspective to decipher the mechanisms that govern

the assembly, interactions and functions of plant-
Living plants are hosts of a complex microbiome, com- associated microbiota, therefore, is a prerequisite to
prising of bacteria, fungi, archaea, protists and viruses facilitate translational research and develop
that internally and externally colonize plant tissues microbiome-based tools to enhance plant productivity
(Hassani et al., 2018; Sapp et al., 2018; Trivedi and agricultural sustainability.
et al., 2020). These beneficial, neutral and pathogenic A panoramic view of the plant microbiota cannot be
plant-associated microorganisms can significantly influ- complete without considering protists as a pivotal com-
ence plant health and performance. The plant hosts ponent. Bacteria dominate the plant microbiota, fol-
and their associated microbiomes are suggested to lowed by fungi, while protists and other organisms
form a ‘holobiont’, where complex plant–microbe inter- (e.g., archaea, nematodes and other soil invertebrates)
actions play crucial roles in regulating and promoting are less abundant, but they were shown to be crucial in
plant growth, biogeochemical cycling, nutrient acquisi- plant health and performance (Leach et al., 2017; Chen
tion, fitness and protection, stress tolerance and dis- et al., 2021). Bacteria and fungi in the rhizosphere are
ease suppression (Hassani et al., 2018; Liu enriched by carbon sources stemming from root exu-
et al., 2019). Plant associated microbiota, in some dates of plants (via bottom-up control), however, they
cases, even contribute more to plant protection and are major microbial prey for protists and thus subject to
stress resistance than the defensive capacity of plant top-down control by protist consumers. Protists, repre-
hosts (Hubbard et al., 2019). A holistic microbiome senting the vast diversity of unicellular eukaryotes,

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2022 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.

Environ Microbiol. 2023;25:229–240. wileyonlinelibrary.com/journal/emi 229

230 NGUYEN ET AL.

function as consumers (main predators of bacteria, the phyllosphere-associated protists (Bamforth, 1973).
fungi and small animals), primary producers (important Protists, especially the phylum Cecozoa consumers,
carbon fixers via photosynthesis), plant and animal par- have been recently identified in the model plant Arabi-
asites, and decomposers (Geisen et al., 2018). The dopsis thaliana (Sapp et al., 2018), sorghum (Sun
contributions of protists to nutrient input, organic matter et al., 2021a), grasses, legumes and forbs (Flues
decomposition and plant health have been previously et al., 2017; Flues et al., 2018), with the ability to
reported (Bonkowski, 2004; Xiong et al., 2020; Geisen improve plant growth and biomass. The phyllosphere,
et al., 2021). Nonetheless, plant-associated protists a habitat of various phages, prokaryotes, protists,
and their functions for plant hosts, compared to bacteria fungi, and visiting insects (e.g., bees, butterflies and
and fungi, have been largely underestimated (Gao herbivores), is supposed to regulated by their complex
et al., 2019; Trivedi et al., 2020). While the importance trophic interactions under direct impacts of environ-
of plant beneficial microorganisms as promising agri- mental changes. Protists shape the community com-
food tools to improve crop production and agricultural position and activities of bacteria and fungi through
sustainability has been increasingly recognized (Chen selective predation (Bonkowski, 2004; Gao
et al., 2021; Hu et al., 2022), the plant–protist–microbe et al., 2019). Notably, the selective predation of pro-
interactions in the above- and below-ground systems tists triggers distinct bacterial strains to produce anti-
are not well understood. microbials, such as 2,4-diacetylphloroglucinol (DAPG)
and pyrrolnitrin (Jousset et al., 2006), or violacein
(Matz et al., 2004), which has been recorded in the
POTENTIAL FUNCTIONS AND interaction between one or a few model protist and
INTERACTIONS OF PROTISTS IN THE bacterial species under in vitro conditions. Hence, pro-
PLANT–SOIL–MICROBE NETWORK tists may stimulate bacteria or fungi to excrete toxic
metabolites to protect plants from air-borne pathogens
Although protists have the great potential to improve or herbivores.
nutrition, suppress pathogens, promote plant growth, Furthermore, protists can potentially select beneficial
and function as bioindicators for plant health traits of microbes through (i) promoting phytohormone-
(Bonkowski, 2004; Xiong et al., 2020), protists coloniz- producing bacteria and ultimately enhancing plant fitness
ing inside plant tissues remain vastly untapped. Most and development; and (ii) regulating the metabolic and
studies on plant-associated protists have focused on functional profiles of bacterial community in the phyllo-
plant pathogenic or parasitic protists causing plant dis- sphere (Figure 1). Some first evidence about phytohor-
eases (Dumack & Bonkowski, 2021) or the below- mone stimulation of protists has been found in plant
ground protist community particularly in the rhizosphere rhizosphere, and their beneficial effects on plant hor-
(Fiore-Donno et al., 2022). Bacteria and fungi are well- mones in the phyllosphere are a fertile area to discover.
characterized plant microbiome components with dis- Recent studies have indicated that bacterivorous amoe-
tinct community compositions across different compart- bae promoted bacteria producing essential phytohor-
ments (e.g., phyllosphere, anthosphere, leaf and root mones (auxin and cytokinin) in the plant rhizosphere
endospheres, rhizosphere, and bulk soils) (Liu though protists alone cannot produce plant hormones
et al., 2019; Trivedi et al., 2020; Sun et al., 2021b). (Bonkowski & Brandt, 2002; Krome et al., 2010). Flues
Given the selective feeding preference of different pro- et al. (2017) revealed that, through a shotgun metage-
tist groups for bacteria and fungi (Dumack et al., 2020), nomic sequencing, the predation of leaf-associated pro-
plant compartments at different developmental stages tists Cercomonas and Paracercomonas strains
may harbour distinct taxonomic and functional diversity, (Cercozoa) dramatically influenced the taxonomic compo-
community structure and functions of protists. In this sition and metabolic functions of leaf-associated bacterial
article, we discuss known functions of protists and pro- community under in vitro conditions, suggesting the
pose their potential roles and activities in different com- strong regulation of protists on the activities and functions
partments of plants (Figure 1; Table 1). of bacteria in the phyllosphere. Many other representa-
tives of leaf-associated Cercozoan consumers (Rhogos-
toma spp.) were found to feed on fungi (here are yeasts)
Phyllosphere-associated protists and algae in the phyllosphere of A. thaliana, and this
grazing activity indicated crucial effects of protists on a
Protists form key members of the plant microbiome wide range of microbes in the phyllosphere.
and an external force shaping the plant microbiome Plants are not passively benefited by microorgan-
assembly (Geisen et al., 2018; Gao et al., 2019), but isms but may proactively use the strategy ‘cry for help’
the diversity and feedback of protists on phyllosphere to recruit beneficial microorganisms to protect them-
microbiome remain surprisingly unknown. The occur- selves under the abiotic (e.g., drought or high tempera-
rence of a protist strain Colpoda cucullus in leaves ture) and biotic stresses (e.g., pathogens or
and stems in the 1970 s is one of early findings about herbivores). The underlying mechanisms and recruited
PROTISTS—PROMISING CANDIDATES FOR AGRIFOOD TOOLS 231

F I G U R E 1 Functions and interactions of protists within the plant-associated microbiota in different plant compartments (phylloshere,
anthosphere, leaf, stem and root endosphere, rhizosphere and bulk soil). (i) Through predation or symbiosis, protists interact with bacteria, fungi
(especially, arbuscular mycorrhiza fungi (AMF)) and other microbes (e.g., archaea) in cycling, uptake and/or translocation of essential nutrients
(e.g., nitrogen, phosphorous, carbon, silicon, calcium, magnesium and iron) for plants and soil organisms in rhizosphere and bulk soils. Protists
can also enhance nutrient input as carbon fixers or through releasing nutrients after the prey consumption; (ii) protists have the potential to form
symbiotic or facilitative relationships with nitrifying and ammonifying bacteria or archaea in nitrogen fixation in plant rhizosphere; (iii) the
predation of protists can trigger the antimicrobial production of bacteria or fungi, inhibiting the infection of air-borne or soil-borne pathogens or
pests in phyllosphere and rhizosphere; (iv) protists may also enhance plant hormone and stress tolerance by directly interacting with plant hosts
or stimulating plant-beneficial traits of microbes.

microorganisms of this strategy, however, are unclear Moreover, endophytic protists colonize root and leaf
and probably distinct across plant compartments. Strik- and stem endosphere, where their interplay with plant
ingly, a board spectrum of bacteria and fungi hosts and other microbes can possibly influence plant
(e.g., yeasts) inhabit the anthosphere (i.e., flowers and hormones, defensive systems and nutrient transloca-
surrounding zones), especially nectar, pollen (Vannette tion to every plant tissue. The stimulation of uptake and
et al., 2013; Schaeffer et al., 2017) and flower surface translocation of nitrogen from rhizosphere soils, plant
(Ushio et al., 2015; Arunkumar et al., 2019), which sig- roots to shoots by protists were reported in wheat
nificantly influence flower-pollinator interactions, plant plants (Clarholm, 1985; Henkes et al., 2018). Notably,
reproduction and yield. Due to the diverse microbes the amoebae Acanthamoeba castellanii promoted the
transmitted from various sources, flowers are poten- phytohormone production (auxins and cytokinin) of bac-
tially dynamic hubs of microbes and pollinators. How- teria in the phyllosphere of cress (Lepidium sativum L.)
ever, the diversity and roles of protists in the and A. thaliana (Krome et al., 2010). Most recent stud-
anthosphere are far from being fully elucidated. ies have attempted to characterize the compositions of
232 NGUYEN ET AL.

TABLE 1 Known beneficial functions of plant-associated protists in different plants

Plants Plant compartments Protist species Functions of protists References

Banana plants Rhizosphere (roots and soil) Cercomonas lenta Plant disease suppression (Guo
strain ECO-P-01 against Fusarium wilt; et al., 2022)
Plant yield
Faba bean (Vicia Rhizosphere (plant roots) Rosculus terrestris Plant disease suppression (Bahroun
faba) S14D1; against Fusarium rot; et al., 2021)
seedlings Bodomorpha sp. Root length and biomass
C10D3; improvement
Cercomonas lenta
C5D5
Cucumber Rhizosphere Protist community Indicators and determinants of (Guo
plant yield and disease et al., 2021)
suppression
Tomato Rhizosphere Protist community Determinants of plant yield and (Xiong
disease suppression et al., 2020)
Wheat Phyllosphere; Consumers Plant uptake and translocation of (Clarholm, 1985)
Rhizosphere inorganic nitrogen
Wheat Phyllosphere and Rhizosphere Acanthamoeba Plant shoot/root biomass; (Henkes
castellanii Nitrogen translocation within root et al., 2018)
system
Arabidopsis Rhizosphere (plant roots) Acanthamoeba Plant uptake of carbon; (Krome
thaliana castellanii Plant uptake of nitrogen; et al., 2009)
Plant growth (root and shoot
biomass) and seed production
Ryegrass Rhizosphere Naked amoebae, Nitrogen availability; (Bonkowski
seedling flagellates and Plant growth et al., 2000)
ciliates
Spruce seedlings Rhizosphere (root systems); Acanthamoeba sp. Plant phosphorus and calcium (Bonkowski
phyllosphere (stems and uptake and translocation; et al., 2001)
needles) Biomass of plant compartments
Rice plants Rhizosphere Acanthamoeba Root growth and shoot biomass; (Herdler
(Oryza sativa castellanii Plant carbon, phosphorus, et al., 2008)
L.) magnesium, and calcium
uptake
Arabidopsis Rhizosphere Acanthamoeba Plant growth (Rosenberg
thaliana castellanii et al., 2009)
Pea seedlings – Amoeba Plant hormone production (auxins (Nikoljuk, 1969)
and related substance);
Plant growth and biomass
Cress (Lepidium Phyllosphere Acanthamoeba Plant hormone production (Krome
sativum L.) castellanii (auxins); et al., 2010)
Shoot growth
Rhizosphere (plant roots) Plant hormone production
(auxins);
Root growth
Arabidopsis Phyllosphere Plant hormone production (auxins
thaliana Rhizosphere (plant roots) and cytokinin);
Root growth
Watercress Rhizosphere (plant roots) Acanthamoeba Plant hormone production (auxin, (Bonkowski &
seedlings castellanii indolyl-3-acetic acid (IAA)); Brandt, 2002)
Root growth and architecture

protists in the plant microbiome (Dumack et al., 2022; Rhizosphere-associated protists

Sun et al., 2021a), hence further insights into the multi-
trophic interactions of protists with plants, microbes, air- In contrast to other plant compartments, protists in the
borne pathogens and insects in the phyllosphere are rhizosphere have received more attention with growing
required. evidence for their crucial roles in (i) plant health and
PROTISTS—PROMISING CANDIDATES FOR AGRIFOOD TOOLS 233

disease control (Xiong et al., 2020), (ii) nutrient cycling comprehensive understanding of protists in the rhizo-
(Clarholm, 1985; Bonkowski, 2004), and (iii) plant hor- sphere and other plant compartments will promote their
mones and growth (Bonkowski & Brandt, 2002). Many applications in plant disease suppression.
bacterial and fungal taxa are well-known producers of Protists are also pivotal contributors to nutrient
antibiotics and toxic metabolites (Hutchings cycling in the rhizosphere (Table 1). Nutrients are tem-
et al., 2019). The selective predation or even the pres- porarily locked up in rhizosphere bacterial and fungal
ence of protists can trigger bacteria to produce specific biomass and can be translocated to protists as micro-
antibiotics as weapons to kill or avoid protists through bial feeders or unlocked by the protists’ predation and
species-specific response (Nguyen et al., 2020). For eventually channelled to benefit plants, which is called
instance, Pseudomonas fluorescens strain SS101 and ‘the microbial loop’ (Clarholm, 1985). Protists directly
Pseudomonas fluorescens strain SBW25 produced release nitrogen and carbon after prey digestion or form
antibiotics massetolide and viscosin, respectively, in a symbiotic relationship with beneficial fungal or bacte-
response to the same bacterivorous amoeba Naegleria rial taxa in cycling essential nutrients (nitrogen, carbon,
americana C1 (Mazzola et al., 2009; Song et al., 2015). iron, silicon or phosphorous) (Geisen et al., 2018; Gao
Fungi also emit antimicrobial volatiles to inhibit the bac- et al., 2019), enhancing soil nutrient input and fertility
terial motility or growth upon bacterial–fungal interac- for nurturing plant growth and rhizo-microbiome. The
tion (Rybakova et al., 2017; Bruisson et al., 2020). great contribution of protists to nutrient cycling has long
However, there is a paucity of effects of protists on the been recognized since 1985, when Clarholm demon-
antibiotic excretion of fungi. The antibiotics produced strated the increasing nitrogen uptake to 75% by plants
by bacteria and fungi are considered as a defensive under the inoculation of protists. The presence of pro-
mechanism to toxify not only protists but also other tists promoted plant phosphorus and calcium uptake
microbial competitors in natural habitats and translocation to stems or needles, as well as modu-
(Święciło, 2016; Cruz-Loya et al., 2019). Through this lated nutrient concentrations (nitrogen, phosphorus,
effect, when plants ‘cry for help’ by sending signals via carbon to nitrogen ratio (C/N ratio), calcium and mag-
root exudates (volatiles, organic acids or others) under nesium) (Bonkowski et al., 2001). Consequently, this
pathogen or pest attacks (Liu et al., 2019), protists may regulation of protists led to the improvement of root
respond by recruiting antibiotic producers to produce growth and architecture as well as biomass of different
antimicrobials to inhibit pathogens or pests for plant compartments (shoots, roots and needles) of spruce
protection. However, this strategy of plants and their seedlings. A similar beneficial effect of protists was
associations with protists are still elusive questions. found in rice plants (Oryza sativa L.) (Henkes
As primary microbial predators, protists can also et al., 2018). Moreover, phototrophic protists contribute
directly consume bacterial and fungal pathogens. The to carbon cycling as carbon fixers via photosynthesis
consumptive effect of protists, typically protistan con- (Schmidt et al., 2016), providing nonnegligible carbon
sumers, can cause fatality of a wide range of bacterial and oxygen inputs to rhizosphere organisms and the
and fungal strains (Chakraborty et al., 1983; Dumack basis for soil life, but their capacity for carbon seques-
et al., 2016). In the rhizosphere of A. thaliana, the diver- tration is still unknown.
sity and abundance of specific bacteria taxa, especially Notably, benefits of protists to plant nutrition are
Betaproteobacteria and Firmicutes, were significantly more efficient when forming symbiosis with other
decreased under the predation of soil amoeba microbes, particularly arbuscular mycorrhizal fungi
A. castellanii. Bahroun et al. (2021) reported that bac- (AMF) that enhance plant nitrogen and phosphorus
terivorous protists alone and their synergistic interac- uptake. Protists might facilitate nutrient acquisition,
tions with bacteria reduced disease severity caused by mineralization and translocation of AMF (Zuccaro
a fungal pathogen Fusarium solani S55 and improved et al., 2014; Henkes et al., 2018), and promote the
root length and plant growth of faba bean (Vicia faba) growth and activities of nitrifying bacteria and other
seedlings (Table 1). Recent studies have indicated bacteria (Bonkowski, 2004), suggesting intimate
important links of protists to soil-borne disease control protist–microbe links in plant benefits. For instance,
and plant health in the rhizosphere of tomatoes (Xiong Bonkowski et al. (2001) indicated that the joint effects
et al., 2020), cucumber (Guo et al., 2021) and banana of protists and mycorrhiza significantly enhanced the
plants (Guo et al., 2022). In particular, numerous Cer- phosphorous uptake from roots to stems, as well as
cozoan and Amoebozoan species can function as affected rhizosphere microbes and essential plant nutri-
important indicators for the health of tomato plants. ents (carbon, phosphorous and trace elements), which
Guo et al. (2022) also revealed that the protistan con- maximized the biomass of different spruce compart-
sumer Cercomonas lenta strain ECO-P-01 substantially ments (shoots, stems and needles). Protists, in rumen
suppressed the density of the fungal pathogen Fusar- ecosystems, were detected to have positive links to
ium oxysporum and increased the disease-suppressive archaea (Solomon et al., 2022), which are key players
bacteria Bacillus in the rhizosphere, and subsequently in the global nitrogen cycle (Hu et al., 2015). However,
improved banana plant growth and yield. Hence, a the contribution of archaea to plant hosts and protist-
234 NGUYEN ET AL.

archaea relationships in nutrient cycle is an intriguing ecosystems (Nguyen et al., 2021), but further research
unexplored topic. Upon nutrient shortage, beneficial is required to disentangle mechanisms for the interplay
protist–microbe interactions may be boosted by the and roles of protists, fungi, bacteria, archaea, and
plant strategy ‘cry for help’, and we cannot have a full viruses in plant-associated microbiota. Beside the
understanding of protists’ roles if ignoring their aforementioned benefits, parasitic protists have nega-
contributions. tive effects on plants, as pathogens have been more
Protists can also significantly influence plant hor- thoroughly characterized than neutral and beneficial
mones and development through regulating the com- protists (Dumack & Bonkowski, 2021). A large number
munity structure and activities of plant-hormone of non-pathogenic endophytic protists inhabit plant tis-
producing rhizobacteria. Plant growth-promoting phyto- sues and across rainforest soil ecosystems (Mahé
hormones auxins (indolyl-3-acetic acid (IAA)) were et al., 2017), but their identity and functions on plant
found in the inoculation of the most studied model spe- hosts remain unknown. Given their high abundance in
cies A. castellanii in bacterial cultures (Nikoljuk, 1969) natural habitats, we suppose that endophytic protists
and rhizosphere of watercress seedlings (L. sativum) have unexplored benefits to plant hosts.
by modulating phytohormone-producing bacteria or rhi-
zobacterial community (Bonkowski & Brandt, 2002).
While root systems are paramount apparatus to take FUTURE FRAMEWORK FOR
up and allocate water and nutrients to every plant tis- UNRAVELLING THE ROLES OF PLANT-
sue for plant growth and environmental adaptation, pro- ASSOCIATED PROTISTS IN PLANT
tists, such as A. castellanii, can trigger phytohormone HEALTH AND PRODUCTION
production (auxins and cytokinin) of bacteria, resulting IMPROVEMENT
in the enhancement of root growth and architecture and
development of plants L. sativum and A. thaliana, more Protists alone or their interactions with other microbes
than bacteria standalone (Bonkowski & Brandt, 2002; are considered to play crucial roles in the plant holo-
Krome et al., 2010). Interestingly, the regulation on biont. It is promising to develop protist-based tools to
phytohormone-producing bacteria strengthens root enhance nutrient availability and plant growth as biofer-
growth and architecture of many crops, including water- tilizers, to control plant disease infection and microbial
cress, pea and cress (Table 1). Hence, it is evident that functions as biocontrol agents, or to promote plant hor-
soil- or rhizosphere-associated protists can significantly mones and nutrient cycling activities and survival of
influence both the above- and below-ground compart- plant beneficial microbes in modern agriculture. Com-
ments of the plant hosts. While microbes are acknowl- pared to other plant microorganisms, the functions, sig-
edged as important hormone producers of plants nalling and feedbacks of protists in multi-organismal
(Nakano et al., 2022), more explorations of protists’ (host–protist, protist–microbe, and protist–visiting
roles in the inter-organismal phytohormone networks insects) interactions with or without the infection of soil-
between plant hosts, protists and other microbes are borne or air-borne plant pathogens and pests are
critical to deploy beneficial protists in improving plant largely unexplored. A more comprehensive under-
immunity and development. standing of the molecular mechanisms and functions of
plant–protist–microbe interactions will enable us to
steer the activities and performance of microbes in the
Protists in bulk soils plant holobiont. Therefore, we propose and discuss
future frameworks to generate a holistic view of plant-
In bulk soils, the diversity of protists is higher than that associated protists and the manipulation and applica-
in the rhizosphere, root and litter (Ceja-Navarro tions of protist-based models in crop production,
et al., 2021; Fiore-Donno et al., 2022), which indicates namely: (i) identification of key factors structuring the
that soil protists function as a ‘microbial seed bank’ for taxonomic and functional traits of plant-associated pro-
plant support and soil functions, as well as the selection tists as well as the core and keystone taxa of protists;
of plants for protist communities. Moreover, protists can (ii) isolation and selection of plant beneficial protists for
influence elemental cycles, soil fertility and soil micro- various crops under different stresses; and
biome by (i) steering the composition and activities of (iii) establishment and applications of protist-based syn-
beneficial microorganisms (e.g., AMF or nitrifying thetic communities (SynComs) to improve plant perfor-
microbes), (ii) excreting nitrogen or carbon sources mance (Figure 2).
after the predation and consumption of prey in bulk Firstly, the identification of key factors structuring
soils, and (iii) mediating the community composition the taxonomic and functional traits of plant-associated
and interactions of soil microbiome via facilitative, sym- protists is a crucial step. To date, most studies have
biotic or predatory relationships between protists and characterized plant-associated protists by conventional
other microbes. The positive relationships between pro- (microscopy-based and direct counting) methods,
tists and bacteria have been identified in soil quantitative PCR or amplicon sequencing. Identifying
PROTISTS—PROMISING CANDIDATES FOR AGRIFOOD TOOLS 235

F I G U R E 2 Proposed framework for future studies of plant-associated protists and harnessing protist-based products for improving crop
production: (1) identification of key factors structuring the taxonomic and functional traits of plant-associated protists and plant core protists in
large-scale investigations; (2) isolation and selection of potential plant beneficial protist candidates through data integration and preliminarily
tests in short-term controlled conditions; (3) establishment and applications of protist-based synthetic communities (SynComs) to improve plant
performance in monoculture or crop mixture. Different SynComs constituted by different inoculants: single protist species, different protist
species, protists-bacteria, protists-bacteria-fungi, and protists-fungi. The integration of ‘omics’ techniques (i.e., metatranscriptomics,
metaproteomics and metabolomics) with integrated data analysis characterizes cellular activities, functions and metabolites of protists, plant host
and other organisms in the plant holobiont. The selection of crop species in this figure is just for illustration.

microbial eukaryotes with high throughput sequencing may dominate in such datasets (Urich et al., 2008; Gei-
techniques, however, is not straight forward, since sen et al., 2015). Furthermore, it is still difficult to esti-
severe primer-biases were identified in previous protist mate exact functioning of protists. Trait databases are
surveys (Lentendu et al., 2014; Hirakata et al., 2019). helpful for the exploration of functioning in microbial
For instance, although many soils are known to be eukaryotes (Dumack et al., 2020), but there is still a
dominated by protists of the taxa Amoebozoa and Cer- lack of a database covering all distinct protistan taxa.
cozoa, the primer-based surveys constantly underesti- The characterization of protists in different plant com-
mate the importance of Amoebozoa (Bonkowski partments (including phyllosphere, leaf, stem and root
et al., 2019). Metatranscriptomics can overcome this endosphere, rhizosphere soil and bulk soil) in large-
issue as they do not rely on primers and, in accordance scale field investigations is important to have full under-
to what is found by morphological surveys, Amoebozoa standings about their taxonomic and functional diversity
236 NGUYEN ET AL.

and community compositions for each plant species. because their performance may be boosted in faculta-
The combination with co-occurrence networks and sta- tive or antagonistic interactions with specific microbes.
tistical modellings will further disentangle the key Thirdly, protist isolates alone or combined with bene-
drivers and principles shaping the protist community ficial bacterial or fungal strains are used to construct dif-
assembly and dynamics in plant microbiome, as well as ferent protist-based synthetic communities to improve
build up a database of key protists that best predict plant health and performance. These SynComs can
plant performance parameters. mimic biological interactions (e.g., competition, predation
Many persistent and abundant members of a spe- or symbiosis) in natural settings, and the diversification
cific host found across wide-range habitats constitute a of trophic interactions (e.g., bottom-up and top-down
core plant microbiota, which carry essential genes to controls or trophic cascade) will boost microbes to pro-
support plant fitness as well as play crucial roles in duce crucial products (e.g., phytohormones, antibiotics
maintaining multiple functions and stability of the host and other compounds), and consequently stabilize the
microbiome (Shade & Stopnisek, 2019). Core bacterial phytobiome and promote crop development. We pro-
taxa, for example, members of the orders Rhizobiales pose to apply the protist-based SynCom models for
and Pseudomonadales, are reported to benefit plant fit- monoculture or mixture of plant species. Neighbouring
ness, growth and resilience under stresses (Trivedi crops in the plant mixture can increase interspecific
et al., 2020). Notably, keystone taxa of protists, highly interactions and functions of beneficial microbes, and
associated members regardless of their abundance, plant uptake of essential resources (nutrients or water),
deserve special attention because they crucially affect with positive consequences for disease suppression and
community structure and functions (Banerjee plant growth (Jing et al., 2022). All SynComs will be
et al., 2018). Therefore, the determination of core and assessed for their efficacy in benefiting plant fitness and
keystone taxa of protists for major crops across differ- growth, nutrient cycling and uptake, disease controls
ent regions, along with core and keystone taxa of bac- and stress tolerance for each plant species. Some
teria and fungi (Banerjee et al., 2018; Trivedi protist-based products, for example, have hit the market
et al., 2020), will leverage our capacity to manipulate and been applied in crop production, such as a protist
plant microbial activities and design optimal SynCom species Nosema Iocustae as a biological control agent
models for maximizing growth and yields of specific in over 90 species of grasshoppers, locusts, and crickets
crops. Crucial for this is a coupling of high throughput in the United States (https://www.gardeninsects.com/
sequencing and metatranscriptomic approaches with grasshopperbait.asp); and 19 biofertilizers developed
subsequent culture attempts, first to identify core sym- from a mixture of beneficial protists, bacteria and fungi
bionts and then to provide them as a culture to enhancing nutrients, plant growth and resilience for a
research. variety of crops in Netherlands (https://ecostyle.nl/
Secondly, to incorporate protists into the agrifood zoeken?query=protozoa).
toolbox, it is paramount to establish a collection of plant In this step, the integration of multiple ‘omics’ tech-
beneficial protists for various crops under different niques (including metatranscriptomics, metaproteomics
stress conditions. It is promising to tailor the high- and metabolomics) with machine learning and statisti-
throughput isolation approach which has proved to be cal modelling, rather than amplicon sequencing or one
effective in isolating bacterial strains from root micro- single method, will enable us to characterize the pano-
biota (Zhang et al., 2021), to characterize and isolate ramic profile of cellular activities, functions, molecular
protists from various plant tissues (e.g., leaf and stem signalling and metabolites of protists, plant host and
endophytes). In the first selection step, the data integra- other organisms in the plant holobiont. The metatran-
tion of plant protists from large-scale investigations with scriptomics elucidate the microbial identification, gene
findings of the high-throughput protist isolation will be a expression and functional profile of protists and other
crucial reference for selection and nomination of prom- organisms, while metaproteomics (e.g., matrix-assisted
ising protist species to establish protist-based Syn- laser desorption-ionization time of flight (TOF)/TOF-
Coms for improving plant performance. In the second mass spectrometry (MS)) is powerful to unravel protein
selection step, the selected protist species, alone or in identification, quantification and origin (Wang
a subset of core and keystone protists, can be prelimi- et al., 2011). Metabolomics can detect and quantify
narily tested for their capacity in performing desired untargeted primary metabolites (e.g., organic acids,
functions, such as suppression of common fungal path- amino acids, and others), such as by gas chromatogra-
ogens and resistance to abiotic stresses, in short-term phy (GC)-MS, and secondary metabolites produced by
controlled laboratory conditions. Core and keystone plant hosts, protists and other associated microorgan-
taxa of protists conferring desired plant-beneficial func- isms, such as by liquid chromatography (LC)-high-
tions will be considered as key members of the protist- resolution MS (LC-HRMS) (Weckwerth, 2010; Sumner
based SynComs. However, other core and keystone et al., 2015). The application of machine learning and
taxa of protists, which do not have the desired features statistical modelling with transcriptomic, proteomic and
in the preliminary tests, should not be discarded metabolomic data can maximize our capacity to identify
PROTISTS—PROMISING CANDIDATES FOR AGRIFOOD TOOLS 237

and predict compound composition, metabolic path- ultimately sustaining healthy agricultural systems.
way, functional traits and activities of protists with the Although the protists’ benefits to plants as bio-fertilizers
plant hosts and other organisms. For instance, the soft- or biocontrol agents have been aware, we still have lim-
ware METABOLIC is an advanced toolkit to profile met- itations on approaches studying the identity and func-
abolic and biogeochemical traits, and functional tions of protists, as well as challenges in how to
networks in microbial communities (Zhou et al., 2022). engineer efficient protist-based SynComs and how to
This integrated strategy will help us to explore and con- maintain their persistence and efficacy in crop produc-
firm the multifunctionality and benefits of plant- tion. The innovation of plant-beneficial products from
associated protists to plant hosts and understand the protists is a daunting task, but it will pay the ways to
complex trophic interactions within the plant–soil sys- accelerate the development of protist-based products
tem in a holistic manner. and to innovate novel mobile molecular technologies to
The development of protist-based SynCom models quickly assess and monitor the activities and commu-
as agrifood tools is potential to improve agricultural pro- nity composition of the applied beneficial microbiome in
duction. It is obvious that there will not be ‘one size fits smart-farming systems and agricultural fields in the
all’ SynComs (Vorholt et al., 2017), hence the construc- near future.
tion of protist-based solutions should target species- or We also highlight some important questions about
tissue-specific SynComs for distinct plants at different plant-associated protists in the plant holobiont: (1) What
developmental stages to optimize the efficacy in plant are biochemical or molecular signals that protists recruit
productivity, like commercial fertilizers or pesticides. or pay partnerships with other plant-associated micro-
Given the above-mentioned contributions of protists to organisms (e.g., bacteria or fungi) in benefiting plant
plants, we advocate for future efforts to target the hosts, as well as interact with insects (e.g., pollinators
development of beneficial protists as novel and sustain- or ants) or herbivores? (2) What individuals or groups
able biofertilizers for improving plant growth and pro- of protists are recruited by the plant hosts? (3) How do
ductivity, biological control agents for enhancing protists at different plant compartments respond to the
pathogenic defence, and biological stimulation strate- strategy ‘cry for help’ of plants under biotic (pathogen
gies for boosting microbial activities and plant- or pest infection) or abiotic (e.g., low/high temperature,
promoting traits for plant health and performance. Bio- drought or salinity) stresses? (4) What are interactions
fertilizers are gaining interests across the agricultural between plant hosts and plant or soil microbiome under
sector, due to the recent rapid increase of fossil fuel impacts of climate change? (5) Beside plant roots, do
price and fertilizer costs, the inoculation and formulation other plant tissues (e.g., leaf or stem) use a similar
of protists into biofertilizers will be powerful to unlock strategy ‘cry for help’ to interact with or recruit benefi-
natural nutrient sources or inorganic and organic fertil- cial protists and other microorganisms for dealing with
izers in soils. Nevertheless, the lack of sufficient knowl- different stresses? (6) How can SynComs and other
edge about the roles of protists in soil ecology limits our protist-based tools be safely introduced and applied to
ability to manage soil health for sustaining crop produc- recipient soils and crops? (7) How can we estimate and
tion. Future research on unravelling the functions and maintain the efficiency and persistence of protist-based
strategies of beneficial plant-associated protists is nec- SynComs and other tools in enhancing plant growth
essary to enhance plant health and production, thus and productivity in recipient soils and crops? The
reducing the application of fungicide and pesticides. answer to these questions is a challenge but also a
great opportunity to leverage our capacity to deploy
plant-associated microbiota to improve crop health and
CONCLUDING REMARKS performance. No single method but the integrated
advanced approaches can help us fully understand
Protists are key members of the plant-associated complex interactions in the plant holobiont.
microbiota. It is evident that their contributions alone or
in combination with other microorganisms significantly AUTHOR CONTRIBUTIONS
benefit plants in not only a single but multiple aspects, Bao-Anh Thi Nguyen: Conceptualization; literature
such as plant nutrition, disease control, plant health investigation; writing – original draft; writing – review
and performance. The interplay between the hosts and and editing. Kenneth Dumack: writing – review and
associated protists in different plant and soil compart- editing. Pankaj Trivedi: Writing – review and editing.
ments is complex and still far from being fully eluci- Zahra Islam: Writing – review and editing. Hang-Wei
dated. Therefore, there are calls to disentangle driving Hu: Conceptualization; funding acquisition; supervi-
factors and key roles of protists in ecological processes sion; writing – review and editing.
and agricultural productivity, which can provide new
insights into the manipulation and applications of bene- AC KN OW LED GE M EN T
ficial protists as biofertilizers and other agricultural This work was supported under the Australian
products in benefiting crop health and productivity and Research Council’s Industrial Transformation Research
238 NGUYEN ET AL.

Program funding scheme (IH200100023). Open access Clarholm, M. (1985) Interactions of bacteria, protozoa and plants
publishing facilitated by The University of Melbourne, leading to mineralization of soil nitrogen. Soil Biology and Bio-
chemistry, 17, 181–187.
as part of the Wiley - The University of Melbourne Cruz-Loya, M., Kang, T.M., Lozano, N.A., Watanabe, R., Tekin, E.,
agreement via the Council of Australian University Damoiseaux, R. et al. (2019) Stressor interaction networks sug-
Librarians. gest antibiotic resistance co-opted from stress responses to tem-
perature. The ISME Journal, 13, 12–23.
C ON F L I CT OF I NT E R E S T Dumack, K. & Bonkowski, M. (2021) Protists in the plant microbiome:
an untapped field of research. In: The plant microbiome.
We declare that we have no competing interests. New York, NY: Springer, pp. 77–84.
Dumack, K., Baumann, C. & Bonkowski, M. (2016) A bowl with mar-
ORCID bles: revision of the thecate amoeba genus Lecythium
Bao-Anh Thi Nguyen https://orcid.org/0000-0001- (Chlamydophryidae, Tectofilosida, Cercozoa, Rhizaria) including
6185-2790 a description of four new species and an identification key. Pro-
tist, 167, 440–459.
Kenneth Dumack https://orcid.org/0000-0001-8798- Dumack, K., Fiore-Donno, A.M., Bass, D. & Bonkowski, M. (2020)
0483 Making sense of environmental sequencing data: ecologically
Pankaj Trivedi https://orcid.org/0000-0003-0173- important functional traits of the protistan groups Cercozoa and
2804 Endomyxa (Rhizaria). Molecular Ecology Resources, 20,
Zahra Islam https://orcid.org/0000-0001-6965-9549 398–403.
Dumack, K., Feng, K., Flues, S., Sapp, M., Schreiter, S., Grosch, R.
Hang-Wei Hu https://orcid.org/0000-0002-3294-102X et al. (2022) What drives the assembly of plant-associated pro-
tist microbiomes? Investigating the effects of crop species, soil
R E F E REN CE S type and bacterial microbiomes. Protist, 173, 125913.
Arunkumar, N., Rakesh, S., Rajaram, K., Kumar, N.R. & Fiore-Donno, A.M., Human, Z.R., Štursova  , M., Mundra, S.,
Durairajan, S.S.K. (2019) Anthosphere microbiome and their Morgado, L., Kauserud, H. et al. (2022) Soil compartments (bulk
associated interactions at the aromatic interface. In: Plant soil, litter, root and rhizosphere) as main drivers of soil protistan
microbe interface. Cham: Springer, pp. 309–324. communities distribution in forests with different nitrogen deposi-
Bahroun, A., Jousset, A., Mrabet, M., Mhamdi, R. & Mhadhbi, H. tion. Soil Biology and Biochemistry, 168, 108628.
(2021) Protists modulate Fusarium root rot suppression by bene- Flues, S., Bass, D. & Bonkowski, M. (2017) Grazing of leaf-
ficial bacteria. Applied Soil Ecology, 168, 104158. associated Cercomonads (protists: Rhizaria: Cercozoa) struc-
Bamforth, S.S. (1973) Population dynamics of soil and vegetation tures bacterial community composition and function. Environ-
protozoa. American Zoologist, 13, 171–176. mental Microbiology, 19, 3297–3309.
Banerjee, S., Schlaeppi, K. & van der Heijden, M.G. (2018) Keystone Flues, S., Blokker, M., Dumack, K. & Bonkowski, M. (2018) Diversity
taxa as drivers of microbiome structure and functioning. Nature of Cercomonad species in the Phyllosphere and rhizosphere of
Reviews Microbiology, 16, 567–576. different plant species with a description of Neocercomonas epi-
Bonkowski, M. (2004) Protozoa and plant growth: the microbial loop phylla (Cercozoa, Rhizaria) a leaf-associated protist. Journal of
in soil revisited. New Phytologist, 162, 617–631. Eukaryotic Microbiology, 65, 587–599.
Bonkowski, M. & Brandt, F. (2002) Do soil protozoa enhance plant Gao, Z., Karlsson, I., Geisen, S., Kowalchuk, G. & Jousset, A. (2019)
growth by hormonal effects? Soil Biology and Biochemistry, 34, Protists: puppet masters of the rhizosphere microbiome. Trends
1709–1715. in Plant Science, 24, 165–176.
Bonkowski, M., Griffiths, B. & Scrimgeour, C. (2000) Substrate het- Geisen, S., Tveit, A.T., Clark, I.M., Richter, A., Svenning, M.M.,
erogeneity and microfauna in soil organic ‘hotspots’ as determi- Bonkowski, M. et al. (2015) Metatranscriptomic census of active
nants of nitrogen capture and growth of ryegrass. Applied Soil protists in soils. The ISME Journal, 9, 2178–2190.
Ecology, 14, 37–53. Geisen, S., Mitchell, E.A., Adl, S., Bonkowski, M., Dunthorn, M.,
Bonkowski, M., Jentschke, G. & Scheu, S. (2001) Contrasting effects Ekelund, F. et al. (2018) Soil protists: a fertile frontier in soil biol-
of microbial partners in the rhizosphere: interactions between ogy research. FEMS Microbiology Reviews, 42, 293–323.
Norway Spruce seedlings (Picea abies Karst.), mycorrhiza (Pax- Geisen, S., Hu, S. & Veen, G. (2021) Protists as catalyzers of micro-
illus involutus (Batsch) Fr.) and naked amoebae (protozoa). bial litter breakdown and carbon cycling at different temperature
Applied Soil Ecology, 18, 193–204. regimes. The ISME Journal, 15, 618–621.
Bonkowski, M., Dumack, K. & Fiore-Donno, A.M. (2019) The protists Guo, S., Xiong, W., Hang, X., Gao, Z., Jiao, Z., Liu, H. et al. (2021)
in soil—a token of untold eukaryotic diversity. In: Modern soil Protists as main indicators and determinants of plant perfor-
microbiology. Cham: CRC Press, pp. 125–140. mance. Microbiome, 9, 1–11.
Bruisson, S., Berg, G., Garbeva, P. & Weisskopf, L. (2020) Volatile Guo, S., Tao, C., Jousset, A., Xiong, W., Wang, Z., Shen, Z. et al.
interplay between microbes: friends and foes. In: Bacterial vola- (2022) Trophic interactions between predatory protists and
tile compounds as mediators of airborne interactions. pathogen-suppressive bacteria impact plant health. The ISME
Singapore: Springer, pp. 215–235. Journal, 16, 1–12.
Ceja-Navarro, J.A., Wang, Y., Ning, D., Arellano, A., Hassani, M., Dura n, P. & Hacquard, S. (2018) Microbial interactions
Ramanculova, L., Yuan, M.M. et al. (2021) Protist diversity and within the plant holobiont. Microbiome, 6, 1–17.
community complexity in the rhizosphere of switchgrass are Henkes, G.J., Kandeler, E., Marhan, S., Scheu, S. & Bonkowski, M.
dynamic as plants develop. Microbiome, 9, 1–18. (2018) Interactions of mycorrhiza and protists in the rhizosphere
Chakraborty, S., Old, K. & Warcup, J. (1983) Amoebae from a take-all systemically alter microbial community composition, plant shoot-
suppressive soil which feed on Gaeumannomyces graminis tritici to-root ratio and within-root system nitrogen allocation. Frontiers
and other soil fungi. Soil Biology and Biochemistry, 15, 17–24. in Environmental Science, 6, 117.
Chen, Q.-L., Hu, H.-W., He, Z.-Y., Cui, L., Zhu, Y.-G. & He, J.-Z. Herdler, S., Kreuzer, K., Scheu, S. & Bonkowski, M. (2008) Interac-
(2021) Potential of indigenous crop microbiomes for sustainable tions between arbuscular mycorrhizal fungi (glomus intraradices,
agriculture. Nature Food, 2, 233–240. Glomeromycota) and amoebae (Acanthamoeba castellanii,
PROTISTS—PROMISING CANDIDATES FOR AGRIFOOD TOOLS 239

protozoa) in the rhizosphere of rice (Oryza sativa). Soil Biology Nikoljuk, V. (1969) Some aspects of the study of soil protozoa. Acta
and Biochemistry, 40, 660–668. Protozoologica, 7, 1–37.
Hirakata, Y., Hatamoto, M., Oshiki, M., Watari, T., Kuroda, K., Rosenberg, K., Bertaux, J., Krome, K., Hartmann, A., Scheu, S. &
Araki, N. et al. (2019) Temporal variation of eukaryotic commu- Bonkowski, M. (2009) Soil amoebae rapidly change bacterial
nity structures in UASB reactor treating domestic sewage as community composition in the rhizosphere of Arabidopsis thali-
revealed by 18S rRNA gene sequencing. Scientific Reports, 9, ana. The ISME Journal, 3, 675–684.
1–11. Rybakova, D., Rack-Wetzlinger, U., Cernava, T., Schaefer, A.,
Hu, H.-W., Chen, D. & He, J.-Z. (2015) Microbial regulation of terres- Schmuck, M. & Berg, G. (2017) Aerial warfare: a volatile dia-
trial nitrous oxide formation: understanding the biological path- logue between the plant pathogen verticillium longisporum and
ways for prediction of emission rates. FEMS Microbiology its antagonist Paenibacillus polymyxa. Frontiers in Plant Sci-
Reviews, 39, 729–749. ence, 8, 1294.
Hu, H.W., Chen, Q.L. & He, J.Z. (2022) The end of hunger: fertilizers, Sapp, M., Ploch, S., Fiore-Donno, A.M., Bonkowski, M. & Rose, L.E.
microbes and plant productivity. Microbial Biotechnology, 15, (2018) Protists are an integral part of the Arabidopsis thaliana
1050–1054. microbiome. Environmental Microbiology, 20, 30–43.
Hubbard, C.J., Li, B., McMinn, R., Brock, M.T., Maignien, L., Schaeffer, R.N., Mei, Y.Z., Andicoechea, J., Manson, J.S. & Irwin, R.
Ewers, B.E. et al. (2019) The effect of rhizosphere microbes out- E. (2017) Consequences of a nectar yeast for pollinator prefer-
weighs host plant genetics in reducing insect herbivory. Molecu- ence and performance. Functional Ecology, 31, 613–621.
lar Ecology, 28, 1801–1811. Schmidt, O., Dyckmans, J. & Schrader, S. (2016) Photoautotrophic
Hutchings, M.I., Truman, A.W. & Wilkinson, B. (2019) Antibiotics: microorganisms as a carbon source for temperate soil inverte-
past, present and future. Current Opinion in Microbiology, 51, brates. Biology Letters, 12, 20150646.
72–80. Shade, A. & Stopnisek, N. (2019) Abundance-occupancy distributions
Jing, J., Cong, W.-F. & Bezemer, T.M. (2022) Legacies at work: to prioritize plant core microbiome membership. Current Opinion
plant–soil–microbiome interactions underpinning agricultural in Microbiology, 49, 50–58.
sustainability. Trends in Plant Science, 27, 781–792. Solomon, R., Wein, T., Levy, B., Eshed, S., Dror, R., Reiss, V. et al.
Jousset, A., Lara, E., Wall, L.G. & Valverde, C. (2006) Secondary (2022) Protozoa populations are ecosystem engineers that
metabolites help biocontrol strain Pseudomonas fluorescens shape prokaryotic community structure and function of the
CHA0 to escape protozoan grazing. Applied and Environmental rumen microbial ecosystem. The ISME Journal, 16, 1187–1197.
Microbiology, 72, 7083–7090. Song, C., Mazzola, M., Cheng, X., Oetjen, J., Alexandrov, T.,
Krome, K., Rosenberg, K., Bonkowski, M. & Scheu, S. (2009) Grazing Dorrestein, P. et al. (2015) Molecular and chemical dialogues in
of protozoa on rhizosphere bacteria alters growth and reproduc- bacteria–protozoa interactions. Scientific Reports, 5, 1–13.
tion of Arabidopsis thaliana. Soil Biology and Biochemistry, 41, Sumner, L.W., Lei, Z., Nikolau, B.J. & Saito, K. (2015) Modern plant
1866–1873. metabolomics: advanced natural product gene discoveries,
Krome, K., Rosenberg, K., Dickler, C., Kreuzer, K., Ludwig-Müller, J., improved technologies, and future prospects. Natural Product
Ullrich-Eberius, C. et al. (2010) Soil bacteria and protozoa affect Reports, 32, 212–229.
root branching via effects on the auxin and cytokinin balance in Sun, A., Jiao, X.Y., Chen, Q., Trivedi, P., Li, Z., Li, F. et al. (2021a) Fertil-
plants. Plant and Soil, 328, 191–201. ization alters protistan consumers and parasites in crop-associated
Leach, J.E., Triplett, L.R., Argueso, C.T. & Trivedi, P. (2017) Commu- microbiomes. Environmental Microbiology, 23, 2169–2183.
nication in the phytobiome. Cell, 169, 587–596. Sun, A., Jiao, X.Y., Chen, Q., Wu, A., Zheng, Y., Lin, Y.X. et al.
Lentendu, G., Wubet, T., Chatzinotas, A., Wilhelm, C., Buscot, F. & (2021b) Microbial communities in crop phyllosphere and root
Schlegel, M. (2014) Effects of long-term differential fertilization endosphere are more resistant than soil microbiota to fertiliza-
on eukaryotic microbial communities in an arable soil: a multiple tion. Soil Biology and Biochemistry, 153, 108113.
barcoding approach. Molecular Ecology, 23, 3341–3355. Święciło, A. (2016) Cross-stress resistance in Saccharomyces cerevi-
Liu, H., Macdonald, C.A., Cook, J., Anderson, I.C. & Singh, B.K. siae yeast—new insight into an old phenomenon. Cell Stress
(2019) An ecological loop: host microbiomes across multitrophic and Chaperones, 21, 187–200.
interactions. Trends in Ecology & Evolution, 34, 1118–1130. Trivedi, P., Leach, J.E., Tringe, S.G., Sa, T. & Singh, B.K. (2020)
Mahé, F., de Vargas, C., Bass, D., Czech, L., Stamatakis, A., Lara, E. Plant–microbiome interactions: from community assembly to
et al. (2017) Parasites dominate hyperdiverse soil protist communi- plant health. Nature Reviews Microbiology, 18, 607–621.
ties in neotropical rainforests. Nature Ecology & Evolution, 1, 1–8. Urich, T., Lanzén, A., Qi, J., Huson, D.H., Schleper, C. & Schuster, S.
Matz, C., Deines, P., Boenigk, J., Arndt, H., Eberl, L., Kjelleberg, S. C. (2008) Simultaneous assessment of soil microbial community
et al. (2004) Impact of violacein-producing bacteria on survival structure and function through analysis of the meta-transcrip-
and feeding of bacterivorous nanoflagellates. Applied and Envi- tome. PLoS One, 3, e2527.
ronmental Microbiology, 70, 1593–1599. Ushio, M., Yamasaki, E., Takasu, H., Nagano, A.J., Fujinaga, S.,
Mazzola, M., De Bruijn, I., Cohen, M.F. & Raaijmakers, J.M. (2009) Honjo, M.N. et al. (2015) Microbial communities on flower surfaces
Protozoan-induced regulation of cyclic lipopeptide biosynthesis act as signatures of pollinator visitation. Scientific Reports, 5, 1–7.
is an effective predation defense mechanism for Pseudomonas Vannette, R.L., Gauthier, M.-P.L. & Fukami, T. (2013) Nectar bacteria,
fluorescens. Applied and Environmental Microbiology, 75, 6804– but not yeast, weaken a plant–pollinator mutualism. Proceedings
6811. of the Royal Society B: Biological Sciences, 280, 20122601.
Nakano, M., Omae, N. & Tsuda, K. (2022) Inter-organismal phytohor- Vorholt, J.A., Vogel, C., Carlström, C.I. & Müller, D.B. (2017) Estab-
mone networks in plant–microbe interactions. Current Opinion in lishing causality: opportunities of synthetic communities for plant
Plant Biology, 68, 102258. microbiome research. Cell Host & Microbe, 22, 142–155.
Nguyen, B.-A.T., Chen, Q.-L., He, J.-Z. & Hu, H.-W. (2020) Microbial Wang, H.-B., Zhang, Z.-X., Li, H., He, H.-B., Fang, C.-X., Zhang, A.-J.
regulation of natural antibiotic resistance: understanding the et al. (2011) Characterization of metaproteomics in crop rhizo-
protist–bacteria interactions for evolution of soil resistome. Sci- spheric soil. Journal of Proteome Research, 10, 932–940.
ence of the Total Environment, 705, 135882. Weckwerth, W. (2010) Metabolomics: an integral technique in sys-
Nguyen, B.-A.T., Chen, Q.-L., Yan, Z.-Z., Li, C., He, J.-Z. & Hu, H.-W. tems biology. Bioanalysis, 2, 829–836.
(2021) Distinct factors drive the diversity and composition of pro- Xiong, W., Song, Y., Yang, K., Gu, Y., Wei, Z., Kowalchuk, G.A. et al.
tistan consumers and phototrophs in natural soil ecosystems. (2020) Rhizosphere protists are key determinants of plant
Soil Biology and Biochemistry, 160, 108317. health. Microbiome, 8, 1–9.
240 NGUYEN ET AL.

Zhang, J., Liu, Y.-X., Guo, X., Qin, Y., Garrido-Oter, R., Schulze-Lefert, P.
et al. (2021) High-throughput cultivation and identification of bacteria How to cite this article: Nguyen, B.-A.T.,
from the plant root microbiota. Nature Protocols, 16, 988–1012.
Dumack, K., Trivedi, P., Islam, Z. & Hu, H.-W.
Zhou, Z., Tran, P.Q., Breister, A.M., Liu, Y., Kieft, K., Cowley, E.S.
et al. (2022) METABOLIC: high-throughput profiling of microbial (2023) Plant associated protists—Untapped
genomes for functional traits, metabolism, biogeochemistry, and promising candidates for agrifood tools.
community-scale functional networks. Microbiome, 10, 1–22. Environmental Microbiology, 25(2), 229–240.
Zuccaro, A., Lahrmann, U. & Langen, G. (2014) Broad compatibility in Available from: https://doi.org/10.1111/1462-
fungal root symbioses. Current Opinion in Plant Biology, 20,
2920.16303
135–145.