*4.4. Microbiome*

Due to technological advances in high-throughput DNA sequencing, more and more studies have examined structures of microbial communities (microbiomes), both in soil ecosystems and in nematodes' guts in order to evaluate other biotic factors potentially involved in mediating interactions between nematodes and fungi [146]. These studies have revealed that while some microbes can exert antagonistic effects on both nematode pests and plant fungal pathogens, others can form mutualistic interactions with plant disease-causing agents. For example, in the root microbiomes of many plants, nematophagous fungi in genera *Clonostachys*, *Dactylellina, Purpureocillium*, *Pochonia*, and *Rhizophydium* often co-occur. However, the nematode-suppressing efficiency in such soils is often limited. Understanding the relationships among members of native anti-nematode microbiome are required for building effective approaches to develop nematode pest-suppressing soil [147]. For example, successive monoculture of soybeans clearly affected the assembly of both bacterial and fungal communities (i.e., the genera *Pseudomonas, Purpureocillium*, and *Pochonia*,) in the rhizosphere and negatively impacted the rhizosphere microbiome in its ability to suppress the soybean cys<sup>t</sup> nematodes [148]. Crop rotation may reverse such negative effects.

On the other hand, studies have revealed that the native microbiomes of nematodes carry a species-rich bacterial community dominated by Proteobacteria such as Enterobacteriaceae and members of the genera *Pseudomonas, Stenotrophomonas, Ochrobactrum,* and *Sphingomonas*. Several studies highlighted the influence of microbiota on *C. elegans* fitness, stress resistance, and resistance to pathogen infection [149]. For example, three *Pseudomonas* isolates were identified to be able to produce an anti-fungal e ffect in vitro and contribute to the worm's defense against fungal pathogens in vivo [146,150]. Another study using *C. elegans* suggested that probiotic yeasts colonizing the nematode gu<sup>t</sup> protected nematodes from infection with non-albicans *Candida* strains and alleviated pathogenic e ffects [151]. Unexpectedly, among the >5000 culturable fungal isolates obtained from the mycobiome of soybean cys<sup>t</sup> nematodes, using in vitro high-throughput screening, a large proportion of these cultured fungi showed bioactivity against nematode egg hatching or showed toxicity toward J2 stage nematodes [152]. Together, these results sugges<sup>t</sup> that supplying one or a few fungi with anti-nematode activities to the soil environments are not su fficient to suppress the nematode populations.

#### **5. Applications of Our Understanding in Fungi–Nematode Interactions in Agriculture: The Control of Phytophagous Nematodes and Soilborne Fungal Pathogens**

The rhizosphere contains a complex of biological and ecological processes. A better understanding of fungi–nematode complexes could benefit the development of ecologically based managemen<sup>t</sup> tools to control important plant pathogen and crop pests. Discoveries of antagonistic interactions between nematodes and some rhizospheric microorganisms can provide the basis for developing control strategies to enhance plant defense against soil-borne plant pathogens and root-knot nematode parasites, including *Meloidogyne* spp., etc. Below we discuss a few potential approaches.

#### *5.1. Nematodes as Biocontrol Agents against Plant Pathogenic Fungi*

Because fungal-feeding nematodes can be attracted to and actively feed on plant pathogenic fungi, these nematodes can potentially be used to reduce the load of fungal plant pathogens and minimize the e ffects of these fungal pathogens on crops.

Some species of the nematode genus *Aphelenchoides* feed on the cytoplasm of fungal hyphae by piercing and sucking using a strong stylet [37]. When mixed with the mycopathogenetic fungus *Trichoderma* spp., these nematodes are able to feed on two plant fungal pathogens, namely *Botrytis cinerea* and *Sclerotinia sclerotiorum* [6], and the combination of *T. harzianum* and *Aphelenchoides* nematode treatment resulted in the best disease control e fficiency against fungal diseases [153]. One species in this nematode genus, *Aphelenchoides hylurgi* is able to parasitize both virulent and hypovirulent strains of the fungus *Cryphonectria parasitica*, the causal agen<sup>t</sup> of chestnut blight [154]. Furthermore, the nematodes were also able to spread propagules of the hypovirulent strain, thus increasing the e fficacy of biological control under field conditions. Another fungal-feeding nematode, *Aphelenchus avenae*, also showed strong abilities to reduce pathogen loads of two root-rot fungi in corn [155], *Rhizoctonia solani* and *Fusarium solani* [156,157], as well as one root-rot fungus, *Fusarium oxysporum*, in beans and peas [158]. However, fungivorous nematodes are often not discriminatory in their food fungal choice. They can also feed on fungi with potential beneficial e ffects to plants. For example, *Trichoderma harzianum*, an extensively studied biocontrol agen<sup>t</sup> against the sclerotium-forming fungus, *Sclerotinia sclerotiorum*, is also a favorite food for the fungivorous nematode *Aphelenchoides saprophilus.* Consequently, *A. saprophilus* can reduce the biocontrol e fficiency of *T. harzianum* against *S. sclerotiorum* [159].

At present, most fungivorous nematodes reported in the literature are those that are easy to propagate in large numbers and can be stored in a dormant stage (anhydrobiosis) for a relatively long time. They have so far not been extensively applied to agriculture or horticulture fields in the form of nematode applications. This is mainly due to the high costs associated with the production, storage, and distribution of fungivorous nematodes for commercial applications. One way to realize such commercial potential is to combine fungivorous nematodes with other agricultural practices

such as crop rotation and the application of other biocontrol agents to reduce the costs and maximize the benefits.

#### *5.2. Biocontrol of Nematodes with Nematophagous Fungi*

It is estimated that, worldwide, plant parasitic nematodes (PPNs) cause a combined >\$150 billion worth of damages to agriculture each year [31]. From an ecological perspective, this group of nematodes is one of many components in the ecosystem that interact with other organisms, contributing to the maintenance and stability of the soil food-web. Over the last 30 years, our understanding of microbial diversity and the multitrophic interactions that are manifested in the rhizosphere, as well as biological control systems as they apply to nematodes, has improved tremendously. Indeed, several environmentally benign strategies have been developed for PPN management. Among PPNs, the root-knot nematodes (RKNs; *Meloidogyne* spp.) represent the most severe challenges to crop production. In a summary of the biocontrol methods evaluated between 2015 and April 2020, 10 microfungi and 3 mushroom species were tested for their e ffectiveness in controlling RKNs [160]. However, most studies were conducted in laboratories and greenhouse settings and their e fficacies in the field are not known. Converting the laboratory successes into equally e ffective field applications represents the next step of the challenge.

#### 5.2.1. Potential for the Discovery of Novel Candidates

It has been estimated that the number of culturable fungal species is between 2.2 and 3.8 million. On the basis of a 1:8.8 ratio between the numbers of cultured fungal species and the number of fungal operational taxonomic units estimated based on metagenome sequencing, there would be approximately 12 million fungal species on earth [161,162]. At present, only ~1.2% (140,000) of these have been described [163]. Thus, many new fungi with potential nematophagous activities await discovery. Even among the known culturable fungi, new compounds with novel mechanisms of nematode-parasite action have been continuously found. For example, in the fungus *Pleurotus ostreatus,* anthelmintic compounds were recently isolated and showed potent activity against a wide range of nematode species. It is possible that there are many novel fungi and novel fungal compounds e ffective at controlling parasitic nematodes of plants, animals, and humans [79].

Over the last few years, high-throughput sequencing of the universal barcode locus for fungi (18S, ITS rDNA) has revealed grea<sup>t</sup> potential for identifying fungi in many ecological niches. However, the fungal community comparisons between niches with di fferent nematodes, especially those with di fferent phytopathogenic nematodes, have been very limited. Microbiome studies of such ecological niches could help reveal the microbial diversities responsible for the di fferential distributions of phytopathogenic nematodes and assist in developing holistic managemen<sup>t</sup> strategies with multi-target modes of action to control these pests. Integration of microfluidics, robotics, and machine learning technologies in interaction studies in microcosms between the microbiome and nematodes could provide novel ways to capitalize on our knowledge about the core microbiomes of pest nematodes. Such knowledge could help increase control e fficiency and stress-resistance of biocontrol applications [164]. On the other hand, novel molecular markers could be developed to analyze the parasitic activities and population dynamics of nematophagous fungi. Such tools could allow us monitoring these fungi and their activities in agricultural fields.

#### 5.2.2. Development and Integration of New Methods

To achieve successful and reproducible biological control, we must understand the ecological interactions a ffecting the control agen<sup>t</sup> and the target. Modern technologies can help us achieve such goals. For example, real-time quantitative PCR provides an e ffective way to quantify and track biocontrol agents after they are applied to soil [165]. Similarly, genetic modifications of the biocontrol agents could be used to help the organisms overexpress traits involved in pathogenicity or nematocidal activity [8,166].

Several studies have demonstrated the effectiveness of using combinations of treatments, including various cultural practices (like soil solarization and soil amendment), chemical nematicides, and biological agents in controlling PPN populations under various conditions [80]. These studies have revealed that soil physical chemical properties can have a significant influence on their control efficacies. Thus, attention should be paid to develop biocontrol protocols that are specific for targeting ecological niches.

The use of nematophagous fungi as endophytes, i.e., rhizosphere colonization by biocontrol agents, is a promising strategy for implementing biocontrol of plant-parasitic nematodes. Endophytes should be relatively easy to apply as inoculants to seeds or seedlings and could therefore be established in the root system before nematodes are attracted to roots [167].

Finally, the unpredictability and relatively low efficacy of nematode antagonists against PPN in field conditions are major obstacles for the application of biocontrol agents for managing plant-parasitic nematodes. Part of the reasons for the differences between laboratory-based and field-based trial results may be related to the intrinsic mechanisms regulating ecosystem stability in field conditions. Application of a large number of a specific organism would disturb the balance of interactions among organisms in native niches, with the target interaction between the applied biocontrol agen<sup>t</sup> and PPN in the field not realized. Thus, understanding how organismal interactions in native niches are regulated could help us develop better applications that take into account native agricultural ecosystems to ultimately produce sustainable methods of crop protection while maintaining biodiversity. Studies that evaluate the effects of coadministration of multiple partners such as nematophagous fungi, mycoparasites of plant pathogens, and plant growth promotors could help generate significant data to allow a systems approach in developing biocontrol measures to minimize the effects of nematode pests and fungal pathogens on agricultural crops [168,169].

**Funding:** This research was jointly funded by the National Natural Science Foundation of China (31760010) to Y.Z., the "Double First Class" Research Project in Yunnan University (C176280107) to J.X., and the "Top Young Talents Program of the Ten Thousand Talents Plan" in Yunnan Province to Y.Z.

**Acknowledgments:** We thank Ivan M. Dubovskiy for the invitation.

**Conflicts of Interest:** The authors declare no conflict of interest.
