Current Utility of Plant Growth-Promoting Rhizobacteria as Biological Control Agents towards Plant-Parasitic Nematodes

Plant-parasitic nematodes (PPN) are among the most economically and ecologically damaging pests, causing severe losses of crop production worldwide. Chemical-based nematicides have been widely used, but these may have adverse effects on human health and the environment. Hence, biological control agents (BCAs) have become an alternative option for controlling PPN, since they are environmentally friendly and cost effective. Lately, a major effort has been made to evaluate the potential of a commercial grade strain of plant growth-promoting rhizobacteria (PGPR) as BCAs, because emerging evidence has shown that PGPR can reduce PPN in infested plants through direct and/or indirect antagonistic mechanisms. Direct antagonism occurs by predation, release of antinematicidal metabolites and semiochemicals, competition for nutrients, and niche exclusion. However, the results of direct antagonism may be inconsistent due to unknown endogenous and exogenous factors that may prevent PGPR from colonizing plant’s roots. On the other hand, indirect antagonism may occur from the induced systemic resistance (ISR) that primes whole plants to better fight against various biotic and abiotic constraints, actuating faster and/or stronger defense responses (adaption), enhancing their promise as BCAs. Hence, this review will briefly revisit (i) two modes of PGPR in managing PPN, and (ii) the current working models and many benefits of ISR, in the aim of reassessing current progresses and future directions for isolating more effective BCAs and/or developing better PPN management strategy.


Introduction
Plant-parasitic nematodes (PPN) are biotrophic parasites that cause billions of dollars in economic damages on major food crops including corn (Zea mays L.), potato (Solanum tuberosum L.), soybean (Glycine max (L.) Merr.), as well as an important fiber crop, cotton (Gossypium hirsutum L.) [1]. Thus, a variety of management strategies using chemical nematicides, cultural control, resistant varieties and biological control (BCA) have long been tested, and employed to manage PPN. Nematicides, however, may not always be environmentally friendly, having hazardous and non-target impacts on flora and fauna including many natural enemies [2]. Although nematicides may initially reduce PPN populations and provide protection from their damage, these plants may allow the increase of populations at the end of the growing season. The following year will require an additional application of costly nematicides [3]. On the other hand, cultural control and resistant cultivars are not always available [4,5]. Hence, increasing attention has lately been drawn to the potential use of BCAs as an effective, inexpensive, and environmentally friendly tools of PPN management [3].

Recent Increases in Agronomic Burden by PPN
PPN, microscopic roundworms, belonging to the phylum Nematoda that are among the most abundant animal on earth; over 4100 species have been found in a variety of environmental conditions. The majority of PPN live in the film of moisture surrounding soil particles and plant roots. PPN have protrusible stylets or mouth spear to enter root tissues [1]. On the basis of their feeding habitats, PPN are chiefly classified into two groups, ectoparasites, and endoparasites. Ectoparasites feed from the outside of root surfaces without entering into plant roots. Thus, they could be more susceptible to environmental stresses and predators, and causing lesser damage to plant roots, than endoparasites. Endoparasites are able to penetrate completely or partly into plant roots during the infection process. This causes physical injury as well as allows secondary damages by bacteria and fungi infected along with or after PPN [113]. Damages caused by endoparasitic nematodes including Meloidogyne spp. (root-knot nematodes; RKN), Heterodera spp., Globodera spp. (cyst nematodes), Pratylenchus spp. (lesion nematodes), and Rotylenchulus spp. (reniform nematodes) are estimated to result in an annual loss of~13% (~$216 billion) worldwide [114]. For instance, two genera of PPN, Meloidogyne spp. and Heterodera spp., alone cause the~10% reduction of major food production including wheat (Triticum aestivum L.), rice (Oryza sativa L.), corn, potato, and sweet potato (Ipomoea batatas L.), which totaled an estimated loss of~$80 to 114 billion per year. Rotylenchulus spp. has also emerged as a major threat in cash crops, cotton, and soybean, over the last decade throughout the southern regions of the US causing an estimated yield loss of over $200 million annually [1,[115][116][117]. Hence, the establishment of effective and sustainable management program to control PPN has become an urgent task across the nations. However, current methods of integrated PPN management are rather restricted, when compared to that for other insect and animal pests, because PPN are microscopic and generally attack the underground portion of plants. The damage caused by PPN also are often not correctly identified because the aboveground symptoms mimic other diseases and environmental root-limiting factors delaying proper management [118,119].

Current Management Method against PPN
The current integrated pest management (IPM) program aimed at PPN largely strategizes the combined uses of crop rotation, tolerant/resistant cultivars, and nematicides. These methods, however, are still inadequate, expensive and/or cause numerous unexpected ecological and social drawbacks [120,121]. Presently, the most common strategy to manage PPN infections and field infestations is the application of nematicides (e.g., aldicarb, fluazaindolizine, fluensulfone, fluopyram, oxamyl, and terbufos) [122,123]. Nematicides have been an effective management tool; however, chemical nematicides are harmful to the environment, dangerous to apply and very costly [118]. Therefore, crop rotation has long been recommended as an alternative and potentially effective cultural practice to reduce nematode population density [5], but is not be a feasible option for all crops and farmers. For example, in the southern areas of the US, one of the most efficient crop rotations schemes for RKN, Meloidogyne incognita (Kafoid and White, 1919) [124] infested fields include the nonhost crop of peanut (Arachis hypogaea L.) [5,125]. However, peanuts require different harvesting equipment compared to cotton, soybeans, and corn, the most prominent row crops in the region. This harvesting equipment can range into the hundreds of thousands of dollars, and therefore may not be an economically feasible strategy.
Resistant cultivars increasingly have become available, preventing PPN from reproducing at as high a level as with susceptible cultivars; however, these cultivars are often accompanied by decreased yield [126]. Thus, some groups have advocated the use of tolerant cultivars that do not decrease the PPN reproduction but have little effect on yield potential [127]. The crucial drawback of tolerant cultivars though is that they cause an increase in the field population density of PPN. Thus, the use of resistant and tolerant cultivars is the most successful when used as a part of IPM system. Hence, alternative strategies must be looked at for effective, sustainable management of PPN. One, emerging option is biological control, which encompasses beneficial microbes such as PGPR that can reduce the impact of PPN, and other biotic attackers and abiotic stresses, as well as synergistically help the plant enhancing growth and development of various plant species [8].

Current utility of PGPR as BCAs of PPN; Mode of actions of PGPR
Recent studies have proposed that biological control could be a cost-effective approach to manage PPN when compared to the conventional methods. Especially, PGPR have emerged as promising BCA candidates [128,129]. PGPR are non-pathogenic bacteria, known to enhance plant growth and development in both non-stressed and stressed conditions by direct and indirect mechanisms [130]. The direct mechanism describes PGPR as bio-fertilizers producing organic compounds that promote plant growth or increase uptake of soil nutrients. Indirect mechanisms refer to PGPR-dependent biocontrol, including the production of antibiotics, Fe chelators (called as siderophores), and external cell wall degrading enzymes (e.g., chitinase and glucanase) that perhaps hydrolyze the pathogen (i.e., fungus) cell wall [131]. In line with this scenario, a number of PGPR, frequently from the genera of Agrobacterium, Bacillus, Paenibacillus and Pseudomonas, have been documented to reduce PPN population density through different mechanisms like parasitism, production of hydrolytic enzymes and antinematicidal metabolites (direct antagonism; Table 1), and inducing systemic resistance (indirect antagonism; Table 2) [130].
Indeed, the exogenous application of P. penetrans demonstrated drastic decreases in the population density of M. javanica (Treub, 1885) [123] in sugarcane (Saccharum officinarum L.), by an average of 97.5% [43]. Thus, a few Pasteuria spp. have been made into commercial products; e.g., Clariva pn, Naviva ST and NewPro (Table 3). However, these products appear to be only active on one or two target species limiting actual practicality and use for field conditions. Producers often look for products that confer a broader spectrum of disease resistance, as well as defense responses against PPN.
On the other hand, a number of PGPR belonging to Bacillus spp. and Pseudomonas spp. produce nematicidal compounds. For example, several strains of Pseudomonas fluorescens (Flüge, 1886) [134] reduce potato cyst nematodes (PCN, Globodera spp.) and RKN (M. incognita) levels in vitro and/or in soil conditions by releasing DAPG, a phenolic antiphytopathogenic metabolite [49]. The wild type (WT) bacterium can reduce the motility of juvenile PCNs by 85%, comparing to 27% by mutant strains that disrupted DAPG productions [49]. On the other hand, P. fluorescens strain CHA0 and P. aeruginosa (Schröter, 1872) [135] produce the volatile compound HCN, an extremely poisonous toxin that adversely affects the proper functions and formations of mitochondria in serval RKN spp. (e.g., M. incognita and M. javanica) [46,50]. Interestingly, P. fluorescens strain CHA0 also releases both DAPG and pyoluteorin, two secondary metabolites that can significantly decrease population density of M. javanica in vitro and in a soil setting [51,53].
However, current attempts to commercialize PGPR for PPN management has had limited success [143]. A major concern has been that PGPR is inconsistent in managing PPN in the field, possibly due to many endogenous and exogenous factors limiting PGPR root colonization. Factors include other plants, variable soil conditions, and various rhizospheric metabolites and organisms [144]. Thus, there is a need to further investigate enhancing and/or optimizing the BCA activity of PGPR. Many studies now have turned into investigate the ability and a potential efficacy of indirect antagonism (i.e., ISR) of PGPR against PPN as an alternative, practical way to ensure the improvement of the defense capacity of plants against PPN, as well as other pathogens and abiotic constraints, and concurrently to enhance plant growth and development.
On the other hand, selective results from earlier studies argued otherwise. Root inoculations of PGPR immediately triggered JA production and signaling transductions in systemic (leaf) tissues, which in turn heighted the state of defense responses throughout the plant [158]. Inoculation of PGPR Serrattia marcescens (Bizio, 1823) [159] strain 90-166 and P. fluorescens WCS417r showed rapid upregulation of several JA-biosynthetic and -responsive genes, e.g., VSP, PDF1.2, HEL, ChiB, LOX, and PAL in systemic (leaf) tissue throughout a number of crop plants, as well as Arabidopsis [61,160]. JA production then coordinates with abscisic acid (ABA) signaling to activate selective stress-responsive transcription factors such as OCP3 (Overexpressor of Cationic Peroxidase 3) and MYB60 [161,162], which in turn regulate stomatal closure, and suppress plant growth and yield [163]. These signaling and metabolic cascades may explain the inhibitory effect of JA in plant growth and development [164]. Indeed, the exogenous application of JA characteristically results in the inhibition of root growth [6,165,166], together suggesting that (i) ISR (and PGPR-induced JA signaling) could compete resource allocations (namely, defense and growth tradeoffs) with PGPR-mediated growth enhancement, or (ii) ISR employs alternative and/or additional (besides JA-responsive) signaling and/or metabolic pathways to prime plant disease resistance without reducing yield potential.

Potential Roles of SA in the Indirect Antagonism, SAR of PGPR against PPN
Recently, selective data argues that PGPR could stimulate SAR (instead of ISR), widely known to be developed by pathogenic microbes but not by PGPR [7,131]. The caveat is that ISR and SAR require mutually antagonistic cellular mechanisms, JA signaling for ISR versus SA signaling for SAR [6,7], which initially conflicted the role of SAR and SA signaling in the indirect antagonism of PGPR. However, a few studies reported that several Bacillus spp. can stimulate SA accumulations and signaling in plants ( Table 2), hypothesizing that a mode of ISR mimics or shares SAR mechanisms. For instance, B. amyloliquefaciens MBI600, active ingredient of the biological fungicide Serifel ® , was characterized to activate SA-dependent, but JA-independent, immunity, and reduce the disease severity of tomato spotted wilt virus in tomatoes [61]. It is notable that independent studies from other groups using similar PGPR strains such as B. amyloliquefaciens, B. mycoides (Flugge, 1886) [167], B. pumilus (Meyer and Gottheil, 1901) [168], and B. subtilis reported an induction of JA signaling (see Table 2). The cause for this contradiction is not understood, but may result from different experimental conditions and/or experimental errors. Alternatively, there may be timing differences to induce JA or SA signaling by PGPR (JA first and SA latter). A recent study using time-resolved transcription analyses indicated that the inoculation of Trichoderma harzianum (Rifai, 1969) [169] rapidly activates JA signaling on a time scale of hours, but later switches to enhance SA activity in 3 to 7 days in both infected and systemic tissues of plants [89]. This finding suggests the presence of complex and/or alternative mechanisms in PGPR-mediated systemic resistance. Further investigations must clarify the combined or antagonistic roles and functions of different plant hormone signaling such as JA, ET, SA, and ABA [170] in conveying defense activation by PGPR.

Crosstalk between SA and JA Signaling
As alluded, JA and SA signaling crosstalk with each other [171,172], and these interactions can be either antagonistic or synergistic. One of the first investigations into their interaction revealed an antagonistic relationship between JA and SA in tomatoes. They observed that aspirin, formulated SA, significantly suppressed the expression of JA marker genes [173]. Similarly, the expression of SA marker genes was inhibited upon the stimulation of JA by Pseudomonas syringae in tomato [174]. Furthermore, the application of exogenous JA also correlated with decreased SA activity, and enhanced JA signaling [175]. A more in depth look into the antagonism demonstrated that SA signaling targets the downstream of promoter in JA-responsive genes, and inhibits any further JA activity [176]. It is hypothesized that the antagonism be-tween the two hormones (JA and SA) helps plants conserve energy, and optimize the defense pathways in the presence of a single pathogen [171,172].
In contrast, a few early studies suggested a potential, synergistic relationship between JA and SA, as the pathogens Botrytis cinerea (Persoon, 1794) [177] and Tobacco mosaic virus stimulate both JA and SA at the same time [178,179]. Mur et al. later suggested that the concentration of hormones determined the interaction between them [180]. If the level of JA or SA is too high there was an antagonistic relationship; however, if the levels of each hormone were lower, the two hormones acted synergistically [180]. The exact concentration where synergism between the two hormones turns to antagonism is yet to be determined.
The goal of biological control research is to optimize the BCA to be as effective and efficient as possible against the target pathogen(s), and determining any manipulation of the JA and SA pathways by the target pathogen is an essential part of this type of research. This can, in turn, determine the best pathway to provide the most effective protection and defense against the target pathogen. This is important to keep in mind when selecting the best BCA in each management strategy.

Commercialization of PGPR
There is still a fundamental question (i.e., reproducibility) to address for the commercialization of PGPR, though multiple strains of PGPR are available on the market as biological nematicides (Table 3). However, the efficacy of these products needs to be further evaluated. To be commercially successful, PGPR products should have a broad application, long shelf life, be safe to use, have a viable market, easy availability, be consistent and have a low investment cost. Indeed, a large number of PGPR strains still remain to be investigated (Table 4). One particularly noticeable genus of PGPR is Bacillus spp. (9 direct antagonism, 7 indirect antagonism, and 17 uncharacterized); considered to be good options because they can quickly replicate and colonize plants, tolerate harsher environments, and easily form endospores. Moreover, they are documented to affect a broad spectrum of plant pathogens including PPN, viruses, bacteria, and fungi [181]. An extra benefit is that many of these Bacillus spp. can promote plant growth and help the plant adapt abiotic stresses, enhancing yield potential [182,183]. Bacillus spp. are the main species of commercialized PGPR due to their hardiness, as described above, compared to other effective PGPR such as Pseudomonas spp. [184]. More studies however are required to assess more effective mechanisms of their modus operandi as BCAs of PPN.

Conclusions
Emerging results have proposed the potential use and value of PGPR as BCAs in managing PPN. Selective strains of PGPR, in particular many Bacillus spp. appear to effectively suppress the growth and infestation of PPN in plants and fields, via direct or indirect antagonistic mechanism. Initially, much effort was invested to isolate (1) predatory PGPR strains and/or (2) PGPR-released nematicidal toxins and hydrolytic enzymes, which readily kill or inhibit PPN. However, their products were often highly host-specific, limiting actual use and practicality for field applications. Thus, recent studies have tried to understand, and improve the indirect antagonism (ISR) of PGPR, especially since PGPR have (i) longer shelf life, (ii) easy availability, (iii) low investment-and production-cost, and are (iv) safe to use. Moreover, PGPR are (v) effective biostimulants facilitating plant growth and yield potential, as well as are able to (vi) enhance plant resistance against a broad range of microbial pathogens, pests, and insect herbivores (i.e., ISR), and (vii) concurrently prime plants to better adapt or acclimate towards various abiotic stresses including drought, salinity, and extreme temperatures. These multifaceted activities, and benefits clearly underpin important merits and practicality for further screening and accessing the activity and efficacy of new PGPR in managing economically damaging PPN. Successful development/commercialization of BCAs will greatly aid in upgrading plants' own survival capacity against various ecological constraints, but without trading off yield potential.