Trichoderma virens and Pseudomonas chlororaphis Differentially Regulate Maize Resistance to Anthracnose Leaf Blight and Insect Herbivores When Grown in Sterile versus Non-sterile Soils

1. Introduction

Maize is the top-yielding cereal crop produced globally and a major source of livestock feed, biofuel, and staple food for many regions worldwide [1]. Pathogen infection and insect herbivory are constant threats to crop production causing over 20% yield losses of maize annually at a global level, resulting in substantial economic impact and food insecurity [2]. The hemibiotrophic fungal pathogen Colletotrichum graminicola is the causal agent of anthracnose leaf blight and stalk rot of maize and accounts for millions of dollars in yield losses annually in the United States [3-5]. Fall armyworm (FAW; Spodoptera frugiperda) originates in the Americas and has recently invaded Europe, Africa, Asia, and Australia causing serious damage [6, 7]. FAW larvae feed on all the aboveground tissue of maize, resulting in several billions of dollars in yield losses globally [6]. Western corn rootworm (WCR; Diabrotica virgifera virgifera) is the most economically important maize pest in the United States and causes losses in excess of US $1 billion annually in the United States and Europe [8-10]. WCR larvae cause the most significant damage through feeding on the corn root while WCR adult beetles damage the silks [8, 11]. Published evidence indicates that the plant phytohormone salicylic acid (SA) plays a critical role in maize defense against C. graminicola [12, 13], while resistance to chewing insects, including FAW, is mainly mediated by the lipid-derived phytohormone jasmonic acid (JA) [14-16]. However, resistance mechanisms against root-feeding WCR remain largely unknown [17].

Microbial biological control agents, including plant growth-promoting rhizobacteria and fungi (PGPR and PGPF, respectively), have been widely used to increase resistance in plants against pathogens and pests [18-20]. Isolates of soil-borne Trichoderma spp. and bacterium Pseudomonas chlororaphis are well-known biocontrol agents and have been extensively studied for their beneficial traits, including growth promotion and enhanced disease resistance of the host plants [21-24]. However, using biocontrol agents against pests has only recently been considered, and little is known about their impact on defense against chewing insects [25, 26]. Several studies have shown that T. virens induces systemic resistance (ISR) against leaf-infecting pathogens, the physiological process tightly regulated by fungal small secreted proteins, Sm1 and Sir1, which play pivotal but contrasting roles in triggering ISR responses [27-30]. Analyses of the T. virens sm1 deletion mutants revealed that they are unable to induce ISR against fungal pathogens in above-ground organs, suggesting that Sm1 peptide is a positive regulator of ISR [27, 28]. In sharp contrast, deletion of SIR1 resulted in much stronger ISR response [29]. Wang et al. (2020)[30] showed that T. virens colonization in maize roots upregulated genes involved in biosynthesis of JA precursors, including 12-OPDA (12-oxo-10(Z),15(Z)-phytodienoic acid) and OPC-4:0 {(Z)-4-[3-oxo-2-(pent-2-en-1-yl)cyclopentyl]butanoic acid}, yet downregulated genes downstream of 12-OPDA for JA biosynthesis. Whether T. virens colonization affects wound-induced JA production in leaves and confers resistance to leaf-feeding FAW and root-feeding WCR larvae remains unexplored.

The principal objective of this study was to investigate the effects of T. virens and its secreted peptide signals on the production of wound-induced JA and other defensive oxylipin metabolites and to test whether the beneficial effects of this endophyte extend to increasing resistance against FAW and WCR. Contrary to our expectation, the results demonstrated that maize seedlings grown in sterile soil amended with wild-type T. virens, the sm1 or sir1 single or the double mutants were found to suppress wound-induced JA biosynthesis, resulting in reduced resistance to FAW. We followed up by testing whether such a detrimental effect on defense against herbivory could be ascribed to another well-studied growth-promoting bacterial endophyte, P. chlororaphis. Colonization of roots by the P. chlororaphis 30-84 also reduced production of JA and defense against FAW. Further analysis showed that while both T. virens and P. chlororaphis induced ISR against C. graminicola when added in sterile or nonsterile soil, suppression of defense against FAW occurred only when maize seedlings were grown in sterile soil amended with these biocontrol agents. In addition, we demonstrated that T. virens reduced survival and growth (weight gain) of WCR larvae, making our study the first showing the potential of T. virens as a biocontrol agent against this devastating pest of maize.

2. Results

2.1. Colonization of Maize Roots by either Δsir1 or Δsm1Δsir1 Enhanced Greater Levels of ISR against C. Graminicola

Previously, we have shown that T. virens colonization triggered ISR against C. graminicola in a Sm1 peptide-dependent manner as the Δsm1 mutant lost its ISR-inducing capability [28, 30]. In contrast, plants treated with the deletion mutant of Sir1 peptide, Δsir1, exhibited enhanced systemic resistance [29, 30]. Moreover, Wang et al. (2020)[30] showed that the expression of SIR1 in Δsm1 remained similar to the level in wild-type T. virens strain (TvWT), while Δsir1 had elevated expression of SM1. These results suggested that one potential reason for increased ISR by Δsir1 is due to elevated SM1 expression in this strain. To test this possibility, we created Δsm1Δsir1 double mutant and tested its capability to induce resistance to C. graminicola. The result showed that colonization of both Δsir1 and Δsm1Δsir1 mutants triggered greater level of ISR against C. graminicola compared to TvWT (32%, 59%, and 56% reduction in lesion areas on TvWT-, Δsir1-, and Δsm1Δsir1-treated plants, respectively, compared to control) (Figure 1). These results suggest that a functional Sir1 suppresses disease resistance to C. graminicola by suppressing not only SM1, but other as yet unknown mechanisms.

2.2. T. virens Colonization Suppressed Insect Defense

Numerous studies have shown that beneficial Trichoderma spp. induce resistance against pathogens [31-33]. However, their effect on inducing insect resistance remains largely unexplored. Here, we tested whether different T. virens stains, including Δsm1, Δsir1, and Δsm1Δsir1, induce differential resistance to the foliar-feeding FAW. Surprisingly, the results showed that FAW larvae gained significantly more weight after feeding on T. virens-colonized plants for 7 days (60% to 95% increase) (Figure 2A) and 3rd-instar larvae consumed more leaf area on T. virens-colonized plants (64% to 83% increase) (Figure 2B). Treatment with T. virens reduced resistance to FAW, regardless of the mutation of these two signaling peptides.

2.3. T. virens Treatment Reduced Wound-Induced JA Accumulation by Suppressing Biosynthesis and Enhancing Catabolism

A previous study showed that treatment with T. virens downregulated JA biosynthesis genes in colonized maize roots [30] and that JA dominantly modulates defense responses against chewing insects [14]. In the present study, we found that T. virens treatment decreased insect defense, which prompted us to test whether T. virens colonization regulates wound-induced JA production in leaves. Phytohormone and oxylipin profiling showed that significantly lower amounts of jasmonates, including JA precursors, 12-OPDA, OPC-4:0, JA, and the bioactive JA derivatives, JA-Isoleucine (JA-Ile), JA-leucine (JA-Leu), and JA-Valine (JA-Val) conjugates, were accumulated in the T. virens-treated plants at 1 and 2 hours post wounding (hpw) (Figure 3A). Specifically, JA accumulation was reduced by 23% to 37% and 33% to 38% and JA-Ile content was reduced by 8% to 37% and 44% to 50% compared to the amounts accumulated in the control plants at 1 and 2 hpw, respectively (Figure 3B,C). Notably, we also found that one of the JA catabolites, 12OH-JA, accumulated to a significantly higher level in the T. virens-treated plants in response to mechanical wounding (83% to 171% and 23% to 82% increase compared to the contents in the control plants at 1 and 2 hpw, respectively) (Figure 3A and 3D). This latter result suggests that one potential reason behind decreased JA content may be due to increased catabolism of JA. To confirm the reduced insect defense by T. virens colonization was modulated by JA signaling, we tested the effect of T. virens on the JA-deficient opr7opr8 mutant [14] to FAW. The result showed that T. virens colonization did not increase susceptibility to FAW on opr7opr8 double mutants, suggesting that attenuated JA signaling plays an essential role in T. virens-reduced insect defense (Figure 4). Interestingly, the levels of the α-ketol, 9,10-KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid), have been significantly reduced at 1 hpw (24% to 37% reduction compared to the contents in the control plants) by all strains of T. virens (Figure 3E). This molecule has been recently shown to play a major JA-dependent signaling role in defense against FAW [34]. Together, these results indicate that T. virens colonization suppressed defense against FAW via downregulating JA biosynthesis and/or enhancing JA catabolism.

2.4. T. virens and P. chlororaphis Colonization Enhanced Growth but Suppressed Insect Defense against FAW in Sterile Soil While Increasing Resistance to C. graminicola in both Sterile and Nonsterile Soil

Bacterial biocontrol agent P. chlororaphis 30-84 was previously reported to promote growth and suppress growth of fungal pathogens [35, 36], yet its role in insect defense against foliar-feeding insects remains mostly unexplored. Therefore, we also tested the impact of P. chlororaphis 30-84 on plant growth promotion, defense against FAW, and pathogen resistance and the effect of combining the two beneficial microorganisms, T. virens and P. chlororaphis 30-84, on these physiological processes. Because the beneficial microorganisms were commonly tested in an agricultural setting, we further tested their effect in both sterile and nonsterile soil mixtures of field soil and potting mix. Similar to T. virens treatment, colonization by P. chlororaphis 30-84 promoted plant growth (12% to 15% increase in plant height) but suppressed defense against FAW (41% to 51% increase in weight gain) in the sterile soil mixture (Figure 5A,B). Plant growth and insect defense were not significantly affected when seedlings were grown in a nonsterile soil mixture upon treatment with T. virens and P. chlororaphis 30-84. However, both T. virens and P. chlororaphis 30-84 were able to increase resistance to C. graminicola in both soil types (53% to 66% reduction and 44% to 55% reduction in lesion areas in nonsterile and sterile soils, respectively) (Figure 5C).

2.5. T. virens and P. chlororaphis 30-84 Colonization Reduced Wound-Induced JA Accumulation by Suppressing Biosynthesis and Enhancing Catabolism in Plants Grown in Sterile Soil

Decreased FAW resistance found with the amendment of P. chlororaphis 30-84 in this study prompted us to investigate whether this biocontrol agent impacts JA homeostasis in response to wounding in sterile soil. Phytohormone and oxylipin profiling revealed that P. chlororaphis 30-84 treatment reduced wound-induced accumulation of jasmonates, including 12-OPDA, JA, JA-Ile, and JA-Leu in sterile soil (Figure 6A). More specifically, treatments with T. virens, P. chlororaphis 30-84, and the mixture of T. virens and P. chlororaphis 30-84 resulted in 25%, 21%, and 19% reduction in JA accumulation and 27%, 32%, and 27% reduction in JA-Ile level at 1 hpw, respectively (Figure 6B,C). Moreover, JA catabolism was increased by 50%, 13%, and 21% in the plants treated with T. virens, P. chlororaphis 30-84, and the mixture of T. virens and P. chlororaphis 30-84, respectively (Figure 6D). Interestingly, suppression of wound-induced JA biosynthesis (Figure 6) and FAW insect defense (Figure 5) by either T. virens or P. chlororaphis 30-84 was only observed in the sterile soil condition. A similar accumulation pattern was observed for 9,10-KODA (17% to 34% reduction) in sterile soil (Figure 6E). Together, these results suggest that T. virens and P. chlororaphis 30-84 suppress foliar-feeding insect defense via regulating JA homeostasis in response to wounding only in plants grown in sterile soil amended with either of these biocontrol agents.

2.6. T. virens Colonization Suppressed WCR Larval Survival and Weight Gain

We also tested whether T. virens plays a role in conferring resistance to root-feeding WCR larvae. The results showed that significantly fewer larvae were recovered (50% fewer) (Figure 7A), and WCR larvae gained significantly less weight after feeding on T. virens-colonized plants (57% less) (Figure 7B). However, T. virens treatment did not reduce tissue consumption (Figure 7C,D). These results suggest that T. virens may be a potent biocontrol agent against WCR by suppressing WCR larval survival and weight gain.

3. Discussion

Soil-borne Trichoderma spp. are ubiquitous in soil worldwide, and have been used as biocontrol agents against pathogens for decades [23, 31, 37-39]. However, the potential utility of Trichoderma spp. as biocontrol agents against insect pests has just begun to be addressed [26], and information on their effects against foliar-feeding and root-feeding insects is scarce. Accumulating evidence suggests that root colonization by Trichoderma spp. triggered ISR against insects, such as aphids [40-43], whiteflies [44], thrips [45], and Lepidoptera [41, 46-50], and also enhanced defense against spider mites [51] and nematodes [52-54]. Previous reports [27, 28, 30] and this study showed that T. virens confers resistance to C. graminicola. In addition, these studies have identified a fungal-secreted peptide, Sm1, as a signal necessary for inducing systemic resistance by showing that the Δsm1 deletion mutant of T. virens is unable to induce ISR [27, 28, 30]. In contrast to Δsm1, another signaling peptide mutant of T. virens, Δsir1 [29, 30], triggered enhanced ISR against C. graminicola. As this mutant overexpressed SM1 transcripts [30], we hypothesized that the increased ISR following colonization by this mutant is due to elevated SM1 expression. We tested this hypothesis by deleting SM1 in the Δsir1 mutant background and found that the Δsm1Δsir1 double mutant still displayed stronger resistance as compared to the wild-type strain. We are suggesting that Sir1 peptide acts to suppress ISR not only by downregulating SM1 expression, but also by employing other as-yet-unknown mechanisms of ISR suppression.

Several reports showed that several Trichoderma spp. directly impact Lepidoptera spp. by producing antifeedant compounds and toxic secondary metabolites [26, 55-59] or indirectly impact them via activation of JA-mediated systemic defense, production of parasitoid-attracting volatile compounds, or disruption of insect gut microbiota and metabolome [41, 42, 46-48, 50, 60]. Here, we demonstrated that the treatment with T. virens suppressed defense against FAW by reducing wound-induced JA production and increased JA catabolism in plants grown in sterile soil. Consistent with our findings, Lelio et al. (2021)[47] showed that when grown at cool temperatures (20°C as compared to 25°C), tomato plants colonized with T. afroharzianum T22 had less negative impact on Spodoptera littoralis larvae and this was accompanied with suppressed JA biosynthesis and signaling pathways. Also consistent with our results, Kinyungu et al. (2023)[49] showed that the treatment with T. atroviride F2S21 strain resulted in a higher leaf defoliation rate by FAW larvae. The fact that some studies showed increased resistance to certain chewing insects due to Trichoderma spp. treatment [41, 46, 50] while others, including the current study, show opposite effect of Trichoderma treatment, suggest Trichoderma species- and strain-specific effects as well an environment-dependent effect on insect defense. Interestingly, our results indicate that the reduced insect defense is independent of the fungal secreted peptides, Sm1 and Sir1, suggesting that fungal signals regulating ISR against pathogens and increased induced susceptibility against chewing insects involve different molecular signals to be identified in the future.

Previous reports showed that root colonization by T. virens induced biosynthesis of the JA precursor, 12-OPDA, and ketols [30, 61]. Both 12-OPDA and ketols were shown to act as xylem-mobile long-distance signals that prime the above-ground organs for greater resistance against pathogens in maize [30, 61]. In contrast to these oxylipins, JA-Ile levels in xylem sap were not significantly increased in response to T. virens treatment and transfusion of maize seedlings with xylem sap supplemented with JA-Ile increased susceptibility to C. graminicola [30]. The role of JA as a susceptibility factor against anthracnose leaf blight caused by C. graminicola was further supported by the studies of Gorman et al. (2020)[12] and Huang et al. (2023)[13]. Moreover, transcriptome analysis showed that maize roots responded to colonization by T. virens by increased expression of 12-OPDA biosynthesis genes but suppressed expression of the genes required for 12-OPDA conversion to JA-Ile [30]. Because JA is widely reported to suppress growth in numerous plant species [62-64], it is tempting to speculate that attenuated synthesis of JAs in roots colonized by T. virens is a potential mechanism accounting for the strong growth promotion effect by this symbiont observed in this study and previous reports. Similar to the T. virens-mediated induction of ISR against pathogens and growth promotion, we showed that root colonization by the bacterial symbiont, P. chlororaphis 30-84, resulted in increased seedling growth and systemic resistance against C. graminicola, suggesting a similar mechanism underlying these two beneficial responses. However, as in the case of T. virens, P. chlororaphis 30-84 treatment reduced JA production and decreased resistance to herbivory by FAW when treated plants were grown in sterile soil. Therefore, it is likely that growth promotion by both beneficial microorganisms, T. virens and P. chlororaphis 30-84, and ISR against the hemibiotrophic pathogen C. graminicola is linked to their ability to suppress JA synthesis and JA-mediated signaling. Interestingly, wound-induced JA accumulation and insect defense were not affected when maize seedlings were treated with T. virens or P. chlororaphis 30-84 in nonsterile soil, suggesting that natural microbial communities dampened JA suppression activities by both endophytes.

WCR root feeding causes substantial economic damage to corn production in the United States and Europe [65, 66] and the insect has adapted to overcome most of the available management techniques, including chemical insecticides and Bacillus thuringiensis (Bt) toxins [66, 67]. So far, there is a lack of efficient management of this notorious pest and, thus, there is an urgent need for a sustainable and economical management approach, such as utilizing efficient biocontrol agents. In this study, we showed that T. virens colonization significantly enhanced mortality and suppressed weight gain of WCR larvae. Our previous report showed that increased WCR mortality was correlated with greater levels of herbivory-induced production of insecticidal death acids and ketols [16]. Therefore, one of the potential mechanisms of T. virens-mediated WCR defense may be to activate the production of insecticidal defensive compounds. The other potential mechanism behind the detrimental effect on larvae survival and growth conferred by T. virens may be the production of some fungus-derived insecticidal metabolites as reported in several studies [26, 55-59]. Further experiments are needed to explore whether T. virens has a direct insecticidal impact on the WCR root-feeding larvae or has an indirect effect due to the induction of the host -mediated synthesis of toxic compounds.

4. Materials and Methods

4.1. Plant, Growth Medium, and Plant-Beneficial Inoculants

The maize (Zea mays) inbred line B73 and opr7opr8 double mutant (Yan et al., 2012)[14] were used in this study. Plants were grown in conical pots (20.5 × 4 cm) filled with autoclaved (121°C for 60 minutes, 2 cycles) commercial potting mix (Jolly Gardener Pro Line C/20) on light shelves at room temperature (22–24°C) with a 16-h light period. To test the efficacy of the beneficial microorganisms in an agricultural setting, maize B73 seeds were grown in a mixture of field soil (collected from Texas A&M University Research Farm in the fall of 2022 after maize was harvested) and potting mix (Jolly Gardener Pro Line C/20) at the ratio of 1 to 3 that was either autoclaved (121°C for 60 minutes, 2 cycles; henceforth referred to as sterile) or untreated (hence referred to as nonsterile) after mixing. Chlamydospores of T. virens strains TvWT (Gv29-8; Baek and Kenerly, 1998)[68], Δsm1 (Djonovic´ et al., 2006; Djonovic et al., 2007)[27, 28], Δsir1 (formerly D77560; Lamdan et al., 2015)[29], and Δsm1Δsir1 (this study) were obtained from liquid cultures grown in molasses-yeast extract medium as described in Wang et al. (2020)[30]. P. chlororaphis 30-84 was grown in Luria-Bertani (LB) medium containing 5 g of NaCl per liter at pH 7 at 28°C with agitation at 200 rpm, as described in Yuan et al. (2020)[36]. Seven days after sowing, seedlings were treated with 0.05 g of the T. virens chlamydospores, or 300 μl of P. chlororaphis 30-84 suspension at OD₆₂₀ = 0.8, or a mixture of both T. virens and P. chlororaphis with the same amount described above (added to the soil at a depth of approximately 2 cm around the seeds), or left untreated.

4.2. Anthracnose Leaf Blight Assay

C. graminicola stain 1.001 was grown on half strength potato dextrose agar (PDA) plates for 2-3 weeks to allow sporulation. Two weeks after being colonized by beneficial microorganisms, the plants were inoculated with 10 μl of conidial suspension (10⁶ conidia/ml) with six droplets of spore suspensions per leaf collected from hemibiotrophic fungal pathogen C. graminicola plates as described in Huang et al. (2023a)[13]. The leaves were scanned and the lesion areas measured using ImageJ software [69] seven days post-inoculation.

4.3. Fall Armyworm Assay

The laboratory strain of fall armyworm (FAW; Spodoptera frugiperda) was purchased from Benzon Research (Carlisle, PA, USA). Two weeks after incubation with the beneficial microorganisms, six FAW neonates were placed to the whorls of maize seedlings contained in individual plastic jars and allowed to move and feed on the plants freely for 7 days, as described in Huang et al. (2023b)[16]. FAW larvae were removed from the plants, and the total weight was determined 7 days post-infestation. To evaluate insect resistance and leaf damaged area, one maize leaf was individually caged in a handmade clip-cage and infested with one 3rd-instar FAW larva per spot for approximately one hour, then moved toward the base of the plant. Leaves were then scanned, and eaten areas were measured with ImageJ software[69].

4.4. Oxylipin Profiling of Wounded Leaf Tissue

For the wounding treatment, the third fully expanded leaf of seedlings grown in sterile or nonsterile soil conditions that were previously colonized with beneficial microorganisms for two weeks or left untreated was wounded seven times using a hemostat, with three wound sites on one side and four on the other side of the midvein with the wound sites approximately 1 cm apart. The wounded regions were then harvested and placed in 2 mL screw-cap Fast-Prep tubes (Qbiogene, Carlsbad, CA, U. S. A.) in liquid nitrogen and stored in a -80 °C freezer. Phytohormone and oxylipin extraction and profiling of wounded leaf tissue were performed as described previously [13].

4.5. Western Corn Rootworm Bioassays

Western corn rootworm (WCR; Diabrotica virgifera virgifera) eggs were provided by USDA-ARS North Central Agriculture Research Laboratory (Brookings, SD, USA). Two weeks after being colonized by beneficial microorganisms, ten WCR neonates were placed on the soil surface in individual cone-tainers with a maize seedling and allowed to burrow and feed for 10 days. WCR larvae were removed from the soil, and the total weight was determined 10 days post-infestation. The fresh weights of shoot and root tissues was measured after the soil was removed and the ratio was analyzed as larvae damaged over control root mass, as described in Huang et al., (2023)[16].

4.6. Statistical Methods

Statistical analyses were performed using the software programs JMP Pro 17 (SAS Institute Inc., Cary, NC, USA). Lesion areas, FAW larval weight, leaf eaten areas, wound-induced accumulation of metabolites, and plant heights were analyzed using One-way Analysis of Variance (ANOVA) with post-hoc Tukey HSD (Honestly Significant Difference). FAW larval weight after feeding on opr7opr8 double mutants and wild-type seedlings treated with T. virens or left untreated and root and shoot ratios after WCR infestation were analyzed using Two-way ANOVA with Tukey HSD. Effect of sterile and nonsterile mixture was analyzed using Student’s t-test.

5. Conclusions

The elevated level of ISR against C. graminicola triggered by both T. virens Δsir1 and Δsm1Δsir1 double mutants revealed that a functional Sir1 signal peptide reduces disease resistance by downregulating not only SM1 (as demonstrated previously by Wang et al. (2020)) but also involves other unknown mechanisms as well. Colonization of plants grown in sterile soil by different T. virens mutants suppressed wound-induced JA accumulation, resulting in reduced defense against FAW, which is independent of both signal peptides, Sm1 and Sir1. When testing both beneficial microorganisms, T. virens and P. chlororaphis 30-84, using both sterile and nonsterile mixtures of field soil and potting mix, both biocontrol agents triggered ISR against C. graminicola in both soil types. However, these agents reduced defense against FAW only when the treated plants were grown in sterile soil due to reduced JA. In contrast to the effect on leaf-feeding FAW, T. virens colonization suppressed root-feeding WCR larvae survival and weight gain, the first report revealing the potential of T. virens as a biocontrol agent against WCR.