First report of Fusarium citri as an entomopathogenic fungus mediating plant resistance against insect pests and phytopathogens

Fusarium citri, has been historically recognized as a phytopathogen but never as an entomophagous fungus (EPF) with plant endogeneity. In the present study, an F. citri strain, FcS1GZL-1, was isolated and identified from diseased Spodoptera litura larvae in a soybean field. The pathogenicity and antagonistic activity of FcS1GZL-1 against five insect pests from Lepidoptera and Hemiptera and a soil-borne pathogen Sclerotinia sclerotiorum, respectively, were assessed, as well as its ability to colonize plants via root irrigation. Induced resistance to insect pests and phytopathogens was also measured. The expression of plant resistance related genes was analyzed using real-time RT-PCR. According to the results, the FcS1GZL-1 strain could not only kill insect pests with high pathogenicity but also inhibited phytopathogen growth in vitro. Furthermore, the FcS1GZL-1 strain could repel insect pest feeding and enhance plant resistance to phytopathogens through endophytic customization following root irrigation, which upregulated 12 genes related to the jasmonic acid, salicylic acid, ethylene, and pathogen-related defense pathways in soybean roots. Herein, we present the first documented case of F. citri naturally infecting insects, and its dual role in controlling insect pests and phytopathogens, with promising biocontrol applications.

Methods
Insects, plants, and phytopathogen strain preparation

Muscardine cadaver larvae of S. litura were collected from a soybean field located in Gongzhuling City, Jilin Province, China (N43° 50′42″, E124° 82′58″).

Tested insects, including Lepidoptera pest larvae, S. litura, Chilo suppressalis, and Ostrinia furnacalis, as well as Hemiptera pest adults, R. padi and Acyrthosiphon pisum, which were maintained on regular artificial diets, were provided by Professor Wenjing Xu from the Plant Protection Institute, Jilin Academy of Agricultural Sciences, Jilin, China.

The Jiyu 303 soybean seeds, developed for commercial use, were cultivated by the Soybean Research Institute of the Jilin Academy of Agricultural Sciences.

The S. sclerotiorum strain, isolated and identified from soybean, was supplied by Professor Jinliang Liu from Jilin University, China.

Isolation and culture of the EPF strain

Muscardine cadaver larvae of S. litura were disinfected by immersion in 75% (v/v) anhydrous ethanol for 30 s, followed by 1% (v/v) sodium hypochlorite for 2 min. They were then rinsed three times with sterile water, and the surface moisture was removed using sterile filter paper. The larvae were sectioned into five pieces with a sterile scalpel and placed on Potato Dextrose Agar (PDA) medium (Sangon Biotech, Shanghai, China) supplemented with 1 mL ampicillin per liter at a concentration of 100 μg/mL. The samples were incubated at 26°C for 5–7 d until mycelial growth was observed. The strain was purified using the single-spore isolation method, as described by Ghalehgolabbehbahani et al., and the purified strain was designated as FcS1GZL-1.

Morphological identification of the EPF strain

The examined isolates were incubated on synthetic nutrient-poor agar (SNA; Nirenberg 1976) at 25 °C for 7 d. Agar pieces measuring approximately 5 × 5 mm were cut from the edges of colonies and transferred onto media for morphological characterization. After 7 d of incubation in the dark on PDA, and SNA, cultural characteristics, such as colony morphology, pigmentation, and odor, were observed. The colors were assessed using the color charts of Fisher et al. [26]. To induce sporodochia, the isolates were incubated under a 12/12-h near-ultraviolet light/dark cycle on SNA and water agar (WA) supplemented with sterilized pieces of carnation leaves at 25 °C. Micromorphological characteristics were examined and documented using water as a mounting medium on an LW300-48LT microscope (Siwei Optoelectronic Technology Co., Ltd., Xi'an, China).

Molecular identification of the EPF strain

Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified CTAB protocol, as described in Guo et al. [29]. Five loci, including the 5.8S nuclear ribosomal RNA gene with the two flanking internal transcribed spacer (ITS), translation elongation factor (EF-1α), calmodulin (CAM), partial RNA polymerase largest subunit (RPB1), and partial RNA polymerase second largest subunit (RPB2) gene regions, were amplified and sequenced. The primer pairs and PCR amplification procedures, according to those in the protocols described by Crous et al. [30], are listed in Table S1. PCR products were sequenced by Sangon Biotech Ltd. (Shanghai, China). The forward and reverse sequencing results were spliced using DANMAN software v6.0.3.99 (Lynnon Biosoft, San Ramon, CA, USA). The sequences of the five genes were submitted to GenBank separately to obtain the accession numbers. The phylogenetic trees were inferred using the maximum likelihood method with a bootstrap consensus tree inferred from 1,000 replicates. The evolutionary distances were computed using the Kimura 2-parameter method. The phylogenetic tree was constructed in MEGA X (MEGA Inc., Englewood, United States). The tree was rooted to F. polyphialidicum NRRL 13459.

Evaluation of virulence against insect pests

Conidia cultivated on PDA medium were suspended in sterile 0.1% (v/v) Tween-80 solution at a concentration of 1 × 108 conidia /mL, with sterile 0.1% (v/v) Tween-80 serving as a control. Uniformly sized second instar larvae of S. litura, C. suppressalis, and O. furnacalis, along with Rhopalosiphum padi and A. pisum adults, were selected for virulence determination through an impregnation assay, as described previously. Following inoculation, S. litura, C. suppressalis, and O. furnacalis larvae were placed in 24-well culture plates, while R. padi and A. pisum were placed in sterile culture boxes. The insects were maintained at a temperature of 25 ± 1°C, with a relative humidity of 70 ± 1% and a 16-h light:8-h dark photoperiod. Each treatment was performed in triplicate with 20 insects per replicate. Insect infection and mortality were assessed every 24 h, along with observations of external characteristics of the infected insects. Insects were considered dead if they exhibited no movement upon gentle probing with sterile forceps. Corrected mortality rates were recorded for each treatment group, and probit linear regression analysis was employed to calculate the median lethal time (LT50) of tested fungi against different insects, as described previously, using the following formula:

Corrected mortality (%) = (mortality of treatment − mortality of control)/(1 − mortality of control) × 100.

Insect cadavers were removed promptly using sterile tweezers and transferred to sterile culture dishes (Φ = 9 cm) lined with sterile filter paper. The insect bodies were incubated in a light-controlled chamber at 25 ± 1°C, with 70 ± 1% relative humidity and a 16-h light/8-h dark photoperiod. Mycelial and conidial growth on the insect bodies was monitored, and the morphology of the mycelia and conidia was observed under a microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany).

In vitro antifungal activity of F. citri

The plate confrontation method was employed to assess the in vitro inhibitory activity of tested fungi against S. sclerotiorum. Discs with S. sclerotiorum pathogen (5-mm diameter) were placed equidistantly (1.5 cm) opposite to the endophyte and incubated in the dark at 25 ± 2°C for 5–7 d. The control group was only inoculated with the fungal pathogen block. Five replicates were included per treatment and the experiments were based on a completely randomized design. In addition, control dishes containing only the pathogen were prepared [35]. At 4t, 5, and 6 days post inoculation (dpi), the radii of the pathogen colonies growing alone (mm) and the pathogen colonies competing against the endophyte (mm) in each treatment were measured using a digital caliper and the colony morphology was photographed using a digital camera.

Colonization of the EPF strain in soybean plants via root irrigation

Soybean plants at the compound leaf stage were selected and irrigated with a conidial suspension (1 × 10⁸ spores/mL) of strain FcS1GZL-1. A 0.1% (v/v) Tween-80 solution acted as the control. Each plant received 20 mL of inoculum at 24-h intervals, applied three times. Each treatment was performed in triplicate with 20 seedlings per replicate. The colonization rate of F. citri in soybean leaves was evaluated 24 h after soil drench inoculation. Fungal colonies were detected by culturing surface-sterilized leaf segments on PDA for 14 d, as outlined in a previous protocol.

In vitro evaluation of plant disease resistance induced by fungal colonization

The resistance of soybean plants to S. sclerotiorum was assessed by measuring the incidence rate and lesion diameter. Soybean leaves were sterilized using 75% ethanol for 30 s, followed by 1% sodium hypochlorite for 2 min, and rinsed twice with sterile water. After 24 h of inoculation with FcS1GZL-1, trifoliate leaves were excised with sterile scissors and placed in culture dishes with moist filter paper, while cotton balls were positioned at the petioles to maintain humidity. For inoculation, 5-mm plugs of S. sclerotiorum, cultured on PDA medium for 5-7 d, were applied to the leaf surface, avoiding the veins. Inoculated leaves were incubated at 25 ± 1 °C with relative humidity above 70%, under a 16-h light and 8-h dark cycle. The experiment included leaves without inoculation as controls, leaves colonized by F. citri alone, leaves inoculated with S. sclerotiorum, and leaves pre-colonized by F. citri followed by S. sclerotiorum inoculation. Each treatment had three replicates of 20 leaves. Lesion diameters were measured at 4 dpi using the cross method and calculated using the following formula:

incidence rate (%) = (number of incidents/total number of inoculated plants) × 100%

Determining the effect of fungal colonization in soybean on insect feeding selectivity

A sterile 14-cm plastic culture dish was lined with moist filter paper, and an appropriate amount of distilled water was added to maintain moisture. In the choice condition, the plate was alternately arranged with five soybean leaves colonized by the fungal strain FcS1GZL-1 and five non-colonized leaves. For the no-choice condition, the plate was either entirely populated with ten colonized or ten non-colonized leaves. Leaves were paired based on length and morphology to control for potential effects of leaf age, and they were spaced evenly around the dish circumference [38]. In the center, 20 second-instar larvae of S. litura were placed, and the dishes were incubated at 26 ± 1 °C, with 60 ± 10% relative humidity and a 14-h light/10-h dark cycle. The experiment was repeated five times. The number of larvae on each leaf was recorded at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6 h after inoculation. The pest feeding rate on soybean leaves was calculated using the following formula:

Feeding rate (%) = (number of insects feeding on soybean leaves/20) × 100

Quantification of plant resistance genes in soybean

The effects of inoculated EPF on soybeans were assessed by measuring the relative expression levels of 12 genes involved in the synthesis of salicylic acid (SA), jasmonic acid (JA), pathogen-related (PR), and ethylene (ET) through qPCR assays. Trifoliate soybean roots from various treatments were collected 48 h after F. citri soil drench inoculation and stored at −80 °C for later analysis. The relative expression levels of resistance genes were analyzed using qPCR with specific primers. Total DNA in each treatment was extracted and the specific primers employed in qPCR are listed in Table S2.

Statistical analysis

All experimental data were analyzed in IBM SPSS Statistics 25 (IBM Corp., Armonk, USA) using one-way Analysis of Variance (ANOVA, P < 0.05) and plotted using GraphPad Prism 8.0.2 (GraphPad Software Inc, San Diego, CA, USA).