DOI: 10.5061/dryad.pzgmsbcx9
title: Data from: First report of Fusarium citri as an entomopathogenic fungus mediating plant resistance against insect pests and phytopathogens

description: 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.
description: <strong><em>Insects, plants, and phytopathogen strain
 preparation</em></strong> Muscardine
 cadaver larvae of <em>S</em>. <em>litura</em> 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, <em>S</em>.
 <em>litura</em>, <em>Chilo suppressalis</em>, and
 <em>Ostrinia</em> <em>furnacalis</em>, as well as
 Hemiptera pest adults, <em>R</em>. <em>padi</em>
 and <em>Acyrthosiphon</em> <em>pisum</em>, 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 <em>S</em>.
 <em>sclerotiorum</em> strain, isolated and identified from
 soybean, was supplied by Professor Jinliang Liu from Jilin University,
 China. <strong><em>Isolation and culture of
 the EPF strain</em></strong> Muscardine
 cadaver larvae of <em>S</em>. <em>litura</em> 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.
 <strong><em>Morphological identification of the EPF
 strain</em></strong> 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).
 <strong><em>Molecular identification of the EPF
 strain</em></strong> 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
 (<em>EF</em>-<em>1α</em>), calmodulin
 (<em>CAM</em>), partial RNA polymerase largest subunit
 (<em>RPB1</em>), and partial RNA polymerase second largest
 subunit (<em>RPB2</em>) 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 <em>F.</em> <em>polyphialidicum</em> NRRL
 13459. <strong><em>Evaluation of virulence
 against insect pests</em></strong> Conidia
 cultivated on PDA medium were suspended in sterile 0.1% (v/v) Tween-80
 solution at a concentration of 1 × 10<sup>8</sup> conidia /mL,
 with sterile 0.1% (v/v) Tween-80 serving as a control. Uniformly sized
 second instar larvae of <em>S</em>.
 <em>litura</em>, <em>C</em>.
 <em>suppressalis</em>, and <em>O</em>.
 <em>furnacalis</em>, along with
 <em>Rhopalosiphum</em> <em>padi</em> and
 <em>A</em><em>.</em> <em>pisum </em>adults, were selected for virulence determination through an impregnation assay, as described previously. Following inoculation, <em>S</em>. <em>litura</em>, <em>C</em>. <em>suppressalis</em>, and <em>O</em>. <em>furnacalis</em> larvae were placed in 24-well culture plates, while <em>R</em>. <em>padi</em> and <em>A</em>. <em>pisum</em> 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). <strong><em>In vitro antifungal activity of F. citri</em></strong> The plate confrontation method was employed to assess the <em>in vitro</em> inhibitory activity of tested fungi against <em>S</em>. <em>sclerotiorum</em>. Discs with <em>S</em>. <em>sclerotiorum</em> 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 4<sup>t</sup>, 5,<sup> </sup>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 <em>F</em>. <em>citri</em> 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. <strong><em>In vitro evaluation of plant disease resistance induced by fungal colonization</em></strong> The resistance of soybean plants to <em>S</em>. <em>sclerotiorum</em> 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 <em>S</em>. <em>sclerotiorum</em>, 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 <em>F</em>. <em>citri</em> alone, leaves inoculated with <em>S</em>. <em>sclerotiorum</em>, and leaves pre-colonized by <em>F</em>. <em>citri</em> followed by <em>S</em>. <em>sclerotiorum</em> 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% <strong><em>Determining the effect of fungal colonization in soybean on insect feeding selectivity</em></strong> 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 <em>S</em>. <em>litura</em> 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 <strong><em>Quantification of plant resistance genes in soybean</em></strong> 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 <em>F</em>. <em>citri</em> 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. <strong><em>Statistical analysis</em></strong> All experimental data were analyzed in IBM SPSS Statistics 25 (IBM Corp., Armonk, USA) using one-way Analysis of Variance (ANOVA, <em>P</em> &lt; 0.05) and plotted using GraphPad Prism 8.0.2 (GraphPad Software Inc, San Diego, CA, USA).
description: # Data from: First report of Fusarium citri as an entomopathogenic fungus
 mediating plant resistance against insect pests and phytopathogens
 [https://doi.org/10.5061/dryad.pzgmsbcx9](https://doi.org/10.5061/dryad.pzgmsbcx9) ## Description of the data and file structure The present study investigated 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. #### Description of the data and file structure We provided data for Processed and Raw, we also provided Software files for Figure 1-8. #### Figure\_2\_5\_genes\_(ITS-EF1a-CAM-RPB1-RPB2)\_squences This file contains all the sequences used to build the multigene phylogenetic tree. #### Growth rate 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). #### Figure\_3\_raw This file contains the original data set of virulence evaluation of Fusarium citri strain FcS1GZL-1 against insect pests.(F) #### Figure\_3\_prosecced This file contains data sets for virulence evaluation of Fusarium citri strain FcS1GZL-1 against insect pests. (F) #### Growth rate  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. #### Mortality Corrected mortality rates were recorded for each treatment group, using the following formula: Corrected mortality (%) = (mortality of treatment − mortality of control)/(1 − mortality of control) × 100 #### Figure\_4\_raw This file contains the original dataset ofconfrontation cultures of Sclerotinia sclerotiorum and *Fusarium citri* in vitro.(A) 3 d. (B) 4 d. (C) 5 d. #### Figure\_4\_prosecced This file contains datasets of confrontation cultures of Sclerotinia sclerotiorum and *Fusarium citri* in vitro.(A) 3 d. (B) 4 d. (C) 5 d. #### Colony diameter The colony diameter of the pathogenic fungi was measured and recorded at 4,5, and 6 dpi. #### Growth rate 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. #### Figure\_5\_raw This file contains the original dataset for colonization detection of Fusarium citri strain FcS1GZL-1 in soybean plants. (C) #### Figure\_5\_prosecced This file contains a dataset for colonization detection of Fusarium citri strain FcS1GZL-1 in soybean plants. (C) #### Colonization rate 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. #### Figure\_6\_raw This file contains the original dataset of feeding selectivity of second instar larvae of Spodoptera litura on Fusarium-soybean symbiosis. (A)  Feeding rate of leaves with and without *F*. *citri* colonization in the selective test. (B) Feeding rate of leaves with and without* F*. *citri* colonization in the non-selective test. #### Figure\_6\_prosecced This file contains a dataset of feeding selectivity of second instar larvae of Spodoptera litura on Fusarium-soybean symbiosis.(A)  Feeding rate of leaves with and without *F*. *citri* colonization in the selective test. (B) Feeding rate of leaves with and without* F*. *citri* colonization in the non-selective test. #### Feeding rate To clarify whether *Fusarium citri* affects the feeding of *Spodoptera litura*. Feeding rate (%) = (number of insects feeding on soybean leaves/20) × 100 #### Figure\_7\_raw This file contains the original dataset of disease resistance induction by Fusarium citri as an endophyte. (A) Incidence rate of different treatments at different times following phytopathogen inoculation. (B) Lesion diameters of different treatments on day 4 following phytopathogen inoculation. #### Figure\_7\_prosecced This file contains the dataset of disease resistance induction by Fusarium citri as an endophyte. (A) Incidence rate of different treatments at different times following phytopathogen inoculation. (B) Lesion diameters of different treatments on day 4 following phytopathogen inoculation. #### Incidence rate incidence rate (%) =(Number of leaves of affected soybean/Total soybean leaves)×100 #### Lesion diameter  Lesion diameters were measured at 4 dpi using the cross method. #### Figure\_8\_raw This file contains the original dataset of resistance-induced marker gene expression in soybean roots.(A) Relative expression of *LOX1*, *GmKTI1*, and *GmG3PA*, in the jasmonic acid (JA) pathway. (B) Relative expression of *GmG3H*, *GmWRKY40*, and *GmNIMIN1* in the salicylic acid (SA) pathway. (C) Relative expression of* PR1*-*1a*, *PR3*, and *PR9* in the pathogen-related (PR) pathway. (D) Relative expression of *ACO3*, *EIN2*, and *ACS7* in the ethylene (ET) pathway. #### Figure\_8\_prosecced This file contains the dataset of resistance-induced marker gene expression in soybean roots.(A) Relative expression of *LOX1*, *GmKTI1*, and *GmG3PA*, in the jasmonic acid (JA) pathway. (B) Relative expression of *GmG3H*, *GmWRKY40*, and *GmNIMIN1* in the salicylic acid (SA) pathway. (C) Relative expression of* PR1*-*1a*, *PR3*, and *PR9* in the pathogen-related (PR) pathway. (D) Relative expression of *ACO3*, *EIN2*, and *ACS7* in the ethylene (ET) pathway. #### Table\_1 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. #### Table\_S1 This file contains informations for Gene primer sequences. #### Table\_S2 This file contains informations for Gene primer sequences. ### Sequence information uploaded to Genbank The file contains links to the five gene sequences in this experiment uploaded to Genbank #### Code/Software The evolutionary distances were computed using the Kimura 2-parameter method. The phylogenetic tree was constructed in MEGA X (MEGA Inc., Englewood, United States). 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). Data are presented in bars as mean  standard error of the mean (SEM). All data were subjected to one-way Analysis of Variance, *P* &lt; 0.05 = *, *P*&lt; 0.01 = **, and *P* &lt; 0.001 = ***. Apart from gene expression data, all data were analyzed using IBM SPSS Statistics 22 (IBM Corp., Armonk, NY, USA), Figures were illustrated using GraphPad Prism 8.0.2 (GraphPad Software Inc, San Diego, CA, USA). 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). All experimental data were analyzed in IBM SPSS Statistics 25 (IBM Corp., Armonk, USA) using one-way Analysis of Variance (ANOVA, *P* &lt; 0.05) and plotted using GraphPad Prism 8.0.2 (GraphPad Software Inc, San Diego, CA, USA).