DOI: 10.5061/dryad.7sqv9s516
title: Data from: Insect hypovirulence-associated mycovirus confers entomopathogenic fungi with enhanced resistance against phytopathogens

description: Mycoviruses can alter the biological characteristics of host fungi
 including decreasing or enhancing virulence or pathogenicity of
 phytopathogens and entomopathogenic fungi (EPF). As an extensively used
 EPF, Beauveria bassiana could not only directly control pests, but also
 improve plant resistance against plant disease through endophytic
 colonization. However, most studies on the mycoviruses found in B.
 bassiana have focused on the effects of the viruses on the virulence of
 host fungi toward insect pests, with relatively few reports on the effects
 to the host fungi with regard to plant disease resistance in hosts. The
 present study investigated the effects of the mycovirus Beauveria bassiana
 chrysovirus 2 (BbCV2) virus infection on host biological characteristics,
 additionally, we evaluated antagonistic activity of BbCV2 against
 phytopathogenic fungi (Sclerotinia sclerotiorum and Botrytis cinerea) in
 vitro and their associated diseases both in in vitro leaves and in pot
 experiments.  Our results showed that the mycovirus, BbCV2,
 enhanced the growth rate, spore production, and biomass of host fungi B.
 bassiana. BbCV2 virus infection enhanced the capacity of host fungi and
 their metabolic products to inhibit phytopathogenic fungi S. sclerotiorum
 and B. cinerea. BbCV2 virus infection reduced the contents of the two
 pathogens in tomato plants significantly, and in turn enhanced the plant
 resistance induced by host fungi colonization against the diseases caused
 by the two pathogens.
description: <strong><em>Evaluation of the effects ofBbCV2 virus
 infection on host biological
 characteristics</em></strong> For the
 growth rate test, 2 μL of <em>B. bassiana</em> suspension at a
 concentration of 5 × 10<sup>4</sup> conidia/mL was added to
 the center of each 20 mL potato dextrose agar (PDA) plate, and cultured in
 a constant temperature incubator at 26±1℃, with 70±1% relative humidity
 (RH), and a 16-h light:8-h dark photoperiod. The colony diameter was
 measured using the cross over method for 12 d, and each strain had five
 replicates. Colony growth rate was calculated according to the following
 formula <sup>[25]</sup>: colony growth rate (mm/d) = (final
 colony diameter - initial colony diameter)/ culture time, and the size and
 morphology of the colony were recorded by taking photos.
 For the spore production test, 100 μL of <em>B.
 bassiana</em> suspension liquid at a concentration of 1 ×
 10<sup>7</sup> conidia/mL was added to the center of each
 20-mL PDA plate. Subsequently, the suspension was blended evenly and
 placed in a constant temperature incubator at 26±1℃ for 15 d under a 16-h
 light:8-h dark photoperiod. Five fungi agar blocks with a 5-mm diameter
 were taken from each plate randomly. A spore suspension was prepared using
 5 mL of 0.1% (v/v) Tween-80 solution. Each strain had three replicates and
 cell number was counted using a blood cell counting plate to determine
 conidia yield per unit area. The calculation formula was as
 follows<sup>[37]</sup>: Conidia yield
 (number/mL) = cell total number × dilution factor × 5 ×
 10<sup>4</sup> To determine the biomass of
 the tested strains, 200 μL of <em>B. bassiana</em> conidia
 suspension was inoculated with 1 × 10<sup>7</sup> conidia/mL
 and cultivated on a PDA plate containing sterile glass paper (20 mL per
 plate), and then incubated in a constant temperature incubator at 26±1℃
 for 10 d under a 16-h light:8-h dark photoperiod. The mycelia were scraped
 and dried in a 60℃ oven for 2 d. The dry weight was measured to determine
 the impact of virus infection on host biomass. Each strain had three
 replicates. Based on the impact of virus infection on
 the biological characteristics of the host fungi, the virus-free strain
 BbOFDH2 was named BbOFDH and the virus-infected strain BbOFDH-CV1 was
 named BbOFDH-CV in subsequent experiments.
 <strong><em> </em></strong><strong><em>Evaluation of antagonistic activity by B. bassiana</em></strong><strong> in vitro</strong> To clarify the direct inhibitory effects of <em>B. bassiana</em> on <em>B. cinerea </em>and <em>S. sclerotiorum </em>phytopathogens, two 5-mm-diameter fungal blocks obtained from the test <em>B. bassiana</em> strains cultured for 7 d were inoculated at a distance of 1.5 cm on both sides of the fungal pathogen block on the same line, whereas the control group was only inoculated with the fungal pathogen block. The plates were cultured at 26℃, observed, and photographed at 3 and 5 d post inoculation (dpi). Each treatment had five replicates. The inhibitory rate was calculated according to the following formula <sup>[38]</sup>: Inhibition rate (%)=[(control group colony diameter - treatment group colony diameter)]/control colony diameter×100% To evaluate the inhibitory effect of fungal metabolites on pathogenic fungi growth, 5-mm diameter <em>B. bassiana</em> blocks were inoculated in SDY (Sabouraud Dextrose Broth with Yeast Extract) medium, and incubated in a constant temperature shaking incubator (180 r/min) at 26℃ for 7 d. The mycelia were removed with sterilized gauze and centrifuged (12000 r/min) at 4℃ for 10 min, and the supernatant was obtained after filtering with a 0.22-μM filter. The supernatant was mixed with PDA culture medium in a volume ratio of 1/5 to obtain the antagonistic culture medium, and a 5-mm pathogen block was placed at the center. The PDA culture medium without supernatant was used as the control. All plates were cultivated in a constant temperature incubator at 26℃. The colony diameter of the pathogenic fungi was measured and recorded at 3 dpi, and the inhibitory rate was calculated using the following formula: inhibition rate (%) = (Cd - Td) /Cd × 100 %, where Cd is the colony diameter on the control PDA plate and Td is the colony diameter on the treated PDA plate <sup>[39]</sup>. Each treatment had five replicates. <strong><em> </em></strong><strong><em>Effect of BbCV2 virus infection on plant disease control effect of host strains in vitro</em></strong> Three treatments were set up in the present study: tomato inoculated with <em>S. sclerotiorum</em> (Ssr) or <em>B. cinerea</em> (Bc) only (Control); tomato inoculated with both BbOFDH and pathogens (BbOFDH); and tomato inoculated with both BbOFDH-CV and pathogens (BbOFDH-CV). Root irrigation was used to achieve <em>B. bassiana</em> colonization, as described by Sui et al. <sup>[36]</sup>. The blastospore suspensions of strains BbOFDH and BbOFDH-CV were prepared at concentrations of 1 × 10<sup>8</sup> conidia/mL for root irrigation. For each seeding, 20 mL of suspension was irrigated every 24 h three times. The colonization rate of <em>B. bassiana</em> was detected using the PDA leaf plate method <sup>[40]</sup>. The second fully unfolded leaves from tomato plants colonized by <em>B. bassiana</em> were used for <em>in vitro</em> disease resistance evaluation, as described by Zhang et al <sup>[41]</sup>. Each treatment had five replicates, with 10 tomato leaves inoculated per replicate. The leaves were sterilized and inoculated with blocks of two phytopathogens, <em>S. sclerotiorum</em> and <em>B. cinerea</em>. The leaves were put into culture dishes and the temperature maintained at 26 ± 1℃ and 70±1% RH The incidence of leaf disease was observed every 24 h for 3 d. The diameters of disease spots on leaves were measured using the cross over method, and photographs were taken to record the morphology of the disease spots. <strong><em>Effect of BbCV2 virus on host strain and plant disease resistance in pot experiments</em></strong> Four treatments were set up in the pot experiments for plant resistance evaluation against both <em>S. sclerotiorum</em> (Ssr) and <em>B. cinerea</em> (Bc) infection, including tomato inoculated without pathogens or <em>B. bassiana</em> strain (Control); tomato inoculated with pathogens (Ssr/Bc); tomato inoculated with both BbOFDH and pathogens (BbOFDH); and tomato inoculated with both BbOFDH-CV and pathogens (BbOFDH-CV). The pots were 15 cm in diameter and 20 cm in height. The tomato seedlings were cultivated in infected soil for Ssr inoculation, and Bc treatments were inoculated with a <em>B. cinerea</em> block at 23°C and 95% RH, according to Sui et al <sup>[42]</sup>. All experiments were conducted on 10 seedlings and repeated three times, yielding a total of 30 seedlings per treatment. The seedlings in the control group and the pathogen inoculation group were irrigated with sterilized 0.1% (v/v) Tween-80 solution. The disease incidence and disease grade of plants were observed and recorded at 9, 12 and 15 d for Ssr, and 3, 5 and 7 d for Bc, after root irrigation. The grading criteria for sclerotinia disease and gray mold disease were evaluated according to Ben and Vallance <sup>[43]</sup> and Sui et al <sup>[42]</sup>, respectively. <strong><em>Evaluation the contents of </em></strong><strong>B. bassiana<em> and phytopathogens in plants</em></strong> To clarify the relationship between the symptoms and contents of phytopathogens and <em>B. bassiana</em>, the quantitative real time PCR (qPCR) method was employed to assess the relative amounts of fungi. On the last day of the disease index investigation (15<sup>th</sup> day for Ssr and 7<sup>th</sup> day for Bc after root irrigation), plant samples were obtained for DNA extraction. Tomato roots were obtained for Ssr and <em>B. bassiana </em>content evaluation, whereas leaves inoculated with Bc were obtained for the evaluation of Bc and <em>B. bassiana </em>contents, using the qPCR method <sup>[42]</sup>. The reaction system was as follows: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 10 s, annealing at 60℃ for 30 s, 40 cycles, extension at 95℃ for 15 s, extension at 60℃ for 60 s, and extension at 95℃ for 15 s. The specific primers for Ssr, Bc, and <em>B. bassiana</em>, as well as the internal reference gene for tomato, are listed in Table S1. <strong><em>Statistical analyses </em></strong> Data are presented in bars as mean ± standard error of the mean (SEM). All data were subjected to one-way Analysis of Variance, <em>P</em> &lt; 0.05 = *, <em>P</em>&lt; 0.01 = **, and <em>P</em> &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).
description: # Insect hypovirulence-associated mycovirus confers entomopathogenic fungi
 with enhanced resistance against phytopathogens
 [https://doi.org/10.5061/dryad.7sqv9s516](https://doi.org/10.5061/dryad.7sqv9s516) The present study investigated the effects of the mycovirus *Beauveria bassiana* chrysovirus 2 (BbCV2) virus infection on host biological characteristics, additionally, we evaluated antagonistic activity of BbCV2 against phytopathogenic fungi (*Sclerotinia sclerotiorum* and *Botrytis cinerea*) *in vitro* and their associated diseases both in *in vitro* leaves and in pot experiments. ## Description of the data and file structure We provided data for Processed and Raw, we also provided Software files for Figure 1-7. #### Figure\_1\_raw This file contains  raw datasets for 'Effects of BbCV2 on the biological characteristics of host fungi. (A) Growth rate. (B) Conidiation per unit area. (C) Biomass.' #### Figure\_1\_prosecced This file contains  prosecced datasets for 'Effects of BbCV2 on the biological characteristics of host fungi. (A) Growth rate. (B) Conidiation per unit area. (C) Biomass.' #### Growth rate The colony diameter was measured using the cross over method for 12 d, and each strain had five replicates. #### Conidiation per unit area Each strain had three replicates and cell number was counted using a blood cell counting plate to determine conidia yield per unit area. #### Biomass The dry weight was measured to determine the impact of virus infection on host biomass. Each strain had three replicates. #### Figure\_2\_raw This file contains  raw datasets for 'Antagonistic activity of different *Beauveria bassiana* strains against phytopathogens. (A) Inhibition activity against *Sclerotinia sclerotiorum* on PDA medium. (B) Inhibition activity against *Botrytis cinerea* on PDA medium. (C) Inhibition rate against *S. sclerotiorum*. (D) Inhibition rate against *B. cinerea*. (E) Colony diameter of *B. bassiana* strains cocultured with *S. sclerotiorum*. (F) Colony diameter of *B. bassiana* strains cocultured with *B. cinerea*.' #### Figure\_2\_prosecced This file contains  prosecced datasets for 'Antagonistic activity of different *B. bassiana* strains against phytopathogens. (A) Inhibition activity against *S. sclerotiorum* on PDA medium. (B) Inhibition activity against *B. cinerea* on PDA medium. (C) Inhibition rate against *S. sclerotiorum*. (D) Inhibition rate against *B. cinerea*. (E) Colony diameter of *B. bassiana* strains cocultured with *S. sclerotiorum*. (F) Colony diameter of *B. bassiana* strains cocultured with *B. cinerea*.' #### Inhibition rate To clarify the direct inhibitory effects of *B. bassiana* on *B. cinerea* and *S. sclerotiorum* phytopathogens, two 5-mm-diameter fungal blocks obtained from the test *B. bassiana* strains cultured for 7 d were inoculated at a distance of 1.5 cm on both sides of the fungal pathogen block on the same line, whereas the control group was only inoculated with the fungal pathogen block. Each treatment had five replicates. Inhibition rate (%)=[(control group colony diameter - treatment group colony diameter)]/control colony diameter×100% #### Colony diameter The colony diameter of the pathogenic fungi was measured and recorded at 3 and 5 dpi. #### Figure\_3\_raw This file contains  raw datasets for 'Antagonistic effect of the secondary metabolites of *B. bassiana* strains on phytopathogens. (A) Inhibition activity against *S. sclerotiorum*. (B) Inhibition activity against *B. cinerea. *(C) Inhibition rate against *S. sclerotiorum*. (D) Inhibition rate against *B. cinerea*.' #### Figure\_3\_prosecced This file contains  prosecced datasets for 'Antagonistic effect of the secondary metabolites of *B. bassiana* strains on phytopathogens. (A) Inhibition activity against *S. sclerotiorum*. (B) Inhibition activity against *B. cinerea*. (C) Inhibition rate against *S. sclerotiorum*. (D) Inhibition rate against *B. cinerea*.' #### Inhibition rate (secondary metabolites of *B. bassiana* strains on phytopathogens) To evaluate the inhibitory effect of fungal metabolites on pathogenic fungi growth, 5-mm diameter *B. bassiana* blocks were inoculated in SDY (Sabouraud Dextrose Broth with Yeast Extract) medium, and incubated in a constant temperature shaking incubator (180 r/min) at 26℃ for 7 d. The mycelia were removed with sterilized gauze and centrifuged (12000 r/min) at 4℃ for 10 min, and the supernatant was obtained after filtering with a 0.22-μM filter. The supernatant was mixed with PDA culture medium in a volume ratio of 1/5 to obtain the antagonistic culture medium, and a 5-mm pathogen block was placed at the center. The PDA culture medium without supernatant was used as the control. All plates were cultivated in a constant temperature incubator at 26℃. Each treatment had five replicates. #### Figure\_4\_raw This file contains  raw datasets for 'Control effects of different *B. bassiana* strains on phytopathogens *in vitro* in leaves. (A) Incidence rates of sclerotinia disease. (B) Lesion diameters of sclerotinia disease. (C) Symptoms of sclerotinia disease. (D) Incidence rates of gray mold disease. (E) Lesion diameters of gray mold disease. (F) Symptoms of gray mold disease.' #### Figure\_4\_prosecced This file contains  prosecced datasets for 'Control effects of different *B. bassiana* strains on phytopathogens *in vitro* in leaves. (A) Incidence rates of sclerotinia disease. (B) Lesion diameters of sclerotinia disease. (C) Symptoms of sclerotinia disease. (D) Incidence rates of gray mold disease. (E) Lesion diameters of gray mold disease. (F) Symptoms of gray mold disease.' #### Incidence rate The incidence of leaf disease was observed every 24 h for 3 d. #### Lesion diameters The diameters of disease spots on leaves were measured using the cross over method, and photographs were taken to record the morphology of the disease spots. #### Figure\_5\_raw This file contains  raw datasets for 'Control effects of different *B. bassiana* strains on sclerotinia disease in pot experiments. (A) Incidence rates at 9 d post root irrigation. (B) Disease index at 9 d post root irrigation. (C) Disease index at 12 d post root irrigation. (D) Disease index at 15 d post root irrigation. (E) Symptoms of tomato seedlings in pots at 15 d post root irrigation. (F) Symptoms of tomato roots at 15 d post root irrigation.' #### Figure\_5\_prosecced This file contains  prosecced datasets for 'Control effects of different *B. bassiana* strains on sclerotinia disease in pot experiments. (A) Incidence rates at 9 d post root irrigation. (B) Disease index at 9 d post root irrigation. (C) Disease index at 12 d post root irrigation. (D) Disease index at 15 d post root irrigation. (E) Symptoms of tomato seedlings in pots at 15 d post root irrigation. (F) Symptoms of tomato roots at 15 d post root irrigation.' #### Disease index The disease incidence and disease index of plants were observed and recorded at 9, 12 and 15 d for Ssr, and 3, 5 and 7 d for Bc, after root irrigation. A disease index was calculated as the areas of the inoculated leaves, and each treatment was applied to 10 plants #### Figure\_6\_raw This file contains  raw datasets for 'Control effects of different *B. bassiana* strains on gray mold disease in pot experiments. (A) Incidence rates at 3 d post root irrigation. (B) Disease index at 3 d post root irrigation. (C) Disease index at 5 d post root irrigation. (D) Disease index at 7 d post root irrigation. (E) Symptoms of tomato seedlings in pots at 7 d post root irrigation.' #### Figure\_6\_prosecced This file contains  prosecced datasets for 'Control effects of different *B. bassiana* strains on gray mold disease in pot experiments. (A) Incidence rates at 3 d post root irrigation. (B) Disease index at 3 d post root irrigation. (C) Disease index at 5 d post root irrigation. (D) Disease index at 7 d post root irrigation. (E) Symptoms of tomato seedlings in pots at 7 d post root irrigation.' #### Figure\_7\_raw This file contains  raw datasets for 'Relative contents of *B. bassiana* and phytopathogens in tomato seedlings. (A) *B. bassiana* strains in the sclerotinia disease control evaluation experiment. (B) *S. sclerotiorum* contents in the sclerotinia disease control evaluation experiment. (C) *B. bassiana* strain contents in the gray mold disease control evaluation experiment. (D) *B. cinerea* contents in the gray mold control evaluation experiment.' #### Figure\_7\_prosecced This file contains  prosecced datasets for 'Relative contents of *B. bassiana* and phytopathogens in tomato seedlings. (A) *B. bassiana* strains in the sclerotinia disease control evaluation experiment. (B) *S. sclerotiorum* contents in the sclerotinia disease control evaluation experiment. (C) *B. bassiana* strain contents in the gray mold disease control evaluation experiment. (D) *B. cinerea* contents in the gray mold control evaluation experiment.' #### Table This file contains  informations for Gene primer sequences. ## Sharing/Access information There is no other ways to access the data, and for linking to sources the data is derived from. ## Code/Software 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).