Insect hypovirulence-associated mycovirus confers entomopathogenic fungi with enhanced resistance against phytopathogens

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.

Methods
Evaluation of the effects ofBbCV2 virus infection on host biological characteristics

For the growth rate test, 2 μL of B. bassiana suspension at a concentration of 5 × 104 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 [25]: 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 B. bassiana suspension liquid at a concentration of 1 × 107 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[37]:

Conidia yield (number/mL) = cell total number × dilution factor × 5 × 104

To determine the biomass of the tested strains, 200 μL of B. bassiana conidia suspension was inoculated with 1 × 107 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.

 Evaluation of antagonistic activity by B. bassiana in vitro

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. 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 [38]:

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 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℃. 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 [39]. Each treatment had five replicates.

 Effect of BbCV2 virus infection on plant disease control effect of host strains in vitro

Three treatments were set up in the present study: tomato inoculated with S. sclerotiorum (Ssr) or B. cinerea (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 B. bassiana colonization, as described by Sui et al. [36]. The blastospore suspensions of strains BbOFDH and BbOFDH-CV were prepared at concentrations of 1 × 108 conidia/mL for root irrigation. For each seeding, 20 mL of suspension was irrigated every 24 h three times. The colonization rate of B. bassiana was detected using the PDA leaf plate method [40].

The second fully unfolded leaves from tomato plants colonized by B. bassiana were used for in vitro disease resistance evaluation, as described by Zhang et al [41]. Each treatment had five replicates, with 10 tomato leaves inoculated per replicate. The leaves were sterilized and inoculated with blocks of two phytopathogens, S. sclerotiorum and B. cinerea. 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.

Effect of BbCV2 virus on host strain and plant disease resistance in pot experiments

Four treatments were set up in the pot experiments for plant resistance evaluation against both S. sclerotiorum (Ssr) and B. cinerea (Bc) infection, including tomato inoculated without pathogens or B. bassiana 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 B. cinerea block at 23°C and 95% RH, according to Sui et al [42]. 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 [43] and Sui et al [42], respectively.

Evaluation the contents of B. bassiana and phytopathogens in plants

To clarify the relationship between the symptoms and contents of phytopathogens and B. bassiana, 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 (15th day for Ssr and 7th day for Bc after root irrigation), plant samples were obtained for DNA extraction. Tomato roots were obtained for Ssr and B. bassiana content evaluation, whereas leaves inoculated with Bc were obtained for the evaluation of Bc and B. bassiana contents, using the qPCR method [42]. 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 B. bassiana, as well as the internal reference gene for tomato, are listed in Table S1.

Statistical analyses

Data are presented in bars as mean ± standard error of the mean (SEM). All data were subjected to one-way Analysis of Variance, P < 0.05 = *, P< 0.01 = **, and P < 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).