
==== Front
Virulence
Virulence
Virulence
2150-5594
2150-5608
Taylor & Francis

39263889
10.1080/21505594.2024.2401978
2401978
Version of Record
Research Article
Research Article
Insect hypovirulence-associated mycovirus confers entomopathogenic fungi with enhanced resistance against phytopathogens
L. SUI ET AL.
VIRULENCE
https://orcid.org/0000-0001-9379-1080
Sui Li a *
Lu Yang a *
Xu Mengnan a b
Liu Jianfeng b
Zhao Yu a
Li Qiyun a b c
https://orcid.org/0000-0001-8822-2107
Zhang Zhengkun a b
a Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Jilin Key Laboratory of Agricultural Microbiology, Key Laboratory of Integrated Pest Management on Crops in Northeast China, Ministry of Agriculture and Rural Affairs , Gongzhuling, Jilin, China
b College of Life Sciences, Jilin Normal University , Siping, China
c College of Agriculture, Jilin Agricultural Science and Technology University , Jilin, China
CONTACT Qiyun Li qyli1225@126.com
Zhengkun Zhang zhangzhengkun@126.com
* These authors contributed equally to this work.

12 9 2024
2024
12 9 2024
15 1 2401978Integra12 9 2024
Integra12 9 2024
09 5 2024
12 8 2024
31 8 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Mycoviruses can alter the biological characteristics of host fungi, including change virulence or pathogenicity of phytopathogens and entomopathogenic fungi (EPF). However, most studies on the mycoviruses found in EPF have focused on the effects of the viruses on the virulence of host fungi towards 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, evaluated antagonistic activity of BbCV2 against two phytopathogenic fungi (Sclerotinia sclerotiorum and Botrytis cinerea), and transcriptome analysis was used to reveal the interactions between viruses and hosts. Our results showed that BbCV2 virus infection increased B. bassiana‘s growth rate, spore production, and biomass, it also enhanced the capacity of host fungi and their metabolic products to inhibit phytopathogenic fungi. BbCV2 virus infection reduced the contents of the two pathogens in tomato plants significantly, and transcriptome analysis revealed that the genes related to competition for ecological niches and nutrition, mycoparasitism and secondary metabolites in B. bassiana were significantly up-regulated after viral infection. These findings indicated that the mycovirus infection is an important factor to enhance the ability of B. bassiana against plant disease after endophytic colonization. We suggest that mycovirus infection causes a positive effect on B. bassiana against phytopathogens, which should be considered as a potential strategy to promote the plant disease resistance of EPF.

KEYWORDS

Mycovirus
Beauveria bassiana
endophytic colonization
plant disease resistance
Jilin Agricultural Science and Technology Innovation Engineering Project CXGC2024JJ011 Jilin Project for Outstanding Talents (Teams) for Innovation and Entrepreneurship in Science and Technology 20230508011RC National Natural Science Foundation of China 10.13039/501100001809 32271683 This project was supported by Jilin Agricultural Science and Technology Innovation Engineering Project [grant number CXGC2024ZY044, CXGC2024JJ011], the Jilin Project for Outstanding Talents (Teams) for Innovation and Entrepreneurship in Science and Technology [grant number 20230508011RC], and the National Natural Science Foundation of China [32271683].
==== Body
pmcIntroduction

Beauveria bassiana is known as a significance entomopathogenic fungus (EPF), extensively used as a fungal insecticide to control various agricultural and forestry insect pests. More and more studies have uncovered that B. bassiana could not only directly infect insects but also colonize plants [1]. Bing and Lewis, for the first time, reported B. bassiana colonization of maize (Zea mays L.) tissue, revealing B. bassiana as an endophyte [2]. In addition, Gurulingappa et al. experimentally demonstrated that B. bassiana could colonize Gossypium hirsutum and Triticum aestivum, thereby reducing the reproduction rate of Helicoverpa armigera, an important agricultural insect pest [3]. Furthermore, B. bassiana colonization of T. aestivum could improve T. aestivum seedling growth significantly [4], and treatment of Solanum lycopersicum and Capsicum annuum roots with B. bassiana could reduce the disease index of Botrytis cinerea phytopathogen [5].

Following inoculation of Nicotiana tabacum seeds with six different B. bassiana strains, the B. bassiana strains could colonize tobacco seedlings and confer a significant increase in resistance to bacterial and fungal pathogens [6]. Moreover, B. bassiana distribution in plant tissues has been observed using green fluorescent protein (GFP)-labelled strains, which has provided technical support for further exploration of B. bassiana endophytic colonization of plant tissues [7]. Following symbiosis establishment with plants, B. bassiana can not only inhibit damage by insect pests and promote plant growth but also minimize the harm caused by phytopathogens to plants, with multiple potential applications in pest biocontrol and growth promotion.

A mycovirus is a type of virus that infects fungi and can replicate within them, and they are widespread in nature. In 1962, Holling first observed viral particles in the fruiting bodies of diseased Agaricus bisporus, reporting these could cause mushroom diseases [8,9], thus attracting attention to mycoviruses. Mycoviruses have varied impacts on host fungi. Following infection with three mycoviruses, the growth rate of a Monilinia fructicola isolate increased 10% when compared with those of virus-free strains [10]. In addition, infection with dsRNA mycovirus Alternaria alternata chrysovirus 1 reduced the growth rate of the host fungi [11], and infection with Penicillium crustosum chrysovirus 1 conferred resistance to fungicides in host fungi [12]. Moreover, the pathogenicity of Sclerotinia sclerotiorum decreased significantly following infection with DNA virus Botrytis gemydayirivirus 1 [13].

Viruses that reduce the pathogenicity of host fungi are known as hypovirulence-associated mycoviruses [9], and they have potential applications in the biological control of plant pathogenic fungal diseases. The mycoviruses Cryptomeria hypovirus 1 and Sclerotinia sclerotiorum hypovirus-associated DNA virus 1 have been applied successfully in biological control in the field [14–17]. In addition, there have been numerous reports of hypovirulence-associated mycoviruses, including Fusarium graminearum hypovirus 2 [18], Botrytis cinerea RNA virus 1 [19], and Rhizoctonia solani, as well as Irus 2, 3, and 4 (RsPV2, RsPV3, RsPV4) [20,21]. Such mycoviruses provide abundant viral resources with potential applications in the biological control of plant diseases caused by phytopathogenic fungi.

Studies on mycoviruses have mainly focused on phytopathogenic fungi, with relatively less attention directed at EPF. At present, more than 20 kinds of B. bassiana viruses have been identified, which are distributed across eight viral families [22–32]. Previous studies have shown that the mycoviruses could affect the biological characteristics and insecticidal virulence of B. bassiana. Beauveria bassiana polymycovirus 1 and 3 (BbPmV-1, BbPmV-3) could regulate the basic metabolic pathways of host fungal strains, thereby affecting spore production and growth [33]. In addition, Beauveria bassiana chrysovirus 2 (BbCV2) could promote the growth of the fungal strain and reduce its pathogenicity to an insect pest, Ostrinia furnacalis [34]. Furthermore, infection with Beauveria bassiana polymycovirus 4 promoted B. bassiana growth, altered its morphology, reduced its spore production, and enhanced its virulence against an insect pest [35]. However, there are few reports on the effects of mycovirus infection in B. bassiana, particularly with regard to its antagonistic activity against phytopathogenic fungi and plant resistance to diseases caused by the mycoviruses.

Most studies have reported that B. bassiana colonization in plants could enhance plant resistance to diseases caused by plant pathogens [5,36], but ignored the effects on disease resistance of mycovirus associated with B. bassiana, though the interaction between mycovirus and B. bassiana has been well documented [33–35]. This leaves an important knowledge gap on how the effects of mycoviruses on EPF concerning plant disease resistance. The aim of the present study was to investigate the effects of BbCV2 virus infection on the biological characteristics of B. bassiana, as well as its antagonistic activity against phytopathogenic fungi and their associated diseases via confrontation, detached leaf, and pot experiments. These findings of the present study could enhance our understanding of the roles of mycoviruses in plant disease resistance induction following B. bassiana colonization, in addition to facilitating the exploration of the biological control potential of mycoviruses in plants.

Materials and methods

Fungal strains

Isogenic strains with and without BbCV2 infection were obtained via the insect coinfection method as described previously [37]. In detail, B. bassiana strain BbOFDH1-5-GFP is a virus-free strain expressed GFP and bar gene, which showed resistance against glyphosate [7], and its three isolates, BbOFDH1, BbOFDH2, and BbOFDH3, were selected randomly by single spore isolation. Three B. bassiana strains infected with BbCV2 virus were obtained by culturing strain BbOFDH1-5-GFP with the virus-infected strain BbOFZK152, which were identified by glyphosate containing culture medium and GFP observation, and named BbOFDH-CV1, BbOFDH-CV2, and BbOFDH-CV3, the three virus-infected strains above were subcultured on potato dextrose agar (PDA) medium for three generations and verified by dsRNA extraction and RT-PCR [34]. The S. sclerotiorum strain was provided by Prof. Jinliang Liu from Jilin University, Changchun, China. The B. cinerea strain was provided by Prof. Wei Li from Hunan Agricultural University, Changsha, China.

Seedlings and soil

The BEAUTY tomato seedling variety, obtained from Jilin Mainland Seed Industry Co. LTD, Gongzhuling, Jilin, China, was used as the plant test material in the present study.

Peat soil, obtained from Pindstrup Mosebrug A/S, Ryomgard, Denmark, was used for grey mould disease pot experiments. The soil used for sclerotinia disease pot experiments was infected soil obtained from the field at the Jilin Academy of Agricultural Sciences, Jilin, China.

Evaluation of the effects of BbCV2 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 centre of each 20 mL PDA plate, and cultured in a constant temperature incubator at 26 ± 1°C, with 70 ± 1% relative humidity (RH), and a 16-h light:8-h dark photoperiod. Colony diameter was measured daily using the cross-method over a period of 12 d, with five replicates per strain. 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 centre of each 20-mL PDA plate. Subsequently, the suspension was blended evenly and placed in a constant temperature incubator at 26 ± 1°C 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 [38]:

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°C for 10 d under a 16-h light:8-h dark photoperiod. The mycelia were scraped and dried in a 60°C 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°C, observed, and photographed at 3 and 5 d post inoculation (dpi). Each treatment had five replicates. The colony diameter of the pathogenic fungi was measured and recorded at 3 and 5 dpi, the inhibitory rate was calculated according to the following formula [39]:

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 rpm) at 26°C for 7 d. The mycelia were removed with sterilized gauze and centrifuged (12000 rpm) at 4°C 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 centre. The PDA culture medium without supernatant was used as the control. All plates were cultivated in a constant temperature incubator at 26°C. 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 [40]. 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; compared with aerial conidia, blastospore had better colonization ability on plants [36], and the blastospore suspensions of strains BbOFDH and BbOFDH-CV were prepared at concentrations of 1 × 108 blastospore/mL for root irrigation. For each seeding, 20 mL of suspension was irrigated every 24 h three times. The colonization of B. bassiana was detected using the PDA leaf plate method [41] and examined under a Leitz Diaplan light microscope [36], B. bassiana could grow from plant leave sections and observed in leaves under light and confocal microscopy (Figure S1).

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. [42]. 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 (a wet cotton ball was placed in the middle of the leaves to moisturize) and the temperature maintained at 26 ± 1°C 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. [43]. 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 grey mould disease were evaluated according to Ben and Vallance [44] and Sui et al. [43], 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 [43]. The reaction system was as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, annealing at 60°C for 30 s, 40 cycles, extension at 95°C for 15 s, extension at 60°C for 60 s, and extension at 95°C for 15 s. The specific primers for Ssr, Bc, and B. bassiana, as well as a single, well-validated housekeeping gene for normalization (the internal reference gene for tomato) was used across all qPCR experiments, and primers are listed in Table S1.

Transcriptome sequencing analysis

The transcriptome sequencing of virus-free and virus-infected isolates of B. bassiana strain BbOFDH1-5-GFP has been performed in our previous study [34], based on which the expression of the pathways and genes related to growth, development and antagonistic activity of B. bassiana was analysed in the present study.

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 analysed 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).

Results

Impact of BbCV2 virus on host biological characteristics

On day 12 of culture, the growth rates of virus-infected B. bassiana strains were significantly higher than those of the three virus-free strains, and BbOFDH-CV2 had the highest growth rate (Figure 1a) (p < 0.05), the spore production of virus-infected B. bassiana strains were significantly higher than those of the three virus-free strains, especially the strain of BbOFDH-CV2 (p < 0.0001) (Figure 1b), and the biomass of virus-infected B. bassiana strains was significantly higher by 88.9%, 188.9% and 150.0 than those of the three virus-free strains, respectively (p < 0.0001) (Figure 1c). Figure 1. Effects of BbCV2 on the biological characteristics of host fungi.

Effect of viral infection on antagonistic activity against B. bassiana pathogens

Confrontation culture results showed obvious antagonistic zones in both the BbOFDH and BbOFDH-CV groups against S. sclerotiorum (Figure 2a) and B. cinerea (Figure 2b). On day 3, the inhibitory rates of BbOFDH and BbOFDH-CV strains against S. sclerotiorum were 25.7 ± 1.1% and 33.1 ± 1.3%, respectively, and on day 5, they were 45.3 ± 0.4% and 51.4 ± 0.9%, respectively, and the inhibitory rates of BbOFDH-CV were significantly higher than those of BbOFDH (p < 0.001) (Figure 2c). On day 3, the inhibitory rates of BbOFDH and BbOFDH-CV strains against B. cinerea were 10.5 ± 2.3% and 19.0 ± 1.6%, respectively (p < 0.001), and on day 5, they were 33.1 ± 1.2% and 39.6 ± 1.0%, respectively, and the inhibitory rates of BbOFDH-CV were significantly higher than those of BbOFDH (p < 0.0001) (Figure 2d). In the pathogen antagonistic experiments, the colony diameter of the virus-infected B. bassiana strain was significantly higher than that of the virus-free strain on both of S. sclerotiorum (Figure 2e) and B. cinerea (Figure 2f). Figure 2. Antagonistic activity of different B. bassiana strains againstphytopathogens.

Regarding the inhibitory effect of secondary metabolites of B. bassiana on phytopathogens, S. sclerotiorum and B. cinerea growth were inhibited obviously at 3 and 5 dpi (Figure 3a,b). The inhibition rate of BbOFDH-CV was significantly higher, by 156.0%, than that of the BbOFDH strain (p < 0.001) (Figure 3c). Similarly, the inhibitory rate of BbOFDH-CV was significantly higher, by 46.4%, than that of the BbOFDH strain (p < 0.001) (Figure 3d). Figure 3. Antagonistic effect of the secondary metabolites of B. bassiana strains on phytopathogens.

Effect of BbCV2 virus on the control effect of host strains on detached leaves

The incidence rates of sclerotinia disease in Ssr, BbOFDH, and BbOFDH-CV groups at 2 dpi were 90.0 ± 0.0%, 88.0 ± 12.9%, and 68.0 ± 12.6%, respectively, and the colonization by the virus-infected strain and virus-free strain of B. bassiana could reduce the disease incidence rates significantly (p < 0.05), while the incidence rates in the BbOFDH-CV treatment group were 20.0% lower than those in the BbOFDH treatment group (p = 0.0729) (Figure 4a). In addition, lesion diameters in the BbOFDH-CV treatment group were significantly lower than those in the BbOFDH treatment group, at 8.5 ± 1.3 mm and 10.2 ± 0.1 mm (p < 0.05), respectively, and those in the Ssr group, at 11.9 ± 0.8 mm, were significantly higher than those in the other two groups (p < 0.05) (Figure 4b,c). Figure 4. Control effects of different B. bassiana strains on phytopathogensin vitro in leaves.

The incidence rates of grey mould disease in the Bc, BbOFDH, and BbOFDH-CV groups at 2 dpi were 66.7 ± 11.5%, 40.0 ± 0.0% and 26.7 ± 5.8%, respectively, and colonization by the virus-infected strain and virus-free strain of B. bassiana could reduce disease incidence rates significantly (p < 0.01). In addition, the incidence rates in the BbOFDH-CV treatment group were 40.0% lower than those in the BbOFDH treatment group (p = 0.0587) (Figure 4d). Lesion diameters in the BbOFDH-CV treatment were significantly lower than those in the BbOFDH treatment group, which were 8.2 ± 0.3 mm and 10.2 ± 0.4 mm (p < 0.05), respectively, and much lower than those in the Bc group, at 11.9 ± 0.8 mm (p < 0.01) (Figure 4e,f).

Effect of BbCV2 virus infection on the plant disease control efficiency of B. bassiana in pot experiments

At 9 d post the root irrigation treatment of B. bassiana strains, the incidence rates of two strains of B. bassiana treatment groups were significantly lower than that of the Ssr treatment under sclerotinia disease (Figure 5a). The results of the disease index evaluation showed that both of the virus-infected and virus-free B. bassiana treatments were decreased significantly compared with that in the Ssr treatment at different days post root irrigation, and the disease index of virus-infected treatment was significantly lower than that in the virus-free treatment at 15 d post root irrigation (Figure 5b–d). There were obvious differences in symptoms of tomato seedlings (Figure 5e) and roots (Figure 5f) in pots at 15 d post root irrigation in different treatments. Figure 5. Control effects of different B. bassiana strains on sclerotiniadisease in pot experiments.

Under grey mould disease infection, the incidence rate was significantly lower in BbOFDH-CV treatment than that of the Bc treatment at 3 d post root irrigation (Figure 6a). The disease index of BbOFDH and BbOFDH-CV treatment were significantly lower than that in the Bc treatment at different days post root irrigation, and the disease index of virus-infected treatment was significantly lower than that in the virus-free treatment at 5 and 7 d post root irrigation (Figure 6b–d). There were obvious differences in symptoms of tomato seedlings in pots at 7 d post root irrigation in different treatments (Figure 6e). Figure 6. Control effects of different B. bassiana strains on gray molddisease in pot experiments.

Relative quantities of pathogens and B. bassiana in tomato seedlings

In the sclerotinia disease experiment, at 15 dpi in B. bassiana strains, the relative B. bassiana contents in the BbOFDH-CV treatment were significantly higher than those in the BbOFDH treatment (Figure 7a), whereas S. sclerotiorum contents were lower than those in the Ssr treatment (Figure 7b). Similarly, in the grey mould disease experiment, at 7 dpi in B. bassiana strains, B. bassiana relative contents in the BbOFDH-CV treatment were significantly higher than those in the BbOFDH treatment (Figure 7c), whereas B. cinerea contents were lower than those in the Bc treatment (Figure 7d). Figure 7. Relative contents of B. bassiana and phytopathogens in tomatoseedlings.

Transcriptome analysis of virus BbCV2 infecting B. bassiana

Transcriptome analysis showed that after viral infection, the expression of pathways and genes related to growth, development, biodegradation enzymes, and metabolism in the host strain was significantly up-regulated. Mitogen-activated protein kinases (MAPK) play a critical role in regulating fungal development, growth, and response to the environment, previous study showed Bbslt2 encodes MAPK, and it was found that the growth of B. bassiana could be affected when the Bbslt2 gene was destroyed [45]. It has been reported that Bbthi and Bbpyr were genes related to pyrimidine synthesis, when these two genes were deleted, the integrity of conidia cell wall was damaged, and the growth rate of mycelium also slowed down [46]. Saccharomyces cerevisiae bcs1 is an ATPase associated with a variety of cellular activities, and its homologues have significant effects on mycovirus growth [47]. The present results showed that Bbslt2 related genes BBA_03334, Bbthi and Bbpyr related genes BBA_03937 and BBA_04964, as well as yeast bcs1 related genes BBA_03426, BBA_10003 and BBA_09074 were significantly up-regulated after BbCV2 infection.

Mycoparasitism is defined as the interrelationship between a fungus parasite and a fungus host [48,49]. It mainly involves penetration of the cell wall due to the production of lytic enzymes that break down cell-wall components, and the release of antibiotics that permeate the perforated hyphae and prevent re-synthesis of the host cell wall. Proteases and carbohydrate active enzymes including chitinase are prevalent in B. bassiana [50]. Our study showed that chitinase-related genes BBA_02153, cellulase-related genes BBA_00417, β-1, 3-glucanase-related gene BBA_00792, as well as protease-related genes BBA_07559, BBA_00619 and BBA_07369 were significantly up-regulated after BbCV2 infection.

Induced systemic resistance, known to be elicited by beneficial microbes and their metabolites, has emerged as an important mechanism by which the whole plant is primed for enhanced defence against a broad range of phytopathogens and insect pests [51]. We found after infection with BbCV2 strain, toxin metabolism related genes BBA_08421, BBA_09720, BBA_02382, BBA_06214 and BBA_08425, antibiotic production related gene BBA_10026, lipopeptide compound production related gene BBA_01877, the acid/threonine protein phosphatase encoding gene BBA_06614, as well as ferriferous transporter related genes BBA_03578, BBA_05816 and BBA_01825 were significantly up-regulated.

Discussion

Mycoviruses can affect the biological characteristics and virulence or pathogenicity of host fungi, especially in the case of phytopathogenic fungi. For example, the virus Aspergillus fumigatus 1 significantly increased the growth rate of the host Aspergillus fumigatus significantly [52]. In addition, Magnaporthe oryzae chrysovirus 1 strains A and impaired growth, altered colony morphology, and reduced pigmentation production in host fungi [53]. Conversely, S. sclerotiorum pathogenicity decreased significantly following infection with DNA virus Botrytis gemydayirivirus 1 (BGDaV1) [13]. The strains infected with the fungal virus R. Necatrix hypovirus 2 (RnHV2) exhibited slowed growth rate and weakened pathogenicity [54]. Mycoviruses are increasingly being discovered in EPF, with significant impacts on their biological characteristics and virulence. Wang et al. [55] identified a novel bipartite virus, Metarhizium majus partivirus 1 (MmPV1), in the pathogenic insect fungus Metarhizium majus RCEF0578 strain. MmPV1 infection orchestrates conidiation, stress response, pathogenicity, and secondary metabolism of the EPF stain M. majus. In addition, Kotta Loizou and Coutts (2017) screened 75 strains of B. bassiana from around the globe, out of which 16 strains contained viruses, and they were identified as BbPV-1 and BbPV-2, BbVV-1, BbSNLV, satellite RNAs, as well as BbPmV-1, BbPmV-2, and BbPmV-3 [25]. BbPmV-1 and BbPmV-3 have been demonstrated to affect the pigment deposition, spore production, and colony growth of their host fungi, which might be attributed to interference with the basic metabolic pathways of B. bassiana following virus infection [33]. In our previous study, BbCV2 infection decreased the virulence of the host fungi B. bassiana to O. furnacalis [34]. In the present study, BbCV2 infection could enhance growth rate, spore production, and the biomass of the host fungi, B. bassiana. These results confirmed that mycoviruses could change the biological characteristics and virulence or pathogenicity of host fungi, and better strain traits such as conidia yield and germination rate may also be beneficial in the interaction between the fungus and its host plant.

Mycoviruses can affect the efficiency of biocontrol fungi against plant diseases. The infection of virus Trichoderma harzianum partitrivirus 1 identified from Trichoderma harzianum, increased the inhibitory effect of the host on Fusarium oxysporum significantly [56], and its infection could alter the colony morphology of the host fungi strain, resulting in slower growth but increased biocontrol activity against S. sclerotiorum [57]. In recent years, it has been reported that B. bassiana could not only kill insects but also establish symbiosis with plants through endophytic colonization, to promote the growth and disease resistance of plants [43,58]. However, there are a few studies on whether infection by mycoviruses affects their capacity to control phytopathogenic fungi. In the present study, BbCV2 infection could enhance the ability of host fungi and its secondary metabolites to inhibit the growth of phytopathogenic fungi, such as S. sclerotiorum and B. cinerea; furthermore, the results of both tomato in vitro leaf experiments and pot experiments indicated that the viral infection resulted in the enhanced plant disease resistance against two phytopathogens of host fungi. Indeed, the underlying mechanisms of the phytopathogens resistance promotion of EPF strains after mycovirus infection are unknown, and require further elucidation.

A study on the resistance mechanisms of B. bassiana to phytopathogens following endophytic colonization demonstrated that it could alter the phyllospheric microorganisms of host plants [59], induce the expression of plant resistance related genes and pathways [43], and inhibit pathogens, based on secondary metabolites [60]. In the present study, antagonistic activity against phytopathogens by metabolites of B. bassiana was promoted after BbCV2 virus infection, and a quantitative analysis of the phytopathogens showed that the contents of B. bassiana infected with the virus were significantly higher than those of virus-free strains following endophytic colonization of plants. The findings are consistent with previous evidence that infection by the virus could increase biomass of host fungi. Furthermore, compared with virus-free fungal strains, virus-infected B. bassiana strain could reduce phytopathogen contents in plants, which may be due to greater metabolism at higher biomass. However, further research is required to identify and evaluate the contents of associated metabolites and their effects on plants.

Previous studies have shown that B. bassiana could endophytically colonize plants, which could provide a nitrogen source, promote plant growth, and regulate plant resistance to biotic and abiotic stress; conversely, plants could provide a carbon source for B. bassiana, fostering a mutually beneficial symbiotic relationship [6,41,61]. BbCV2 infection has been demonstrated to reduce the pathogenicity of host fungi to insect pests so that the virus is beneficial to insects but not to plants [34]. However, in the present study, notably, the antagonistic activity against phytopathogens of B. bassiana was promoted after virus infection and conferred plants with higher resistance to phytopathogen infection, which means that B. bassiana continues to maintain its protective effect on plants in a different way. Although the mechanisms responsible for the beneficial effects of BbCV2 virus on B. bassiana remain to be investigated, our results indeed confirmed the beneficial relationship between B. bassiana and its host.

EPF can directly suppress plant pathogens through competition for ecological niches and nutrition, mycoparasitism, and by production of secondary metabolites [62]. In this study, we found that the virus BbCV2 infection significantly increased the growth rate of host B. bassiana, and significantly up-regulated the expression of growth-related genes, such as Bbslt2, Bbthi, Bbpyr and yeast bcs1 related genes, this result indicated that strain BbCV2 had more dominant in competition with phytopathogens and thus established better disease resistance; furthermore, our results showed that the expression of genes related to chitinase, cellulase, β-1, 3-glucanase and protease were significantly up-regulated after BbCV2 infection, which indicated that compared with virus-free strain, virus infection led to more biodegradable enzymes production, then had stronger parasitic ability on phytopathogens; In addition, the antagonistic activity of supernatant solution cultured by virus-infection B. bassiana strains was significantly higher than that of virus-free strains, which means that the secondary metabolites production is stimulated by viral infection. The results of transcriptome analysis also showed that the expression of genes related to metabolites in B. bassiana were up-regulated after viral infection. As transcriptome analysis is an important means to study the interaction between mycovirus and host, our results showed that the virus BbCV2 infection significantly regulated the expression of genes and pathways related to growth, development, biodegradation enzymes, and metabolism on host strain, indicating that the mycovirus infection is an important factor to enhance the ability of B. bassiana against plant disease after endophytic colonization. Researches could be more focused on the functional verification of critical genes and pathways in the future.

Conclusion

The data provided evidence of the effects of mycovirus on EPF, which demonstrated that BbCV2 virus infection could increase the biomass and disease resistance of host fungi B. bassiana in endophytically colonized plants; transcriptomic studies were employed to explain the preliminary mechanism involving enhanced pahytopathogens resistance caused by viral infection on B. bassiana. These findings highlight the potential application of mycoviruses in the biological control of plant fungal diseases such as B. bassiana, and inform subsequent studies of the interaction mechanism between mycovirus and B. bassiana.

Supplementary Material

Figure S1.jpg

Table.docx

Acknowledgements

We would like to thank the native English speaking scientists of the Elixigen Company (Huntington Beach, California) for editing our manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, Zhengkun Zhang, Li Sui, and Qiyun Li; methodology, Yang Lu, Mengnan Xu and Yu Zhao; data curation, Mengnan Xu and Li Sui; software, Mengnan Xu; validation, Zhengkun Zhang, Li Sui and Yang Lu; formal analysis, Mengnan Xu and Jianfeng Liu; writing original draft preparation, Zhengkun Zhang, Li Sui and Mengnan Xu; writing review and editing, Yang Lu and Qiyun Li; visualization, Zhengkun Zhang and Li Sui; funding acquisition, Zhengkun Zhang and Li Sui. All authors have read and agreed to be published.

Data availability statement

All metadata to replicate the findings of this study are provided in the methods, and data could be made available at [Dataset]. Dryad. https://doi.org/10.5061/dryad.7sqv9s516.

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2024.2401978.
==== Refs
References

[1] Mascarin GM, Jaronski ST. The production and uses of Beauveria bassiana as a microbial insecticide. World J Microbiol Biotechnol. 2016;32 (11 ):177. doi: 10.1007/s11274-016-2131-3 27628337
[2] Bing LA, Lewis LC. Suppression of Ostrinia nubilalis (Hübner) (Lepidoptera: pyralidae) by endophytic Beauveria bassiana (Balsamo) Vuillemin. Environ Entomol. 1991;20 (4 ):1207–14. doi: 10.1093/ee/20.4.1207
[3] Gurulingappa P, Sword GA, Murdoch G, et al. Colonization of crop plants by fungal entomopathogens and their effects on two insect pests when in planta. Biol Control. 2010;55 (1 ):34–41. doi: 10.1016/j.biocontrol.2010.06.011
[4] Gurulingappa P, Mcgee PA, Sword G. Endophytic Lecanicillium lecanii and Beauveria bassiana reduce the survival and fecundity of Aphis gossypii following contact with conidia and secondary metabolites. Crop Prot. 2011;30 (3 ):349–353. doi: 10.1016/j.cropro.2010.11.017
[5] Barra-Bucarei L, Iglesias AF, González MG, et al. Antifungal activity of Beauveria bassiana endophyte against Botrytis cinerea in two Solanaceae crops. Microorganisms. 2019;8 (1 ):65. doi: 10.3390/microorganisms8010065 31906060
[6] Qin X, Zhao X, Huang S, et al. Pest management via endophytic colonization of tobacco seedlings by the insect fungal pathogen Beauveria bassiana. Pest Manag Sci. 2021;77 (4 ):2007–2018. doi: 10.1002/ps.6229 33342046
[7] Tong S, Yuan M, Liu Y, et al. Ergosterol-targeting fusion antifungal peptide significantly increases the verticillium wilt resistance of cotton. Plant Biotechnol J. 2021;19 (5 ):926–936. doi: 10.1111/pbi.13517 33217142
[8] Hollings M. Viruses associated with a die-back disease of cultivated mushroom. Nature. 1962;196 (4858 ):962–965. doi: 10.1038/196962a0
[9] Ghabrial SA, Castón JR, Jiang D, et al. 50-plus years of fungal viruses. Virology. 2015;479-480 :356–368. doi: 10.1016/j.virol.2015.02.034 25771805
[10] Tran TT, Li H, Nguyen DQ, et al. Co-infection with three mycoviruses stimulates growth of a Monilinia fructicola isolate on nutrient medium, but does not induce hypervirulence in a natural host. Viruses. 2019;11 (1 ):89. doi: 10.3390/v11010089 30669656
[11] Okada R, Ichinose S, Takeshita K, et al. Molecular characterization of a novel mycovirus in Alternaria alternata manifesting two-sided effects: down-regulation of host growth and up-regulation of host plant pathogenicity. Virology. 2018;519 :23–32. doi: 10.1016/j.virol.2018.03.027 29631173
[12] Wang S, Yang Z, Zhang T, et al. Molecular characterization of a chrysovirus isolated from the citrus pathogen Penicillium crustosum and related fungicide resistance analysis. Front Cell Infect Microbiol. 2019;9 :156. doi: 10.3389/fcimb.2019.00156 31157173
[13] Khalifa ME, Macdiarmid RM. A mechanically transmitted DNA mycovirus is targeted by the defence machinery of its host, botrytis cinerea. Viruses. 2021;13 (7 ):1315. doi: 10.3390/v13071315 34372522
[14] Heiniger U. Biological control of chestnut blight in Europe. Annu Rev Phytopathol. 1994;32 (1 ):581–599. doi: 10.1146/annurev.py.32.090194.003053
[15] Yu X, Li B, Fu Y, et al. Extracellular transmission of a DNA mycovirus and its use as a natural fungicide. Proc Natl Acad Sci USA. 2013;110 (4 ):1452–1457. doi: 10.1073/pnas.1213755110 23297222
[16] Tian B, Xie J, Fu Y, et al. A cosmopolitan fungal pathogen of dicots adopts an endophytic lifestyle on cereal crops and protects them from major fungal diseases. Isme J. 2020;14 (12 ):3120–3135. doi: 10.1038/s41396-020-00744-6 32814863
[17] Zhang H, Xie J, Fu Y, et al. A 2-kb mycovirus converts a pathogenic fungus into a beneficial endophyte for brassica protection and yield enhancement. Mol Plant. 2020;13 (10 ):1420–1433. doi: 10.1016/j.molp.2020.08.016 32998002
[18] Li P, Zhang H, Chen X, et al. Molecular characterization of a novel hypovirus from the plant pathogenic fungus Fusarium graminearum. Virology. 2015;481 :151–160. doi: 10.1016/j.virol.2015.02.047 25781585
[19] Yu L, Sang W, Wu MD, et al. Novel hypovirulence-associated RNA mycovirus in the plant-pathogenic fungus Botrytis cinerea: molecular and biological characterization. App Environ Microbiol. 2015;81 (7 ):2299–2310. doi: 10.1128/AEM.03992-14
[20] Lyu R, Zhang Y, Tang Q, et al. Two alphapartitiviruses co-infecting a single isolate of the plant pathogenic fungus rhizoctonia solani. Arch Virol. 2018;163 (2 ):515–520. doi: 10.1007/s00705-017-3627-3 29101540
[21] Li Y, Li S, Liang Z, et al. RNA-seq analysis of Rhizoctonia solani AG-4HGI strain BJ-1H infected by a new viral strain of Rhizoctonia solani partitivirus 2 reveals a potential mechanism for hypovirulence. Phytopathology. 2022;112 (6 ):1373–1385. doi: 10.1094/PHYTO-08-21-0349-R 34965159
[22] Yie SW, Khalifa ME, Hahn T, et al. Molecular characterization of a novel victorivirus from the entomopathogenic fungus Beauveria bassiana. Arch Virol. 2014;159 (6 ):1321–1327. doi: 10.1007/s00705-013-1938-6 24327093
[23] Koloniuk I, Hrabáková L, Petrzik K. Molecular characterization of a novel amalgavirus from the entomopathogenic fungus beauveria bassiana. Arch Virol. 2015;160 (6 ):1585–1588. doi: 10.1007/s00705-015-2416-0 25854690
[24] Kotta-Loizou I, Sipkova J, Coutts RH. Identification and sequence determination of a novel double-stranded RNA mycovirus from the entomopathogenic fungus Beauveria bassiana. Arch Virol. 2015;160 (3 ):873–875. doi: 10.1007/s00705-014-2332-8 25577168
[25] Kotta-Loizou I, Coutts RH. Studies on the virome of the entomopathogenic fungus Beauveria bassiana reveal novel dsRNA elements and mild hypervirulence. PloS Pathog. 2017;13 (1 ):e1006183. doi: 10.1371/journal.ppat.1006183 28114361
[26] Shi N, Yang G, Wang P, et al. Complete genome sequence of a novel partitivirus from the entomogenous fungus Beauveria bassiana in China. Arch Virol. 2019;164 (12 ):3141–3144. doi: 10.1007/s00705-019-04428-1 31598844
[27] Gilbert KB, Holcomb EE, Allscheid RL, et al. Hiding in plain sight: new virus genomes discovered via a systematic analysis of fungal public transcriptomes. PLoS One. 2019;14 (7 ):e0219207. doi: 10.1371/journal.pone.0219207 31339899
[28] Shi N, Hu F, Wang P, et al. Molecular characterization of two dsRNAs that could correspond to the genome of a new mycovirus that infects the entomopathogenic fungus Beauveria bassiana. Arch Virol. 2021;166 (11 ):3233–3237. doi: 10.1007/s00705-021-05239-z 34535823
[29] Li L, Kang Q, Zhang S, et al. The complete genome sequence of a novel chrysovirus from the entomopathogenic fungus Beauveria bassiana Vuillemin. Arch Virol. 2021;166 (12 ):3443–3447. doi: 10.1007/s00705-021-05215-7 34553285
[30] Kang Q, Li L, Li J, et al. A novel polymycovirus with defective RNA isolated from the entomopathogenic fungus Beauveria bassiana Vuillemin. Arch Virol. 2021;166 (12 ):3487–3492. doi: 10.1007/s00705-021-05238-0 34623502
[31] Najie S, Fei H, Ping W, et al. Molecular characterization of two dsRNAs that could correspond to the genome of a new mycovirus that infects the entomopathogenic fungus Beauveria bassiana. Arch Virol. 2021;166 (11 ):3233–3237. doi: 10.1007/s00705-021-05239-z 34535823
[32] Xu M, Liu H, Jia X, et al. The complete genome sequences of a negative single-stranded RNA virus and a double-stranded RNA virus coinfecting the entomopathogenic fungus Beauveria bassiana Vuillemin. Arch Virol. 2024;169 (3 ):42. doi: 10.1007/s00705-024-05985-w 38332318
[33] Filippou C, Diss RM, Daudu JO, et al. The polymycovirus-mediated growth enhancement of the entomopathogenic fungus Beauveria bassiana is dependent on carbon and nitrogen metabolism. Front Microbiol. 2021;12 :606366. doi: 10.3389/fmicb.2021.606366 33603722
[34] Zhang Z, Guo W, Lu Y, et al. Hypovirulence-associated mycovirus epidemics cause pathogenicity degeneration of Beauveria bassiana in the field. Virol J. 2023;20 (1 ):255. doi: 10.1186/s12985-023-02217-6 37924080
[35] Kang Q, Ning S, Sui L, et al. Transcriptomic analysis of entomopathogenic fungus beauveria bassiana infected by a hypervirulent polymycovirus BbPmV-4. Fungal Biol. 2023;127 (3 ):958–967. doi: 10.1016/j.funbio.2023.02.003 36906386
[36] Sui L, Lu Y, Zhu H, et al. Endophytic blastospores of Beauveria bassiana provide high resistance against plant disease caused by botrytis cinerea. Fungal Biol. 2022;126 (8 ):528–533. doi: 10.1016/j.funbio.2022.05.007 35851145
[37] Ning SY, Kang Q, Liu HY, et al. Interspecific spread of dsRNA mycoviruses in entomogenous fungi Beauveria spp. Virus Res. 2022;322 :198933. doi: 10.1016/j.virusres.2022.198933 36165923
[38] Ding JL, Lin HY, Feng MG, et al. Mbp1, a component of the MluI cell cycle box-binding complex, contributes to morphological transition and virulence in the filamentous entomopathogenic fungus beauveria bassiana. Environ Microbiol. 2020;22 (2 ):584–597. doi: 10.1111/1462-2920.14868 31743555
[39] Zhang Z, Tian Y, Sui L, et al. First record of Aspergillus nomiae as a broad-spectrum entomopathogenic fungus that provides resistance against phytopathogens and insect pests by colonization of plants. Front Microbiol. 2023;14 :1284276. doi: 10.3389/fmicb.2023.1284276 38260878
[40] Nzabanita C, Zhang L, Zhao H, et al. Fungal endophyte Epicoccum nigrum 38L1 inhibits in vitro and in vivo the pathogenic fungus Fusarium graminearum. Biol Control. 2022;174 :105010. doi: 10.1016/j.biocontrol.2022.105010
[41] Sui L, Zhu H, Xu W, et al. Elevated air temperature shifts the interactions between plants and endophytic fungal entomopathogens in an agroecosystem. Fungal Ecol. 2020;47 :100940. doi: 10.1016/j.funeco.2020.100940
[42] Zhang Z, Sui L, Tian Y, et al. Metarhizium rileyi with broad-spectrum insecticidal ability confers resistance against phytopathogens and insect pests as a phytoendophyte. Pest Manag Sci. 2024;80 (7 ):3246–3257. doi: 10.1002/ps.8027 38358040
[43] Sui L, Lu Y, Zhou L, et al. Endophytic Beauveria bassiana promotes plant biomass growth and suppresses pathogen damage by directional recruitment. Front Microbiol. 2023;14 :1227269. doi: 10.3389/fmicb.2023.1227269 37664126
[44] Ben Abdeljalil NO, Vallance J. Bio-suppression of sclerotinia stem rot of tomato and biostimulation of plant growth using tomato-associated rhizobacteria. J Plant Pathol Microbiol. 2016;7 (02 ):11. doi: 10.4172/2157-7471.1000331
[45] Luo X, Keyhani NO, Yu X, et al. The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus beauveria bassiana. Fungal Genet Biol. 2012;49 (7 ):544–555. doi: 10.1016/j.fgb.2012.05.002 22587950
[46] Jin D, Sun B, Zhao W, et al. Thiamine-biosynthesis genes Bbpyr and Bbthi are required for conidial production and cell wall integrity of the entomopathogenic fungus Beauveria bassiana. J Invertebr Pathol. 2021;184 :107639. doi: 10.1016/j.jip.2021.107639 34139258
[47] Hou J, Ding JL, Peng YJ, et al. Genome-wide identification of BCS1 domain-containing proteins reveals the mitochondrial bcs1 essential for growth, stress response, and virulence of the filamentous entomopathogenic fungus Beauveria bassiana. Microbiol Res. 2023;267 :127262. doi: 10.1016/j.micres.2022.127262 36450212
[48] Barnett HL. The nature of mycoparasitism by fungi. Annu Rev Microbiol. 1963;17 (1 ):1–44. doi: 10.1146/annurev.mi.17.100163.000245
[49] Jeffries P. Biology and ecology of mycoparasitism. Can J Bot. 1995;73 (S1 ):1284–1290. doi: 10.1139/b95-389
[50] Xiao G, Ying SH, Zheng P, et al. Genomic perspectives on the evolution of fungal entomopathogenicity in Beauveria bassiana. Sci Rep-UK. 2012;2 (1 ):483–493. doi: 10.1038/srep00483
[51] Pieterse CM, Zamioudis C, Berendsen RL, et al. Induced systemic resistance by beneficial microbes. Annu Rev Phytopathol. 2014;52 (1 ):347–375. doi: 10.1146/annurev-phyto-082712-102340 24906124
[52] Ejmal MA, Holland DJ, Macdiarmid RM, et al. The effect of Aspergillus Thermomutatus Chrysovirus 1 on the biology of three Aspergillus species. Viruses. 2018;10 (10 ):539. doi: 10.3390/v10100539 30279352
[53] Urayama S, Kato S, Suzuki Y, et al. Mycoviruses related to chrysovirus affect vegetative growth in the rice blast fungus Magnaporthe oryzae. J Gen Virol. 2010;91 (Pt 12 ):3085–3094. doi: 10.1099/vir.0.025411-0 20797967
[54] Arjona-López JM, Telengech P, Suzuki N, et al. A moderate level of hypovirulence conferred by a hypovirus in the avocado white root rot fungus, rosellinia necatrix. Fungal Biol. 2021;125 (1 ):69–76. doi: 10.1016/j.funbio.2020.10.007 33317778
[55] Wang P, Yang G, Shi N, et al. A novel partitivirus orchestrates conidiation, stress response, pathogenicity, and secondary metabolism of the entomopathogenic fungus metarhizium majus. PloS Pathog. 2023;19 (5 ):e1011397. doi: 10.1371/journal.ppat.1011397 37216409
[56] Chun J, Yang HE, Kim DH. Identification of a novel partitivirus of Trichoderma harzianum NFCF319 and evidence for the related antifungal activity. Front Plant Sci. 2018;9 :1699. doi: 10.3389/fpls.2018.01699 30515186
[57] You J, Hu Z, Li C, et al. The effect of trichoderma harzianum hypovirus 1 (ThHV1) and its defective RNA ThHV1-S on the antifungal activity and metabolome of Trichoderma koningiopsis T-51. J Fungi (Basel). 2023;9 (2 ):175. doi: 10.3390/jof9020175 36836290
[58] Zhu H, Fu J, Wang H, et al. Fitness consequences of oviposition choice by an herbivorous insect on a host plant colonized by an endophytic entomopathogenic fungus. J Pest Sci. 2023;96 (2 ):745–758. doi: 10.1007/s10340-022-01527-y
[59] Chang Y, Xia X, Sui L, et al. Endophytic colonization of entomopathogenic fungi increases plant disease resistance by changing the endophytic bacterial community. J Basic Microbiol. 2021;61 (12 ):1098–1112. doi: 10.1002/jobm.202100494 34738230
[60] Yun HG, Kim DJ, Gwak WS, et al. Entomopathogenic fungi as dual control agents against both the pest Myzus persicae and phytopathogen Botrytis cinerea. Mycobiology. 2017;45 (3 ):192–198. doi: 10.5941/MYCO.2017.45.3.192 29138624
[61] Moraga EQ. Entomopathogenic fungi as endophytes: their broader contribution to IPM and crop production. Bio Sci Technol. 2020;30 (9 ):864–877. doi: 10.1080/09583157.2020.1771279
[62] Jaber LR, Ownley BH. Can we use entomopathogenic fungi as endophytes for dual biological control of insect pests and plant pathogens? Biol Control. 2018;116 :36–45. doi: 10.1016/j.biocontrol.2017.01.018
