Effects of Isaria fumosorosea on TYLCV (Tomato Yellow Leaf Curl Virus) Accumulation and Transmitting Capacity of Bemisia tabaci

Tomato yellow leaf curl virus (TYLCV) is transmitted by the Bemisia tabaci pest Middle East-Asia Minor 1 (MEAM1) in China. Isaria fumosorosea is a fungal pathogen of B. tabaci. However, the effects of fungal infection on TYLCV expression and transmission by MEAM1 are unclear. In this study, potted tomatoes containing second instar nymphs of MEAM1 were treated with I. fumosorosea IfB01 strain and the relationship between fungal infection in MEAM1 and its TYLCV transmission capacity was investigated. The results indicated that a significantly (p < 0.05) decreased incidence of transmission of TYLCV-infected plants (ITYPs) transmitted by second instar nymphs of MEAM1 infected with fungus. Further, we found a negative correlation between fungal conidial concentrations and eclosion rates of MEAM1, and a positive correlation between ITYPs and eclosion. In addition, when each plant was exposed to three adults treated with fungus, a significantly decreased transmission of TYLCV (TYTE) was observed in the infected group. However, the incidence of TYLCV-carrying MEAM1 adults (ITYAs) was not significantly different in the infected and control groups (p < 0.05). Nevertheless, a significant decrease in viral accumulation using TYLCV AC2 gene as a marker was observed in the fungus-infected MEAM1. In conclusion, the results suggested that I. fumosorosea infection decreases TYLCV accumulation in MEAM1 and subsequently reduces its transmission. Our study provides new insights into the relationship between host plant, plant virus, insect vector, and entomopathogenic fungus.


Introduction
Bemisia tabaci, is an important insect species comprising harmful pests: Middle East-Asia Minor 1 (MEAM1, i.e., B-biotype) and Mediterranean species (MED, i.e., Q-biotype) [1,2]. B. tabici transmits the tomato yellow leaf curl virus (TYLCV), which is a begomovirus infecting plant species, resulting in agricultural damage [1,2]. In China, TYLCV was identified in the 1980's. However, its prevalence increased only after 2006, resulting in serious decrease in rpm for 3 min and the supernatant was discarded. The broth containing pellets was transferred to an injector for TYLCV inoculation. When the tomato potted-plants grew to the 3-to 4-leaf stage, a 0.2 mL of TYLCV inoculum was injected at the stem base of each tomato plant. The plants were further cultured to a 6-7-leaf stage. The plants showing apparent symptoms were selected and subjected to PCR testing to validate the TYLCV infection. The TYLCV-infected plants (TY-plants) were maintained in 60×60×60 cm cages at 25±1°C and exposed to 14h dark/10h light cycle.
The basic and interior leaves of the TY-plants were initially covered with a plastic pocket, and two leaves were retained for MEAM1 infestation. Forty MEAM1 adults were introduced from B. alboglabra plants into each TY-plant. After laying eggs for 1 d, the adults were driven away. When the insects developed to 2 nd instar nymphs, their numbers were counted to ensure adequate number in each TY-plant (others were removed).

Fungal treatment experiment 1
The I. fumosorosea IfB01 strain (China Center for Type Culture Collection access number: CCTCC M 2012400) was used. The slant was inoculated on a PDA plate and cultured at 26°C for 2 to 3 weeks. The conidia were collected and suspended with 0.02% Tween 80 into a stock solution containing 10 8 spores/mL. The different working dilutions were prepared from the stock using 0.02% Tween 80 solution.
The fungal treatments were conducted using three different concentrations of IfB01 conidia: 100.0 × 10 6 , 10.0 × 10 6 , and 1.0 × 10 6 spores/mL. The leaf immersion method (China standard NY/T 1154.14-2008) was used for the bioassay. Before treatment, the potted plants were examined to ensure the presence of adequate number of second instar nymphs of MEAM1. During the treatment, the leaf with MEAM1 nymphs was dipped into each conidial working suspension for 30 s. The control treatment was 0.02% Tween-80. After drying, the potted plants were transferred to cages. Three cages contained three types of plants: 3 TY-plants and 3 normal plants (3/3 cage), 2 TY-plants and 6 normal plants (2/6 cage), 1 TY-plants and 5 normal plants (1/5 cage) (Fig 1). A total of 300 nymphs were allotted to each cage, i.e., 100 nymphs per TYplant in the 3/3 cage, 150 nymphs for each TY-plant in the 2/6 cage and 300 nymphs targeting each TY-plant in the 1/5 cage (Fig 1). The culture conditions were same as above. All the treatments were performed in triplicate, and the same experiment was repeated twice in 2011-2013 and 2014-2015. The treatments were inspected for insect growth. The nymphs, pupae and adults were counted daily. The eclosion rate was evaluated using the equation: eclosion rate (%) = 100 × puparium number / total nymphs number The incidence of TY-plants (ITYP) was surveyed on post-eclosion days 3 and 8 after all the pupae molted. The TYLCV in each plant was detected as described previously [26]. In brief, leaves weighing up to 150 mg were cut from the top row of normal plants using a clean scissors, and homogenized. The genomic DNA was extracted with DP3111 kit (BioTeke Corporation, Beijing, China) according to the manufacturer's protocol. The primers TYLCV-F (5'-ACGCATGCCTCTAATCCAGTGTA-3') and TYLCV-R The results were analyzed using analysis of variance (ANOVA), Duncan's multiple range test (DMRT) and regression with SPSS V17.0 statistical software (IBM, USA).

Fungal treatment experiment 2
The second instar nymphs of MEAM1 generated on TY-plants were treated with an IfB01 conidial suspension of 10×10 6 spores/mL as described above. After eclosion, the MEAM1 adults were transferred to normal potted plants. Three adults were introduced to each potted plant to transmit TYLCV for three days. All the adults were collected separately and stored at -20°C for TYLCV tests. Each treatment included 10 potted plants and the experiment was performed in triplicate (30 plants). The ITYPs were investigated similarly after treatment on days 3 and 8 post-treatment.
To evaluate the incidence of TYLCV-carrying MEAM1 adults (TY-adults), the TYLCV in each adult was detected [27]. Initially, the total DNA was extracted from each adult with an OSR-M401 TGuide Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) according the manufacturer's protocol. The TYLCVSF543 fragment was amplified with PCR as described above. Thirty adults were tested. The MEAM1 adults testing positive with PCR served as TYadults. The incidence of TY-adults (ITYA) was evaluated according to the following equation: ITYA (%) = 100 × TY-adults number / 30, where "30" denotes the total number of MEAM1 adults tested.

Effect of I. fumosorosea on MEAM1 eclosion
The eclosion rates of MEAM1 were decreased with the fungal conidial dose after the 2 nd instar nymphs were treated by I. fumosorosea IfB01 strain (Table 1). At the fungal conidia concentrations of 100.0, 10.0 and 1.0 (×10 6 spores/mL), eclosion rates of 11.11%, 25.89% and 43.67% were recorded, respectively. A significantly higher value of 84.89% was observed in the CK (p < 0.05) ( Table 1). The ANOVA results indicated significantly different eclosion rates (p < 0.01) of MEAM1 following exposure to varying concentrations of fungal conidia. The differences were insignificant (p > 0.05) when TY-plants were compared with normal plants, and conidia concentrations in TY-plants were compared with normal plants ( Table 2). The results suggested that the conidial concentration of IfB01 strain significantly affected the eclosion of MEAM1. However, the comparison of TY-plants with normal plants and their conidial concentrations was not feasible. Regression analysis revealed a negative relationship between eclosion rate and the logarithm of fungal conidial concentration (Fig 2A).

Effect of I. fumosorosea on TY-plants
Based on the results of fungal treatment experiment 1, the incidence of TY-plants (ITYP) on MEAM1 post-eclosion days 3 and 8 were significantly decreased (p < 0.05) depending on the fungal conidial dose after the 2 nd instar nymphs were treated with I. fumosorosea IfB01 strain (Table 3). ANOVA indicated significantly different (p < 0.01) ITYPs with various concentrations of fungal conidia and the TY-plants /normal plants. However, the differences in exposure to the conidial concentrations of TY-and normal plants were insignificant (Table 4). These results suggested that the conidial concentration of IfB01 strain and the TY-plants/normal plants showed a significant effect on ITYPs. However, the interaction of conidial concentrations with TY-plants /normal plants was not observed. Further regression analysis showed a negative relationship between eclosion rate and the logarithm of fungal conidial concentration (Fig 2A).
In the fungal treatment (experiment) 2, in which each normal plant was exposed to 3 adults, the fungus infected MEAM1 showed lower ITYPs (Fig 3). The T-test indicated that on posttreatment day 3, the ITYP in the treatment group was 16.67%, which was significantly (P < 0.05) lower than the ITYP of the control group (30%). However, ITYPs after the treatment (40.00%) and control (46.67%) on post-treatment day 8 were not significant. Obviously, the increased ITYP values on post-treatment day 8 were not attributed to transmission because all the MEAM1 adults were removed on post-treatment day 3. Probably, TYLCV levels were inadequate in plants on the post-treatment day 3. However, after 5 days of proliferation, the TYLCV was substantial and detectable.

Effects of I. fumosorosea on TYLCV transmission by each MEAM1 adult (TYTE)
The TYTEs were evaluated according to the fungal treatment experiment 1 (Fig 4). The TYTE values were closely affected by the fungal concentrations, the transmission times (days of post-  For example, treatment of T/N2/6-C100 with 6 adults per plant yielded a 0.13% TYTE, which was remarkably larger than 0.05% TYTE detected with T/N2/6-CK involving 44 adults per plant on post-treatment day 3 (Fig 4). Nevertheless, the significantly lower ITYP of 27.78% related to 75.00% control was recorded in the same treatment ( Table 3), suggesting that the TYTEs in fungal treatment experiment 1 does not reflect the true viral transmission capacity of insects.
In an additional experiment, in which every normal plant was only introduced to three adults, the TYTE was significantly reduced with the fungal treatment compared with the control on post-treatment day 3 ( Fig 3B). Obviously, this result was contrary to the data obtained from the first experiment. Therefore, the actual TYTEs were masked by the large numbers of adults on each plant in experiment 1. The second experiment carrying relatively fewer numbers (3 adults/plant) offered more realistic TYTE values.

Effects of I. fumosorosea on the incidence of TY-adults (ITYA)
The TYLCV AC2 genes of MEAM1 adults on post-eclosion day 3 were amplified using PCR in the fungal experiment 2. The results indicated that TYLCVs were found in 17/30 adults in the treatment group and 18/30 adults in the control group (Fig 5), which indicated that the ITYAs in the treatment and control groups were not statistically different.

Effects of I. fumosorosea on accumulation of TYLCV in individual MEAM1 adult
The cumulative TYLCV levels in individual MEAM1 adults were evaluated by qPCR amplification of AC2 gene in the TYLCV-carrying adults of the fungal treatment 2. The results indicated that the cumulative levels of TYLCV in each adult in the treatment group were apparently lower than in the control group (Fig 6) suggesting that the cumulative levels of TYLCV in TYadults were likely reduced after the MEAM1 nymphs were infected by I. fumosorosea.  * indicating significant differences between the two means by T-test. In this pot-cultured tomato in cage, the conidial suspension contained the IfB01 strain of I. fumosorosea. The MEAM1 2 nd instar nymphs in TY-plants were exposed to a conidial concentration of 10×10 6 spores/mL via leaf immersion. After eclosion, each of the normal tomato plants was infected with three adults. The control was treated with healthy MEAM1 adults. doi:10.1371/journal.pone.0164356.g003

Discussion
The incidence and epidemic of insect-transmitted pathogenic viral disease is influenced by complex factors associated with the host plant, virus, vector and environment. Recognition of the interaction between host, virus, insect vector, and entomopathogenic fungus is essential to enhance our knowledge of plant viral disease and insect pests. In this study, we first focused on the relationship between tomato, TYLCV, MEAM1, and I. fumosorosea. In order to determine the role of fungal infection in the insect in TYLCV transmission by MEAM1, we conducted fungal experiment 1 to survey the variation in the incidence of TY-plants at different dosages of fungal treatment in nymphs and the incidence rates in TY-plants / normal plants. The results indicated that the incidence of TY-plants (ITYP) was substantively decreased after the plant carrying nymphs was treated with I. fumosorosea, suggesting that I. fumosorosea infection decreased the eclosion of MEAM1, and reduced the ITYPs. We investigated the TYLCV transmission efficiencies of each adult (TYTEs) to evaluate the relationship between ITYPs and MEAM1 capacity of TYLCV transmission. However, we found that TYTEs declined with reduced concentrations of fungal treatment in the first experiment. Further analysis revealed that the excessive number of adults in each plant significantly reduced the TYTEs and was within the limits of every adult. Apparently, the actual TYTEs  were masked by the higher numbers of adults in each plant. Therefore, the TYTE failed to reflect the insect's true viral transmission capacity.
Therefore, the fungal experiment 2 introduced three MEAM1 adults on each plant. As expected, significantly decreased ITYPs and TYTEs were observed with fungal treatment (10×10 6 spores/mL) compared with the control on post-treatment day 3. Apparently, the different ITYPs were related to fungal infection but not to reduced eclosion. Further, the TYTE values in the second experiment were more realistic.
Furthermore, we inspected the TYLCV in each MEAM1 adult in the second experiment. The results indicated that ITYAs in the treatment and control groups were not statistically different. However, the apparently lower TYLCV accumulation of TY-adults in the treatment group than in the control group was validated by further qPCR tests on post-treatment day 3. Thus, it is reasonable that the lower ITYPs in fungal treatment relate to smaller TYLCV levels in the MEAM1 adults and therefore, smaller TYLCV titers in the tomato plants. As a result, the virus levels are undetectable. However, after 5 days (on post-treatment day 8), the virus proliferation is significant and substantial for PCR detection.
ITYPs are attributed to viral transmission by TY-adults. However, in this study, after MEAM1 nymphs are inoculated with I. fumosorosea, most of the nymphs are infected by fungus and finally die from fungal disease. However, the other nymphs uninfected by the fungus eventually develop into adults, which are the main reservoir of TY-adults. Additional TY-adult resource includes infected nymphs, which develop into adults. Therefore, we investigated the gene fragments of fungal rDNA-ITS in MEAM1 adults by PCR, and unfortunately, the fungus was not found (data unpublished). This result suggests that fungus was not found in surviving MEAM1 adults. However, the limited number of few fungal cells in the adults was undetectable through PCR.
The lower levels of TYLCV in MEAM1 adults might be related to specific substances produced by the fungi, which decrease feeding or interfere with TYLCV replication and transcription. In fact, entomopathogenic fungi usually produce a few compounds and specific proteins that inhibit other micro-organisms in the host [30,31,32]. For example, Metarhizium anisopliae secretes destruxins to suppress host immunity and viral activity [33]. Specific materials isolated from I. fumosorosea inhibited the virus, suggesting possible control of TYLCV by reducing the viral titers in MEAM1. Therefore, the management of insect-transmitted plant viruses is facilitated via control of viral levels in insect vectors. The reduction of TYLCV in MEAM1 adults is probably related to host plants and endophytic fungal entomopathogens. Several reports of endophytic fungal entomopathogens were related to Beauveria bassiana, Metarhizium anisopliae, Purpureocillium lilacinum (Formerly Paecilomyces lilacinus) and Lecanicillium lecanii, which affect insect growth, development and reproduction [34,35,36,37]. If Isaria fumosorosea was endophytic after inoculation on tomato plants, additional investigation is desirable.
In conclusion, ITYPs and TYTEs were reduced by MEAM1 nymphs treated with I. fumosorosea IfB01 strain. Fungal treatment of MEAM1 nymphs decreased the eclosion rates and the cumulative levels of TYLCV in MEAM1 TY-adults. Consequently, infection of nymphs with I. fumosorosea IfB01 strain eventually reduces the adult numbers and decreases the TYLCV transmission capacity of TY-adults. Our study provides new insight into interactions between host plant, phytopathogenic viruses, insect vectors and entomopathogenic fungi.

S1 Data. The original data of experiments (supplementary materials).
(XLS)