A Sensitive Method for Detecting Beauveria bassiana, an Insecticidal Biocontrol Agent, Population Dynamics, and Stability in Different Substrates

Beauveria bassiana is a well-known insecticidal biocontrol agent. Despite its broad field applications, its survival, colonization, and stability under field conditions remained unclear, mainly due to the lack of a quick and reliable detection method. In this study, we developed a quantitative real-time PCR technology to monitor the stability and population dynamics of B. bassiana in different substrates (water, soil, and on the cotton leaves surface), different spores of B. bassiana applied on Chinese cabbage leaves surface, and the lethality of Pieris rapae spraying with different spores of B. bassiana. Our results showed a decreased concentration of B. bassiana DNA in all three substrates from the 1st day till 9th day of post inoculation (dpi) period, possibly due to the death of B. bassiana. After this decrease, a quick and significant rebound of B. bassiana DNA concentration was observed, starting from the 11th dpi in all three substrates. The B. bassiana DNA concentration reached the plateau at about 13th dpi in water and 17th dpi in the soil. On cotton leaves surface, the B. bassiana DNA concentration reached the highest level at the 17th dpi followed by a small decline and then stabilized. This increase of DNA concentration suggested recovery of B. bassiana growth in all three substrates. We found that the most suitable killing effectiveness of P. rapae was the 1.0 × 107 spores/mL of B. bassiana. In summary, we have established a detection technology that allows a fast and reliable monitoring for the concentration and stability of B. bassiana under different conditions. This technology can benefit and help us in the development of proper management strategies for the application of this biocontrol agent in the field.


Introduction
Beauveria bassiana is an entomopathogenic fungus that can infect more than 700 insect species belonging to 149 diferent families in 15 diferent orders by causing white muscardine disease in them [1][2][3].To date, entomopathogenic fungi have been used as biological control agents for the management of various insect and mite pests [4].B. bassiana is the most well studied entomopathogenic fungi and has been frequently used as a commercial mycoinsecticide in the feld [5,6].For example, B. bassiana was used to control insect pests, including Pissodes castaneus, Ostrinia nubilalis, and Rhynchophorus ferrugineus, infeld through direct spraying [7][8][9].It is known that B. bassiana is safe to human, natural benefcial insects, and benefcial microorganisms.During feld applications, it can also be used together with other commercial chemical pesticides [10][11][12].More importantly, there is no evidence that insect pest(s) has evolved resistance against B. bassiana.Currently, B. bassiana is widely used to control insect pests in the order of Lepidoptera and Coleoptera, including many forest pests [13,14].
It was reported that B. bassiana could secrete many biologically active compounds like lactide and poisonous proteases during its infection in insect hosts [15,16].
B. bassiana is also known to hijack nutrients and water from the infected insects to accelerate their death process [17].B. bassiana spores can be formulated to produce wettable powder for commercial use in agriculture as well as in forests [18][19][20][21].A separate study conducted recently showed that the survival and successful recolonization of the B. bassiana in feld depended largely on environmental factors [22].We reasoned that the understanding of the survival and growth dynamics of this fungus in nature is necessary for the integration of this biocontrol agent into a more efective and safe plant protection strategy and to reduces public concerns on environment conservation [23,24].
Peng and coworkers investigated the growth and survival of Metarhizium anisopliae in oriental migratory locust [25].In this study, we used a similar strategy to establish an accurate quantitative real-time PCR technology to determine the stability of B. bassiana in various substrates (i.e., water, soil, and cotton leaves surface) at 28 °C.With this new technology, we can now monitor B. bassiana population dynamics and regrowth in feld samples and develop more efective management strategies using this and other biocontrol agents.

DNA Extraction from B. bassiana.
DNA was extracted from B. bassiana containing water, soil, and plant leaf samples using a DNA extraction kit (Tiangen Biochemical Technology, Beijing, China).Concentration and quality of each DNA sample were measured by using a spectrophotometer (Termo Fisher Scientifc, Massachusetts, USA).For each DNA sample, three technical replicates were analyzed during real-time fuorescent PCR with a SYBR Green I reagent (TransGen Biotech, Beijing, China) as instructed by the manufacturer (TransGen Biotech).

Synthesis of B. bassiana Specifc Primers.
Te initial PCR amplifcation was carried out using a set of bacterial universal primers (Baker.2003) and the DNA isolated from B. bassiana.Te resulting PCR products were cloned and sequenced by the TSINGKN Biotech (TSINGKN Biotech, Beijing, China).Conserved DNA sequences obtained from the PCR products were analyzed using the DNAMAN software, and six pairs of real-time PCR primers were designed based on the obtained sequences using the Primer 5.0 software in the DNAMAN (LynnonBiosoft, USA).In the subsequent real-time PCR assays, diluted B. bassiana genomic DNA or double distilled water (ddH 2 O) were used as the positive and negative control sample, respectively.Te six designed primer sets were frst tested by gradient PCR with three diferent annealing temperatures (52, 54 and 56 °C) and later by PCR amplifcations.Te resulting PCR products were visualized in agarose gels through electrophoresis.Te primer set giving a single specifc PCR product was considered to be the optimal B. bassianareal-time PCR primer set and was used in further experiments.

Preparation of Real-Time PCR Standard Curve.
According to a previously described method [26], we frst generated a recombinant plasmid DNA carrying a fragment of B. bassiana genomic DNA and then measured its concentration using a spectrophotometer.Te copy number of the recombinant plasmid DNA was calculated as previously reported [27] and then further diluted prior to the real-time fuorescent PCR on a CFX96 ™ Real-Time System (Bio-Rad, California, USA), and the standard curve was established using the concentration logarithm and the Ct values.

Determination of Detection Limit Using Purifed
B. bassiana DNA.B. bassiana was homogenized, dried, and diluted 1 : 99 (w/v) in sterilized water.Te diluted sample was incubated at 28 °C inside an incubator and sampled once every two days (one mL sample per sampling time) till the 29 th day of postincubation (dpi) period.Te collected samples were centrifuged at 12,000 rpm/min for 1 minute and the supernatants were discarded prior to the extraction of DNA from the pellets.For real-time PCR reactions, three technical replicates were analyzed and used to represent a specifc DNA sample.

Detection of B. bassiana DNA in Soil
Samples.One gram dried B. bassiana powder was mixed with 99 grams sterilized black soil, incubated at 28 °C in an incubator and then sampled once every two days (1 g soil sample per sampling time) as described above.Te collected soil samples (0.1 g each) were used for DNA extraction as described above.Real-time PCR was then performed using three technical replicates per sample.

Detection of B. bassiana DNA in Cotton (Gossypium spp.)
Leaves Samples.One gram dried B. bassiana powder was diluted in 99 grams sterilized water and then spread onto the surface of cotton leaves.After air drying, three to four cotton leaf pieces were randomly collected once every two days.Te sampled cotton leaf tissues were grounded in liquid nitrogen and 0.1 g powder from each leaf sample was used for DNA extraction.For real-time PCR, three technical replicates were used to represent a specifc sample.

Detection of Diferent Number Spores of B. bassiana DNA on Chinese Cabbage (Brassica rapa Pekinensis) Leaves.
Te B. bassiana with 1.0 × 10 2 (A), 1.0 × 10 4 (B), 1.0 × 10 6 (C), 1.0 × 10 7 (D), and 1.0 × 10 8 (E) spores/mL were sprayed evenly on Chinese cabbage leaves at the same growth level at In this study, all the experiments were repeated twice to ensure the repeatability of the results.

Data Analysis
Data obtained from three independent experiments were combined and analyzed by using one-way ANOVA program in Excel (Microsoft ® Ofce Excel 2003, USA) and the SPSS 13.0 statistical software package (SPSS, Inc., Chicago, IL, USA).Post hoc multiple comparisons were conducted at the 5% level of probability using Duncan's multiple range test (DMRT).

Determination of Optimal PCR Primers and Standard
Curve of Real-Time PCR.Gradient PCR was frst used to determine the optimal annealing temperature for individual primer sets listed in supplementary Table 1.Te recombinant plasmid DNA was diluted based on the copy number of plasmid DNA estimated using the following equation: copy numbers/μL � (6.0 × 10 14 copies × plasmid concentration (g)/ μL)/(number of bases × 660 Dalton/base).Te efciency of each primer set was determined through PCR reactions and the resulting PCR products were visualized in agarose gels through electrophoresis.Results of the assay showed that, when the annealing temperature was set at 54 °C, four of the six primer sets gave strong and correct sized PCR product bands.At 52 °C, three primer sets gave positive PCR products of diferent size, and at 56 °C, only two primer sets gave positive PCR products (Supplementary Figure 1).Consequently, the 4 th primer set (e.g., 98F 5′GGCATCGATGAA GAACGCAG3′ and 333R 5′GTATTACTGCGCAGAGGT CG3′) was used for the following real-time PCR assays.Trough PCR amplifcation assays, a standard curve of PCR was determined as Y � −3.26X + 11.25, R 2 � 0.99965, and the PCR amplifcation efciency � 1.03 (Supplementary Figure 2).Using serially diluted plasmid DNA samples, it was observed that by the 10-fold dilution of DNA samples; the Ct values increased by 3-4.Te concentration of standard plasmid DNA was found to be closely correlated with the Ct values, leading to single melting curves (Supplementary Figure 3).

Stability of B.
bassiana in Water at 28 °C.Dried B. bassiana powder was diluted in water and incubated at 28 °C for several days followed by DNA isolation.Stability of B. bassiana DNA in water was determined as the concentrations of B. bassiana DNA in water over time by real-time PCR.Te result of the assay showed that the amount of B. bassiana DNA in assayed samples stored for 1 and 3 days was similar (5.646 × 10 6 and 5.622 × 10 6 copies of B. bassiana DNA/μL, respectively) (Figure 1).Te amount of B. bassiana DNA decreased quickly to 3.762 × 10 6 copies of B. bassiana DNA/μL on the 7 th day of postinoculation period followed by an increase up to 6.619 × 10 6 copies of B. bassiana DNA/ μL on the 13 th day of postinoculation period.B. bassiana DNA decreased again at the 17 th dpi and maintained at a similar level till 29 th dpi.In this study, no B. bassiana DNA was detected in the negative control sample.Chinese cabbage leaves surface showed roughly the same trend, which showed a decreasing trend from the 1 st dpi to the 9 th dpi (except (A), (B), and (E)), all concentrations showed an increasing trend from the 11 th dpi to the 19 th dpi and then remained stabilized at 21 th dpi to the 29 th dpi.Tis experiment was repeated twice.B. bassiana DNA was not detected in the negative control samples.3.0×10 6   4.0×10 6   5.0×10 6   6.0×10 6   7.0×10 6  Canadian Journal of Infectious Diseases and Medical Microbiology (D) spores/mL reached 50% at the 13 th dpi and reached 90% at the 21 st dpi; the concentration of 1.0 × 10 8 (E) spores/mL reached 50% at the 14 th dpi, and reached 90% at the 19 st dpi.Tis experiment was repeated twice.B. bassiana DNA was not detected in the negative control samples.

Conclusion and Discussion
In Diferent number spores of B. bassiana applied on Chinese cabbage result shown that the stability of B. bassiana DNA are consistent when plants treated with 1.0 × 10 6 , 1.0 × 10 7 , and 1.0 × 10 8 spores/mL.But the mortality rate of P. rapae treated with diferent spores at 21 st , respectively, reached 15%, 50%, 65%, 90%, and 95%, the concentration of 1.0 × 10 6 spores/mL reached 65% at the 19 th dpi; the concentration of 1.0 × 10 7 spores/mL reached 50% at the 13 th dpi and reached 90% at the 21 st dpi; the concentration of 1.0 × 10 8 spores/mL reached 50% at the 14 th dpi, and reached 90% at the 19 st dpi.With the increase in the initial spores of B. bassiana, the mortality of P. rapae gradually increased.At low spores (1.0 × 10 2 spores/mL and 1.0 × 10 4 spores/mL), the death rate was approximately 15-50%.At high spores (1.0 × 10 6 , 1.0 × 10 7 , and 1.0 × 10 8 spores/mL), the death rate was 65-95%.Te results indicated the efect of killing insects was obviously increased with increasing the concentration of B. bassiana, whereas considering the cost-efectiveness, the 1.0 × 10 7 spores/mL of B. bassiana was the most suitable.
Since the invention of real-time PCR, this technology has been widely applied to molecular biology studies [28].Currently, PCR, and real-time PCR are the most popular technologies for plant pathogen diagnosis [29,30] and host gene expression analysis.Te potential of this technology for the assays on the stability of the inoculated biocontrol agents remained largely unknown.Te main reason for this is mainly due to the lack of specifc PCR primers.Current studies on B. bassiana feld applications focused mainly on the development of antibiotic-resistant strains [31][32][33].Te development of antibiotic-resistant B. is, however, time-consuming and the resulting resistance strain(s) might become attenuated after a few generations.In contrast, understanding the environmental impacts on B. bassiana stability and recolonization behavior in the feld can facilitate the design and establishment of more efective insect pest management strategies and thus achieving a better control efect.Tis PCR detection technology may also be modifed for other biocontrol agents.In summary, this study has established a fast, low-cost, and reliable method for the determination of B. bassiana stability and recolonization in various substrates under the controlled conditions.experiments.WZ and QP provided some reagents/materials.ZG, LC, XL, and LY wrote the paper.
Soil at 28 °C.Stability of B. bassiana DNA in soil was also determined by real-time PCR.Results shown in Figure 2 demonstrated that B. bassiana DNA concentration declined quickly from the 1 st dpi (5.253 × 10 6 copies of B. bassiana DNA/μL) to the 9 th dpi (4.530 × 10 5 copies of B. bassiana DNA/μL).As shown in Figure 2, B. bassiana DNA concentration rebound at the 11 th dpi and continued to increase till 17 th dpi (4.776 × 10 6 copies of B. bassiana DNA/μL).After this increase, B. bassiana DNA concentration remained relatively stable till 29 th dpi, suggesting a reestablishment of B. bassiana population in soil samples.Tis experiment was repeated twice.4.4.Stability of B. bassiana DNA on Cotton Leaves at 28 °C.Dried B. bassiana powder was diluted in water and sprayed onto the surface of cotton leaves.After incubation at 28 °C for several days, the leaf samples were analyzed for B. bassiana DNA concentrations by real-time PCR.Te results shown in Figure 3 indicated that the concentration of B. bassiana DNA was at 3.202 × 10 5 copies of B. bassiana DNA/μL at the 1 st dpi and then declined to 2.777 × 10 4 copies of B. bassiana DNA/μL at the 9 th dpi.Te concentration of B. bassiana DNA rebound at the 11 th dpi and reached 2.975 × 10 5 copies of B. bassiana DNA/μL at the 17 th dpi.Te B. bassiana DNA concentration decreased slightly again and reached 2.126 × 10 5 copies of B. bassiana DNA/μL at the 21 st dpi, and then remained stabilized.Tis experiment was repeated twice.B. bassiana DNA was not detected in the negative control samples.4.5.Stability of Diferent Number Spores of B. bassiana DNA on Chinese Cabbage Leaves at 28 °C.To investigate the dynamics of diferent amounts of B. bassiana spores over time, we selected diferent concentrations of spores to spray on Canadian Journal of Infectious Diseases and Medical Microbiology Chinese cabbage leaves and then detected the stability of B. bassiana DNA.Results shown in Figure 4 indicated that the concentration of B. bassiana DNA was at 7.976 × 10 7 (A), 1.100 × 10 8 (B), 1.308 × 10 8 (C), 1.391 × 10 8 (D), and 1.460 × 10 8 (E) copies of B. bassiana DNA/μL at the 1 st dpi and then declined to 1.724 × 10 7 (A), 5.261 × 10 7 (B), 1.021 × 10 8 (E) copies of B. bassiana DNA/μL at the 9 th dpi and 8.292 × 10 7 (C), 9.076 × 10 7 (D) copies of B. bassiana DNA/μL at the 7 th dpi.Te concentration of B. bassiana DNA rebound at the 11 th dpi and reached 5.381 × 10 7 (A), 1.156 × 10 8 (B), 1.474 × 10 8 (C), 1.553 × 10 8 (D), and 1.615 × 10 8 (E) copies of B. bassiana DNA/μL at the 21 st dpi and then remained stabilized.Although the concentration of B. bassiana was diferent, the stability of B. bassiana DNA on

Figure 2 :
Figure 2: Stability of B. bassiana DNA in soil at 28 °C.Te stability of B. bassiana DNA was determined by qRT-PCR.Tree technical replicates were used for each sample, and the experiment was repeated twice.Statistical signifcances were determined by DMRT, p ≤ 0.05.

Figure 3 :Figure 4 :
Figure 3: Stability of B. bassiana DNA on cotton leaves surface at 28 °C as determined by qRT-PCR.Tree technical replicates were used for each sample, and the experiment was repeated twice.Statistical signifcances were determined by DMRT, p ≤ 0.05.

Figure 1 :
Figure 1: Stability of B. bassiana DNA in water and at 28 °C.Tree technical replicates were used for each sample and the experiment was repeated twice times.Statistical signifcances were determined by DMRT, p ≤ 0.05.

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Canadian Journal of Infectious Diseases and Medical Microbiology 28 °C and 60% humidity.Te bufer solution used for the dilution of the spores of B. bassiana was set as a blank control.Te leaf samples were analyzed for B. bassiana DNA concentrations by real-time PCR after 24 hours of spraying, three to four pieces of cabbage leaf were randomly collected once every two days.For real-time PCR, three technical replicates were used to represent a specifc sample.× 10 8 (E) spores/mL were used to spray uniformly on the cabbage leaves.After spraying 12 hours, cabbage leaves were fed to 40 heads of P. rapae selected at normal active fourth stage larvae, the P. rapae larvae were cultured in a net cage at 28 °C and 90% humidity.Te bufer solution used for the dilution of the spores of B. bassiana was set as a blank control.After feeding for 24 hours, one P. rapae were randomly collected to check for the B. bassiana DNA concentrations by real-time PCR.Te selected P. rapae was rinsed with water to avoid the presence of B. bassiana outside the body, which will afect the experimental results.For real-time PCR, three technical replicates were used to represent a specifc sample.
this study, we identifed a pair of PCR primers specifc for the B. bassiana 16s rDNA and established a sensitive and reliable PCR and a real-time PCR method for the detection of B. bassiana in various substrates.Te sensitivity of the detection technology was 4.337 × 10 4 copies of plasmid/μL.Using this method, we can now reliably monitor B. bassiana population dynamics in B. bassiana preinoculated soil or cotton leaves samples.Our results indicated that during the 1 st to the 9 th dpi, B. bassiana DNA concentration declined in all substrates.According to our understanding, this decline was caused by the massive death of initially inoculated B. bassiana.Te DNA concentration started to increase sharply after the 9 th till 11 th dpi.It is possible that this increase represents a rapid regrowth of B. bassiana, survived from the treatments.From the 11 th to the 29 th dpi, B. bassiana DNA concentrations remained high in all three substrates, suggesting that the B. bassiana population had reached the maximum level under the assayed conditions.In this study, B. bassiana DNA was not detected in any negative control samples and thus, the identifed PCR primers produced the B. bassiana DNA specifc bands when electrophoretically analyzed.