Background

The bean flower thrips, Megalurothrips usitatus Bagnall (Thysanoptera: Thripidae), is a major pest of cowpea causing considerable economic losses to this crop by damaging cow pea leaves, flowers and pods (Mound and Walker, 1987). M. usitatus damage can cause leaf wrinkling, growth point atrophy, pod scab, and other effects on cowpea plant reducing the market value of crop (Tang et al.2015). The flowering stage is known to be the most vulnerable to M. usitatus attack and hundreds of M. sitatus (nymphs or adults) can be seen per flower during peak pest ultimately leading to reduced yield through premature flower loss (Tang et al.2015). Currently, chemical insecticides are being used as the principal control agent against M. usitatus. They are not effective enough due to the cryptic habits of M. usitatus (such as staying within flowers and their short life cycles) (Tang et al.2015). The injudicious use of chemical insecticides has resulted in development of insecticide resistance in M. usitatus (Immaraju et al.1992). Therefore, the development of ecologically sound and sustainable control measures for M. usitatus management is required on urgent basis (Elimem et al. 2018).

Entomopathogenic fungi (EPF) have received considerable attention as biological control agents of thrips and several studies have demonstrated the effectiveness of them (such as Beauveria bassiana, Metarhizium anisopliae, Paecilomyces fumosoroseus, A. attenuatus, Aschersonia aleyrodis) against different thrips species (Skinner et al.2012 and Wright et al., 2016). Ugine et al. (2006) reported that application of B. bassiana significantly reduced Frankliniella occidentalis populations by 81%, especially at the flowering stage of crops. The Verticillium lecanii (V3450, Vp28) is a well-known species of EPF against thrips and some isolates of this species are being developed as commercial biopesticides (Ainsworth et al., 2008).

The potential use of B. bassiana, C. fumosorosea, and A. attenuates have been demonstrated on many insect pests (Ali et al.2018 and Zimmermann 2018). And the insecticidal attribute of B. bassiana and V. lecanii developed its use in biopesticide industry (Singh et al. 2015). Isaria fumosorosea is mainly used to control agricultural pests such as Bemisia tabaci, aphids and thrips. A. nomiu is a facultative pathogen of many plants and animals (St Leger et al., 2000).

The present study was designed to isolate, identify, and describe different strains. The fungal conidia and crude extract were also tested for their pathogenicity against M. usitatus.

Materials and methods

Sample collection and fungal isolation

The soil samples were collected from farmers’ fields from 6 localities (Yunan, Hunan, Guangxi, Guangdong, Fujian and Hainan) of Southern China. Fungal isolation was carried out following the method described by Gottel et al. (2008) and Imoulan et al. (2011). Briefly, 3 g soil sample was added to 30 ml of ddH2O containing 0.05% Tween 80. The mixture was vortexed for 15 min and 1 ml suspension was layered onto potato dextrose agar (PDA) plates. The plates were incubated at 26 ± 6 °C, 70 ± 5% RH and 16:8 h L:D photoperiod for 7 days. The growth of the fungi was observed after 7 days and were re-inoculated by inoculation of individual fungus on new PDA plates. In this way, several rounds of inoculation were performed until a purified strain based on the phenotypic properties and fungal morphology was obtained (Saito and Brownbrideg, 2016 and Du et al.2019)

DNA extraction, PCR amplification, and construction of phylogenetic trees

Genomic DNA of fungal isolates, purified after the isolation process, was extracted by using a fungal genomic DNA extraction kit supplied by Ezup, Sangon Biotech, Shanghai, China Internal transcribed spacer region (ITS) of fungal DNA was amplified in order to identify the species of each strain (Imoulan et al.2016). The reaction mixture for PCR amplification consisted of 25 μl 2 × TapPCR Master Mix, 1 μl of each primer (10 μM), 1 μl genomic DNA, and 22 μl ddH2O. The ITS regions were amplified using the primers (Table 1). The thermal profile of the PCR condition for primer included an initial denaturation at 94 °C for 5 min, followed by 35 cycles with a new denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 1 min, and with a final extension of 10 min at 72 °C. The PCR products were assayed for fragment size by agarose gel electrophoresis. Then, the PCR products were sent to the Shanghai Majorbio Bio-pharm Technology (Shanghai, China) for sequence analysis. The obtained ITS gene sequences of all strains were sequence-spliced with Genious version 7.1.4 (Goloboff and Catalano, 2016), then aligned in National center for biotechnology information (NCBI), the aligned sequences were downloaded, and then all sequences were constructed with MEGA Version 7.0 to construct a maximum likelihood (ML) tree (Kumar et al.2016).

Table 1 Primers used in this study
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Virulence of the fungi in the laboratory

Insect colony

Adults of M. usitatus were collected from a cowpea field at South China Agricultural University, Guangzhou, China during 2017. The collected insects were subsequently reared under laboratory conditions by the bean pod method (Espinosa et al.2005). The insect colonies were kept at 26 ± 6 °C, 70 ± 5% RH and 16:8 h L:D photoperiod in a climate control chamber. Newly emerged adult females were used for fungal bioassay studies.

Fungus culture, conidia suspension, and crude extract preparation

The tested strains (SCAUDCL-53, SP016, SP535, SB009, SB010, and SCAUN-1) were cultured on PDA plates for 7 days under laboratory conditions. Conidia were harvested from culture plates by scraping the surface of the mycelia with sterile cell scrapers into sterile deionized water containing 0.05% Tween-80. The conidia were counted under compound microscope (Ningbo Shunning Instruments Co. Ltd) at × 40 magnification by using hemocytometer (Qian Yihua Glass Instruments Co. Ltd.) to calibrate a concentration of 1 × 108 conidia ml−1. Freshly prepared conidial suspensions were used for all experiments.

Fifty milliliters of freshly prepared sabouraud dextrose broth medium (SDB) was added to 250-ml Erlenmeyer flasks followed by sterilization at 121 °C for 30 min. Five milliliters of freshly prepared fungal suspension (1 × 107 conidia ml−1) of different fungal isolates was added to culture medium. The flasks were incubated in a rotary shaker at 120 rpm, 28 °C for 5 days. After 5 days, fungal cultures were filtered through Whatman filter paper (Qian Yihua Glass Instruments Co. Ltd) to obtain the supernatant for subsequent studies. The protein concentrations of crude extracts were quantified by using bouvine albumin serum as substrate (Bradford 1976). The protein concentration of all the crude extracts was adjusted to 0.4 mg mL−1 by following Quesada et al. (2006).

Bioassay method

Laboratory bioassays were conducted to evaluate the toxicity of different fungal isolates identified during this study against the M. usitatus adults by using centrifuge tube residual bioassay (Du et al.2019). The centrifuge tubes (9 ml) and bean pods (1 cm) were immersed in conidial suspension (1 × 108 conidia ml−1) and crude extract for 2 h, followed by drying under sterilized conditions. The centrifuge tubes and bean pods treated with ddH2O having 0.05% Tween-80 served as control. After drying, adult females of M. usitatus (100 individuals) were transferred to the tubes having bean pods treated with same fungal treatment. Each centrifuge tube was sealed with a cotton plug to prevent thrips from escaping. The whole experiment was repeated thrice with fresh batches of insects at 26 ± 1 °C, 75% RH, and 16:8 L:D photoperiod. The insects were observed on daily basis to record the mortality data following Du et al. (2019).

Microscopic examination

Newly emerged M. usitatus adults were inoculated by the conidial suspension (1 × 108 conidia mL−1) and the crude protein extract (0.4 mg ml−1) of all the 6 fungal isolates and were incubated at 25 °C and 75% RH in the dark. The infected M. usitatus (5 individuals) were sampled at 1, 2, 3, 4, and 5 days after inoculation. Gross changes in the appearance of the infected M. usitatus were directly monitored at different times after inoculation under a D850 camera (Nikon Co. Ltd. Japan).

Statistical analysis

Mortality data were percent transformed and were subjected to probit analysis. All the analysis were performed through SAS software v9.1 (SAS et al., 2000).

Results and discussion

Identification of the fungi

The present study reported 6 fungal isolates belonging to 4 fungal species from isolated soil samples collected from different localities of southern China. The result showed that the strains SB009, SB010, and SP016 were identified as B. bassiana (NR_111594). Strain SP535 was identified as C. fumosorosea (AF461747). Strain SCADDCL-53 was consistent with the A. attenuatus; MH558279 and isolate SCAUN-1 was identified as A. nomius (NR_121218) (Fig. 1).

Fig. 1
figure 1

Majority rule consensus phylogram from the maximum likelihood (ML) tree based on the sequences of the ITS region for six strains (SB009, SB010, SP016, SP535, SCAUDCL-53, and SCAUN-1). Metarhizium robertsii and Metarhizium anisopliae were used as an outgroup

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The identification of entomopathogenic fungi, based on morphological features, can lead to ambiguous results (Ziółkowska et al.2015). Nucleic acid sequence analysis is commonly used method for the identification and classification of fungi (Funk et al.1983). It compares the correlation between homologous molecules by determining the composition of nucleotide sequences in the primary structure of nucleic acid (Diaz et al.2012). Sequencing of the ITS region of rRNA is currently regarded as the standard method for phylogenetic analyses and identification of fungal species (Schmidt and Moreth, 2002; Cafarchia et al.2013 and Kawasaki 2011) (Table 2). In this study, the comparison of ITS sequences of the isolated fungi showed that they were closely related to B. bassiana (isolates SB009, SB010, SP016), C. fumosorosea (isolate SP535), A. attenuatus (isolate SCAUDCL-1), and A. nomius (isolate SCAUN-1).

Table 2 Reference entomopathogenic fungi from GeneBank used for the phylogenetic analysis
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Virulence of the fungi in the laboratory and greenhouse

Analysis of mortality data showed that B. bassiana isolate SB010 was the most effective fungal isolate (except A. attenuatus isolate SCAUDCL-53) against M. usitatus adults when compared with other isolates used in this study as conidial suspension. The crude extract of this isolate caused 90 and 93.3% mortality rates of M. usitatus adults (Figs. 2 and 3). The conidial suspension of SCAUDCL-53 strain showed highest mortality rate (100%), whereas lowest mortality (25%) was observed for conidial suspension of SP535. The probit analysis of the pathogenicity data revealed significantly lower LT50 values for conidial suspension as well as crude protein extracts of A. attenuatus isolate SCAUDCL-53 and B. bassiana isolate SB010 than other isolates used in this study (Tables 3 and 4). The lowest LT50 value of conidial suspension (1.36 days) was observed for A. attenuatus isolate SCAUDCL-53, while in case of crude protein extract the lowest LT50 was observed for B. bassiana isolate SB010 with a mean value of 2.85 ± 0.16 days. Significant differences in the pathogenicity of different fungal isolates against M. usitatus were observed during this study. The conidial suspension (1 × 108 conidia ml−1) of A. attenuatus isolate SCAUDCL-53 was proved to be the most virulent showing (100%) mortality of M. usitaus after 5 days of application.

Fig. 2
figure 2

Corrected mortality rates of Megalurothrips usitatus with different time of conidial suspensions

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Fig. 3
figure 3

Corrected mortality rates of Megalurothrips usitatus with different time of crude protein extract

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Table 3 LT50 values for fungal strains against Megalurothrips usitatus adults (1 × 108 conidia ml−1)
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Table 4 LT50 values for fungal crude extracts against Megalurothrips usitatus adults (0.4 mg ml−1)
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Several investigations have reported the susceptibility of different thrips species to EPFs. Ravensberg et al. (1990) reported that sprayings V. lecanii at weekly intervals reduced the F. occidentalis incidence by 90%. Wang (2012) screened out different isolates of Lecanicillium lecanii for the management of Gynaikthrips ficorum and showed more than (85.56%) mortality of G. ficorum by L. lecanii isolateV16063. The LT50 values (1.36 days) of A. attenuatus isolate SCAUDCL-53 against M. usitaus at a concentration of 1 × 108 conidia mL−1 observed during this study are a little lower than those observed by Du et al. (2019) who observed LT50 of 3.5 days for A. attenuatus isolate SCAUDCL-38. B. bassiana is known as an effective biological control agent of several insect species (Rezende et al. 2009). We observed high pathogenicity of B. bassiana (SB010) towards M. usitaus under laboratory conditions, suggesting that it has high capacity in M. usitaus control. In the current study, conidial suspension (1 × 108 conidia mL−1) of B. bassiana isolate SB010 exhibited 90% mortality of M. usitaus after 5 days of fungal application whereas the median lethal time (LT50) of conidial suspension was 3.79 days. However, the isolates SB009 and SP016 of B. bassiana just inhibited 36.25% and 36.21% of M. usitaus. Jacobson et al. (2001) reported that B. bassiana showed remarkable pathogenicity on F. occidentalis and reported 87% mortality. The differences in the values of mortality compared to those obtained in the present study could be explained by the different B. bassiana species and experimental insects hosts (Jacobson et al., 2001). At present, I. fumosorosea is mainly used to control agricultural pests such as whiteflies, aphids, and thrips (Faria and Wraight, 2007; Kabaluk and Gazdik, 2005). In this study, M. usitaus were less susceptible to the strain SP535 isolated of C. fumosorosea, showing 25% mortality.

Although fungal conidia are commonly used to control insect pests, this application method has limitation as fungal conidia can be susceptible to environmental conditions (Skinner et al. 2012; Rangel et al. 2008). The current study also examined the biological activities of crude fungal protein extracts. In the current study, crude extracts of all the six fungal isolates (used at 0.4 mg mL−1 protein concentrations of crude extract) showed significantly different rates of M. usitaus mortality. The crude extracts of B. bassiana isolate SB010 was the most virulent showing 93.3% mortality of M. usitaus after 5 days of application. Previous studies have reported similar dose-dependent insecticidal effects of fungal secondary metabolites against different insect species. Muzammil and Shoaib (2018) reported that 10 mg protein/ml concentration of crude B. bassiana (isolate Bb-01) extract showed (91%) mortality on Musca domestica L. (Diptera: Muscidae). The other major impediment in the development of fungal insecticides is the time required to control an insect pest (St Leger and Screen, 2001). The concentration of crude B. bassiana extract used in the present experiments was much lower than those used by Muzammil and Shoaib (2018) which confirmed the higher pathogenic potential of B. bassiana isolate SB010.

Microscopic examination

The 6 fungal isolates induced similar infection symptoms in M. usitatus adults. In the prophase of post-infection, M. usitaus’ head generated white hyphae, along with the post-infection of time, the hyphae developed continuously around the insects’ body white hyphae covered the whole insect body and the activities of insects were abnormal (Fig. 4). However, the insects were treated by crude protein extracts generated hyphae earlier than conidial suspension. Furthermore, the density of hyphae was also higher, following the crude protein extracts application when compared with the application of conidial suspension (Fig. 5).

Fig. 4
figure 4

Images of Megalurothrips usitatus infected with conidia suspension and observed through a dissecting microscope. A1–F1 Infected Megalurothrips usitatusat SB009, SB010, SP016, SP535, SCAUDCL-53, and SCAUN-1, respectively

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Fig. 5
figure 5

Images of Megalurothrips usitatus infected with crude extract and observed through a dissecting microscope. A2–F2 Crude extract infected Megalurothrips usitatus at SB009, SB010, SP016, SP535, SCAUDCL-53, and SCAUN-1, respectively

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Conclusion

Successful isolation and molecular characterization of 6 different strains belonging to 4 fungal species were reported. The pathogenic potential of these isolates was also observed through bioassays of conidial suspension and crude protein extracts against M. usitatus adults. B. bassiana isolate SB010 and A. attenuatus isolate SCAUDCL-53 were the most effective against M. usitatus. These findings will provide baseline information about the screening of effective biological control agents against M. usitaus. Further field trials as well as studies on identification of effective fungal toxins are still required to design efficient and environmentally sustainable biopesticides for M. usitaus management.