Isolation and efficacy of the endophytic fungus, Beauveria bassiana (Bals.) Vuillemin on grapevine aphid, Aphis illinoisensis Shimer (Hemiptera: Aphididae) under laboratory conditions

The endophytic fungus, Beauveria bassiana has been found to occur endophytically in hundreds of plant species tissues and has been inoculated for establishment in different plants. It has negative effect on piercing-sucking insects in grapevine plants. Thus, the present study aimed to detect the endophytic fungus, B. bassiana throughout ITS genes analysis in the grapevine plant, Vitis venifera cv. Taify and assess its potential for controlling the grapevine aphid, Aphis illinoisensis Shimer (Hemiptera: Aphididae). The obtained 5 isolates were DNA sequenced for ITS region. Data analysis showed that there were 2 different isolates (accession number: MN900613 for isolate Bb-Taif1 and MN900614 for isolate Bb-Taif2). The values of LC50 were 6.041 × 104, 3.199 × 105 spores/ml while those of LC90 were 6.13 × 106 and 7.474 × 107 spores/ml after 3 days of treatment by Bb-Taif1 and Bb-Taif2, respectively. These values showed that the virulence of Bb-Taif1 isolate was higher in its efficacy than the Bb-Taif2 isolate. Further investigations are needed to evaluate its efficacy on other piercing sucking or chewing insect pests throughout conidia spraying, soil inoculation, and/or root immersion for grapevine plants.


Background
The fungal endophytes (living within plant tissues) play important roles in plant community ecology (Wani et al. 2015). The study of endophytic microorganisms is defined as "endophytology" (Unterseher et al. 2012). They are called endophytes because they colonize the healthy plant tissues in some periods of their life cycle without causing damage for the plants (Azevedo et al. 2000). The distinction between pathogenic and endophytic microorganisms depends on the stage of the interaction of the microorganism with the host plant (Strobel et al. 2004). Many authors have proposed that the studies of endophytic fungi can be called "endophytism" (Suryanarayanan 2013), and others define these fungi as "mycoendophytes" (Rai et al. 2014). These endophytic fungi have negative effects on different insect pests and plant pathogens by producing toxic compounds or by modifying the host plant defense response to enable pest and pathogen resistance (Backman and Sikora 2008;Saikkonen et al. 2010). There are different beneficial effects of these endophytic fungi on host plants as plant growth promoting, increasing and induction of plant defense mechanisms, disease severity reduction, and producing of different anti-herbivore products (Yang et al. 2016).
The important group of endophytic microorganisms is the entomopathogenic fungi of the order Hypocreales, known to cause infections in insects. Beauveria spp. are common terrestrial entomopathogens, infecting numerous insect and arachnid taxa in various regions of the world (Vega 2008). Beauveria bassiana has been recognized to occur endophytically in hundreds of species in plant tissues and has been inoculated for establishment in different valuable crops and vegetable plants (Vega 2008;Vidal and Jaber 2015;and Gonzalez et al. 2016). The lethal effect of these endophytic fungi on insects is shown by eating the plant parts in chewing insects or by oral ingestion of plant sap in sucking insects (Batta 2018).
Thus, there is much interest in how to detect and use endophytic fungi specially Beauveria and Metarhizium with insect pathogenic capabilities to induce plant tolerance to insect pests (Vega 2008). These fungi are globally distributed and commercially available as biological control agents (Lacey et al. 2015). Many investigations were carried out on the inoculation of 13 plant species belonging to 15 families with various entomopathogenic fungi such as B. bassiana. These studies have examined seed dressing, seed soaking, foliar sprays, mycosed insects placed in the soil, soil drenching, and fungal plugs placed in the soil (Vega 2018). However, the endophytic B. bassiana present in grapevine has negative effect on piercing-sucking insects, while it reduces infestation rate and growth of the vine mealy bug, Planococcus ficus (Signoret) in young plants and induces high reduction of infestation with grape leafhopper, Empoasca vitis (Goethe) (Rondot and Reineke 2018).
Till now, there is no negative impact of the presence of endophytic B. bassiana on various host plants in a range of investigations (Zimmermann 2007;Klieber and Reineke 2016). Endophytic B. bassiana has been reported for maintaining systemic protection against various piercing-sucking insect pests or for inhibiting insect development (Rondot and Reineke 2018). Moreover, presence of endophytic B. bassiana reduces disease symptoms induced by various fungal pathogens (Cosoveanu et al. 2014;Jaber 2015).
The detection and identification of endophytic fungi is important to determine their distribution in host plants and their beneficial effects. Detection of endophytic microorganisms is characteristically attained through various techniques, including isolation of fungal material directly from plant tissues to growth media (culturedependent), microscopy, and molecular recognition of endophytic DNA from different plant materials, using PCR (culture-independent) (McKinnon et al. 2017). Another recent technique is the fluorescence microscopy, which can be used to detect endophytes inside plant tissues (Pacifico et al. 2019). In general, the internal transcribed spacer-1 (ITS1), the internal transcribed spacer-2 (ITS2), and 5.8 S regions of the nuclear ribosomal DNA are commonly utilized for molecular identification of B. bassiana (Mondal and Baksi 2018).
Grapevine (Vitis vinifera L. cv. Taify) is the second most important economical fruit in Saudi Arabia and it is consumed as table grapes, grape juice, or raisins. This cultivar has been reported as the best quality for chemical composition comparing to other cultivars cultivated in Saudi Arabia (Fahmi et al. 2012). The grapevine trees are infested with different insect pests such as lepidopterous insects, mealy bugs, leafhopper, thrips, scale insects, and aphids. The grapevine aphid, Aphis illinoisensis (Shimer) is a grapevine pest infesting young terminal shoots, the lower surface of young leaves (Blackman and Eastop 2006) and fruit clusters causing drops of some grape berries (Pfeiffer and Schultz 1986). It is one of such invasive species recorded since the 2000s in Southern Europe and in the North African countries (El-Gantiry et al. 2012).
The present study aimed to detect and assess the potential of endophytic fungus, B. bassiana in the grapevine, V. venifera cv. Taify for controlling the grapevine aphid, and A. illinoisensis under laboratory conditions.

Fungi isolation
After sterilization of leaves surface, 3 leaf discs (1 cm in diameter) were obtained from each leaf. Leaf discs were placed on selective medium of EPF (DOC2-PDA medium), which prepared as follow: 2 mg crystal violet, 0.2 g CuCl 2, 39 g Potato Dextrose Agar (PDA) dissolved in 1 liter distilled water, while pH was adjusted to 4.0 with HCl and autoclaved for 20 min at 120°C (Shin et al. 2010). Plates were incubated at 20°C with a 12:12 h light: dark photoperiod. After 10-14 days, leaf discs were examined visually for the presence of endophytic B. bassiana that grew from internal plant tissues of surface sterilized leaf discs with white dense mycelia and creamy color at the edge (Humber 1997).

Genomic DNA isolation
For final detection of endophytic B. bassiana, mycelia samples were analyzed with molecular techniques. DNA was extracted from fungal tissues, using the fungi/yeast genomic DNA extraction mini Kit (Favorgen Biotech Corporation, Taiwan) according to the manufacturer's instructions. The extracted DNAs were stored at − 20°C for using as templates for PCR.

PCR for ITS region and sequencing
The PCR amplification was carried out to amplify a partial region of ribosomal DNA (ITS) containing 18S gene (partial sequence), internal transcribed spacer-1 (complete sequence), 5.8S gene (complete sequence), internal transcribed spacer-2 (complete sequence), and 28S gene (partial sequence). The amplification was done using specific primers (F: 5-GGAAGTAAAAGTCGTAA-CAA-3 and R: 5-TCCTCGCTTATTGATATGA-3) (Sayed et al. 2018). PCR amplification was carried out in a final volume of 50 μl by mixing 2 μl of DNA with 1 μl of each primer (10 pmol), 25 μl of M7123 green master mix (Promega, USA), and 22 μl autoclaved double distilled water. The conditions of PCR amplification were 3 min at 94°C followed by 35 cycles of 40 s at 94°C, 40 s at 52°C, and 40 s at 72°C with a final extension of 7 min at 72°C. PCR products were checked by electrophoresis on a 1.5% agarose gel. After electrophoresis, the PCR products were cut out, cleaned and purified by the use of BioFlux BioSpin Gel Extraction kit (Bioertechnology Co., Ltd), and sequenced using the same primers by an automated DNA sequencer (Macrogen, Korea). Then, each sequence was checked in comparison to the GenBank database of the NCBI website. Finally, the sequences were deposited in GenBank of the NCBI website.

Insects and fungi isolates
Fresh leaves of grapevine plants infested with the aphid, A. illinoisensis were harvested on the same experimental day from the vineyard at Taif. Both fungal isolates were prepared as suspensions in six concentrations of 1 × 10 4 , 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 spores/ml. Then, 0.02% Tween 80 was added to each suspension for dispersing the conidia uniformly in the suspension (Selvaraj et al. 2012). The suspensions were vortexed for 5 min to obtain homogeneity in the suspension.

Experimental method
Five replicates (each contained 40 aphid individuals) with totally 200 aphid individuals were used for each concentration. For the bioassay, 1 μl of every single concentration was dropped straight on the aphid body (Eidy et al. 2016). Every Petri dish was filled by a thick layer (3-4 mm) of agar (0.1%). The individuals of adult aphid (1 day old) were carefully relocated by a fine camel hairbrush from the leaf of grapevine to Petri dishes. In the control, aphid adults were treated with 1 μ of distilled water with Tween 80 (0.02%) for each one. The plant leaves were changed daily with other fresh leaves for feeding aphids. The experiment was carried out at (25 ± 1°C, 65-70% RH and a photoperiod of 16 L: 8D). The aphid mortality was checked daily till the 3rd day.

Statistical analysis
Abbott's formula (Abbott 1925) was used with the purpose of correcting the mortality data in the treatments with that in the control. The LC 50 and LC 90 values for both isolates were measured employing Probit analysis with the SPSS software program (SPSS 2015). The LC 50 and LC 90 values, 95% confidence intervals (lower bound and upper bound), slope, intercept, and chi square of both isolates were subjected to t test with a P = 0.05.

Results and discussion
Seventy-five leaf discs from each location were examined for detection of the endophytic B. bassiana. Some leaves had fungal growth (Fig. 1). All obtained mycelia having the same characteristics of B. bassiana were analyzed for ITS amplification with B. bassiana specific primers. Therefore, the endophytic B. bassiana was detected in 3 plant samples of location-1 and 2 plants of location-2, with a PCR product of 593 bp (Fig. 2). The 5 products were sequenced and the sequences showed that the 3 specimens from location-1 were identical. Moreover, the sequences of the 2 specimens from location-2 were also identical; meanwhile, they were differed than the specimens in location-1. Consequently, the 3 specimens from location-1 were considered as unique isolate and were named as Bb-Taif1, while the 2 specimens from location-2 were considered as the second isolate and were named as Bb-Taif2. The alignment of ITS sequences for both isolates resulted in identity of 99%. In this way, less geographical distances were related with less genetic variability where the isolation by distance of B. bassiana assumes a significant role in its genetic variation (Rehner et al. 2006). The differences between both isolate were in 5 nucleotides as follows: 2 nucleotides in the ITS1 and 3 in the ITS2 (Fig. 3). These 2 sequences were submitted in the NCBI GenBank database with the accession numbers of MN900613 for isolate Bb-Taif1 and MN900614 for isolate Bb-Taif2. In this sense, isolate Bb-Taif1 was identical in the sequence of ITS with previous isolate (accession number: LC338054), which isolated from the soil at the same location (Sayed et al. 2018). In our opinion, this finding means that the same isolate in the soil was colonized in the grapevine plants. This suggestion is in accordance with other investigations, while soil inoculation was used for endophytic   (Greenfield et al. 2016). Also, grapevine inoculation trials via soil inoculation or root dipping resulted in endophytic colonization of B. bassiana but not at all plants (Rondot and Reineke 2018). The values of LC 50 and LC 90 for the 2 tested isolates of endophytic B. bassiana against A. illinoisensis after 3 days of treatment are presented in Table 1. The LC 50 value of isolate Bb-Taif1 (6.041 × 10 4 ) was significantly lower than of isolate Bb-Taif2 (3.199 × 10 5 spores/ml) (t = 2.767, P = 0.024). Also, there was a significant difference between the lowest bound of 95% confidence interval of LC 50 values (t = 2.278, P = 0.052), while this difference was significant in the upper bound (t = 3.476, P = 0.008). The LC 50 value of isolate Bb-Taif1 is compatible with Sayed et al. (2019) (6.46 × 10 4 spores/ml) for an isolate, which was isolated from the soil at the same location. This finding, coupled with the molecular conformity, reinforce the previous   Akmal et al. (2013) who found that the LC 50 values were 6.28 × 10 5 and 6.76 × 10 6 spores/ml against Brevicoryne brassicae (Linn.) and Schizaphis graminum L., respectively. On the same context of LC 50, in the present study, the LC 90 value of isolate Bb-Taif1 (6.13 × 10 6 spores/ml) was significantly lower than of the isolate Bb-Taif2 (7.474 × 10 7 spores/ml) (t = 2.739, P = 0.025). Also, both of the lowest and highest bound of 95% confidence interval of LC 90 values were differed significantly (t = 2.332, P = 0.048 and t = 3.075, P = 0.015, respectively). Meanwhile, the intercepts and χ 2 were not differed significantly (t = 0.726, P = 0.488 and t = 1.072, P = 0.315, respectively), while the difference in slope was significant (t = 3.263, P = 0.011).
Generally, the virulence of the 2 tested endophytic isolates indicated that the isolate Bb-Taif1 was higher in its virulence than the Bb-Taif2 isolate. Moreover, the findings of molecular characteristic and the virulence in this study and the findings of Sayed et al. (2018Sayed et al. ( , 2019 showed that the isolate (Bb-Taif1) is endemic in the soil and establishment in the grapevine tissues as an endophytic fungus.

Conclusion
Using the endophytic indigenous isolate of B. bassiana for controlling aphids in vineyards achieved a high mortality rate in aphids' populations. Also, the isolate (Bb-Taif1) could be used as conidia spraying, soil inoculation, and/or root immersion for grapevine plants. Further investigations are needed on this isolate for its efficacy on other piercing sucking or chewing insect pests.