Effect of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, on Thrips tabaci Lindeman (Thysanoptera: Thripidae) populations in different onion cultivars

Background: Thrips tabaci Lindeman (Thysanoptera: Thripidae) is the key pest of onions that causes economic yield losses in commercial onion production in Pakistan. In this study, potential of the entomopathogenic fungi (EPF), Beauveria bassiana and Metarhizium anisopliae, as a bio agent was evaluated to manage buildup of thrips population on onion crop. Results: Efficacy tests for EPF were conducted against T. tabaci infesting 3 different onion varieties (Phulkara, Swat 1, and Virio 7). Commercial formulations of B. bassiana strain GHA and M. anisopilae strain ESC-1, were evaluated at 4 different concentrations (10, 10, 10, and 10 conidia/ml) under field conditions for 2 years. The efficacy was assessed 3, 5, 7, and 10 days after spray application of the whole onion plant. Efficacy expressed as T. tabaci (nymphs and adults) percent population reduction in comparison to controls. Maximum corrected percent population reduction was observed in onion plants treated with B. bassiana 10 conidia/ml, i.e., 86.62, 84.59, and 86% in Phulkara, Swat 1, and Virio 7 onion varieties respectively, after 10 days of spray application. While onion plants treated with M. anisopliae 10 conidia/ml showed minimum corrected percent population reduction, i.e., 69.42, 68.45, and 69.11% in Phulkara, Swat 1, and Virio 7 onion varieties respectively, after 10 days of spray. Conclusions: Beauveria bassiana could significantly reduce thrips population and could provide a better long-term management of T. tabaci on onion. B. bassiana had a high toxic effect against offspring production of the T. tabaci under field conditions than M. anisopliae.


Background
Growth and yield of the onion crop is significantly stressed by various factors including sap sucking insects. The onion thrips (Thrips tabaci Lindeman, Thysanoptera: Thripidae) cause the main pest for onion crops whose feeding results in reduced growth and yield for onion plants (Ananthakrishnan, 1993). The onion thrips (Thrips tabaci Lindeman.) cause extensive economic losses to onion crops in field and greenhouse vegetable production (Diaz-Montano et al. 2011). At nymphal and adult stages, thrips cause direct damage by feeding/sucking cell sap through their modified mouthparts and indirectly disseminate/vector viral pathogens which cause diseases such as yellow spot viral disease (Gent et al. 2004).
In Pakistan, sucking insect pests is the major reason of decrease in onion production. These pests include thrips as major pest and onion maggot (Delia antiqua), leaf minors (Lyriomyza spp.), and cutworm (Agrotis ipsilon) as minor pests (Khan et al. 2015). The demand for organic horticulture products that are safe for environment and consumers are increasing. Non-chemical methods such as biotechnical methods and intercropping (Trdan et al. 2006), late planting, physical barriers, and mulching are identified control methods for T. tabaci on onion crop (Gawande et al. 2010). However, all the above tactics could form one component of integrated pest management. Biological control in onion fields faces an immense difficulty because onion is treated intensively with insecticides. In organic horticulture, biological control is recognized as a basic component of IPM in which microbial control is a preferred technique due to its positive attributes such as amenable to production, broad spectrum effectiveness, and long-term storage (Dinesh 2017).
Among entomopathogenic microbial agents, fungal pathogens EPF isolated from different thrips species and proven to be pathogenic to T. tabaci include Metarhizium anisopliae (Metschn.) Sorokin., Beauveria bassiana (Bals.) Vuill., Neozygites cucumeriformis, Zoophthora radicans, Entomophthora thripidum, Verticillium lecanii, and Paecilomyces fumosoroseus (Butt and Brownbridge 1997). EPF are developed for the management of onion thrips T. tabaci, western flower thrips Frankliniella occidentalis and legume thrips Megalurothrips sjostedti in leguminous crops, ornamental plants, and vegetables (Maniania et al. 2002). Biopesticide usage for integrated crop pest management has increased in last few years (Sahayaraj and Namasivayam 2008). Various EPF have been industrialized as formulated products such as (i) Beauveria as BotaniGard® ES, Beauverin®, and Mycotrol® WPO, Betel®; (ii) M. anisopliae as Met52® EC, Bio-Catch-M® SL, and Green Muscle® SU; (iii) M. flavoviride as Biogreen® L; and (iv) Isaria fumosorosea as Preferal® WG and Priority® WP (Faria and Wraight 2007). These formulated products are being used to manage a wide range of pests such as thrips, whiteflies, aphids, mealybugs, psyllids, plant bugs, scarab beetles, and weevils (Copping 2009). These EPF have potential to be used in integrated insect pest management programs due to their less persistent nature, low mammalian toxicity, and natural occurrence (Lee et al. 2016). Although EPF formulated products have been developed, there is a little or no information about their evaluation in Pakistan farmer's greenhouse and field conditions. The present study focused on determining the efficacy of these EPF against T. tabaci under field conditions, in order to generate knowledge for their use as a component in organic farm production.

Experimental site
Field trials were conducted at Chak Shahzad, Islamabad, where vegetables are grown through the year. This site is situated at longitude 33°40 N, latitude 73°8.9 E and with elevation 499 meters ASL. The minimum and maximum average temperature of the area is 10 and 38°C, respectively. Crops grown in the surroundings of the experimental area were wheat in the South, brassica and canola in the West, cabbage in the East, and bamboos in the North.

Nursery sown
Seeds of onion varieties (Phulkara, Swat 1, and Virio 7) were acquired from Ayub Agriculture Research Institute, Faisalabad. Nursery of these three varieties was raised after soil preparation during the October 2018 and 2019. Fertilizer was applied in 3 split concentrations: at the time of nursery raising, at transplanting stage, and at bulb initiation stage for better yield. Nursery was transplanted in December 2018 and 2019 for the winter season at 4-5 leaf stage after 8 weeks of emergence.

Experimental layout
The plot layout was a randomized complete block design for onion varieties Phulkara, Swat 1, and Virio 7 with 3 replications. Each experimental plot consisted of (3 m × 3 m) with distance of 10 cm between seedlings with 30 cm distance between rows. To avoid contamination and drift hazards among treatments, each experimental plot was separated by a distance of 1.5 m.

Formulations of entomopathogenic fungi
The B. bassiana WP and M. anisopliae WP formulations contained active ingredient based on 2.01 × 10 10 cfu/g of product. Commercial formulations of B. bassiana strain GHA and M. anisopliae strain ESC-1 were added in distilled water to make spray solution (Maniania et al. 2003). Spore germination rates of these EPF were tested on PDA at 25°C after 24 h for 80% germination. The conidial concentration of EPF was determined by a hemocytometer.
EPF were applied after the 7th week of transplantation. Treatments were sprayed to onion plants infested with thrips with the help of Solo 418-One Hand Pressure Sprayer. Surfactant (0.02% tween 80) was mixed to the spray solution to enhance the adjuvant ability of solution and for better spread of entomopathogens. Treatments were sprayed during the evening times to lessen the ultraviolet radiation adverse effects on spore germination (Morley et al. 1996) and providing better conditions regarding humidity and temperature for fungal growth. Experimental plots were irrigated before spray of EPF to maintain relative humidity. Entomopathogens and insecticide were sprayed with the help of separate hand sprayer to avoid contamination. Five randomly selected onion plants were observed for thrips population density in each experimental plot before and after application of treatments. Following experimental treatments were prepared: 1.5 l + 0.02% tween 80 (Ganga and Krishnamoorthy 2012) Control plants were sprayed by water and surfactant as a negative control and recommended insecticide (Bifenthrin) as positive control. Entomopathogens were applied in the evening.

Statistical analysis
Conidial application of these EPF was made 3 times at 10 days interval as experiment replication. The results were presented as onion thrips (%) population reduction pooled means of these replications. Thrips population reduction percentage in comparison to control treatments were calculated by the Henderson-Tilton's formula (Henderson and Tilton 1955).
Thrips counts before treatment application were used in population reduction percentage calculation by Henderson-Tilton's formula for each treatment. Pretreatment and post-treatment means were analyzed by using the statistical software (Statistix 8.1) for ANOVA. Means were compared at 0.05 probability levels by Tukey's Honest Significant Difference test (HSD).

Results
The present study was carried out, using 2 EPF, with 4 different concentrations on 3 onion varieties, i.e., Phulkara, Swat 1, and Virio 7. Effect of EPF on thrips population's percentage in 3 different onion varieties is shown in Fig. 1. Maximum thrips population reduction percentages were recorded in Swat 1 onion variety after application.

Onion thrips corrected population reduction percentage on Phulkara variety
Effect of EPF on thrips population reduction percentage during 2019 at Phulkara onion variety is presented in Table 1. High corrected population reduction percentage was recorded in case of high concentrations of EPF. Among all the treated onion plots, the highest reduction in thrips population (89.14 %) was observed in bifenthrin-treated plots. Results revealed that B. bassiana treatment of onion plots at the concentration of 10 11 conidia/ml showed the highest thrips population reduction (22.03%), which is at par with M. anisopliae at Thrips population significantly reduced after 5 to 7 days of EPF application. After 5 days, all the fungal treatments significantly reduced onion thrips population in comparison to control plots. Population reductions between 35.47 and 41.15% were observed in the treatments M. anisopliae 10 10 and B. bassiana 10 11 conidia/ml, respectively. After 7 days of EPF application, the highest reduction in populations was observed in B. bassiana at the concentration of 10 11 conidia/ml (73.33%) treated onion plots. Significant differences (F = 5.21; P = 0.00) were observed among treatments after 7 days of EPF application. After 10 days, the highest population reduction percentage found was induced by B. bassiana 10 11 conidia/ml (86.62%) treatment, followed by M. anisopliae 10 11 conidia/ml (81.66%) and B. bassiana 10 10 conidia/ml (81.31%) treatments (F = 5.88; P = 0.00). A significant difference in population reduction percentage was observed at all the treatments at different level of entomopathogen concentrations for T. tabaci.
Effect of EPF on thrips population reduction percentage during 2020 in Phulkara onion variety is presented in Table 2. The thrips density recorded before treatment varied from 10 to 36 nymphs/plant. Large differences among treatments were observed in the start-up populations of the thrips. The rate of reduction in thrips population was significantly higher in insecticide-treated plots after 24 h of treatment application as compared to the EPF treatments. Application of B. bassiana caused a high reduction in the thrips population after 7th and 10th day than the other EPF treatments.To PO.PNGys of EPF applications, thrips population was significantly reduced among all the tested entomopathogenic fungi treatments. The highest population reduction was observed in B. bassiana 10 11 conidia/ml (F = 4.98; P = 0.00). Results showed that after 3 days of application,  Reduction of thrips population was recorded up to 35.48% by the application of B. bassiana at the concentration of 10 11 conidia/ml. Thrips population further reduced after 5 and 7 days of EPF application. B. bassiana 10 11 conidia/ml induced the population reduction (74.20%), followed by B. bassiana 10 10 conidia/ml (70.63%) and M. anisopliae 10 11 conidia/ml (67.79%) after 7 days of EPF application (F = 0.59; P = 0.77). Maximum reduction in thrips population was observed after 10 days of EPF application. For treated onion plots, B. bassiana at the concentration of 10 11 conidia/ml showed the highest thrips population reduction 79.19% which is at par with M. anisopliae 10 11 conidia/ml (77.16%) (F = 2.46; P = 0.06) after 10 days of EPF application. Population counts were undertaken before treatment application ranged between 69 and 135 thrips per 5 plants. Thrips densities reduced in both insecticide-and EPF-treated plots in comparison to untreated control plots during the trials (Table 2).

Onion thrips population reduction percentage on Swat 1 variety
The population reduction percentage after the application of EPF on Swat 1 variety during 2019 is presented in Table 3 and Fig. 1. After 3 days of EPF application, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (24.43%), followed by B. bassiana 10 10 conidia/ml (22.36%) treatments. Significant difference in population reduction percentage of onion thrips was observed after 5 days of spray. The highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (40.83%) followed by B. bassiana 10 10 conidia/ml (37.04%) and M. anisopliae 10 11 conidia/ml (36.45%) treatment (F = 7.91; P = 0.00) after 5 days of spray. Significant differences were observed among treatments after 7 days of application. After 7 days, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (76.50%), followed by B. bassiana 10 10 conidia/ml (65.22%) treatments. After 10 days of EPF application, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (84.59%), followed by M. anisopliae 10 11 conidia/ml (84.16%) treatment and B. bassiana 10 10 conidia/ml (82.90%) treatments (F = 6.38; P = 0.00). A significant difference in population reduction percentage was observed at all the treatments at different level of concentrations for T. tabaci. After EPF applications, thrips numbers were reduced in treated plots than the untreated control plots. However, significant differences were observed in the treatments applied at different concentration.
Significant differences (F = 21.68; P = 0.00) in corrected population reduction percentage of onion thrips were observed after 3 days of treatment application on Swat 1 variety ( Table 4). The highest population reduction percentage (89.46%) was observed in 3 days after spraying with bifenthrin. After 5 days, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (60.62%), followed by B. bassiana 10 10 conidia/ml (59.89 %) treatments. After 7 days, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (87.92%), followed by B. bassiana 10 10 conidia/ml (81.22%) and M. anisopliae 10 11 conidia/ml (78.70%) treatments (F = 3.62; P = 0.01). After 10 days of entomopathogenic application, the highest percent population reduction observed was induced by B. bassiana 10 11 conidia/ml (76.58%) followed by B. bassiana 10 10 conidia/ml (75.76%) treatment (F = 9.61; P = 0.04). A significant difference in population reduction percentage was observed for all the treatments at different level of doses for T. tabaci on Swat 1 onion variety. Thrips population before spray ranged between 65 and 170 thrips/5 plants. After 1 week of treatment applications, decline in thrips population density was more in EPF treatments than in insecticide-treated plots (Table  4). Average hourly temperature and RH measurements ranged from 23.2 to 31.3°C and 28 to 76%, respectively, during the study.

Onion thrips population reduction percentage on Virio 7 variety
A high population reduction percentage was recorded at high concentrations of EPF during 2019 (Table 5). Results revealed that the maximum population reductions were 23.74% and 40.97% after 3 and 5 days of B. bassiana 10 11 conidia/ml application, respectively. Further population reduction of thrips was observed after 7 days of spray. B. bassiana at the concentration of 10 11 conidia/ml treated onion plants showed the highest population reduction 71.91% which is at par with M. anisopliae 10 11 conidia/ml (67.14%) (F = 20.40; P = 0.00). Significant differences were observed in treatments after 7 days of application. After 10 days, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (86.00%), followed by M. anisopliae 10 11 conidia/ml (82.70%) and B. bassiana 10 10 conidia/ml (80.58%) treatments (F = 6.97; P = 0.00). A significant difference in population reduction percentage was observed at all the treatments at different level of doses for T. tabaci.
Significant difference in population reduction percentage of onion thrips was observed after 3 days of application on Virio 7 variety ( Table 6). The highest population reduction percentage (80.78%) observed was induced by application of bifenthrin (F = 7.47, P = 0.00) after 3 days of spray. After 5 days, the highest population reduction percentage observed was induced by B. bassiana 10 11 conidia/ml (49.93%) followed by M. anisopliae 10 11 conidia/ml (45.53%) treatment (F = 0.92; P = 0.52). Thrips generations showed peak populations after 5 to 6 weeks in control treatments. There were significant differences  observed in treatments after 5 days of application. After 7 days of application, the highest population reduction percentage was induced by B. bassiana 10 11 conidia/ml (68.65%) followed by M. anisopliae 10 11 conidia/ml (65.51%) treatment..

Discussion
Obtained results highlighted the prospective of EPF for controlling onion thrips. In particular, more than 80% thrips population reduction was recorded by B. bassiana and M. anisopliae application field trials. The EPF species used against T. tabaci varied in their efficacy to reduce pest's populations. B. bassiana concentration (1 × 10 11 conidia/ml) tested was more effective than any M. anisopliae treatments. The results are in agreement with Neves and Alves (2000). B. bassiana is an efficient alternative method for use in biocontrol against the onion thrips (Maniania et al. 2003). In this study, B. bassiana showed 86.62 ± 1.43 population reduction percentage after 10 days of treatment application which are similar to Ansari et al. (2008) who stated that Beauveria spp. were the most efficient, causing 54-84% onion thrips corrected population reduction percentage after 11 days of application. Low population reduction percentage by M. anisopliae application might be due to the more time required for conidial germination on insect body as compared with filtrate. Some other factors like viability of conidia, rate of germination, hyphae growth rate, and environmental factors such as temperature, humidity, and UV light could also affect spore production and virulence of fungal isolates on different insects (Molenaar 1984).
Results showed that EPF induce an immediate effect on thrips populations to obtain 2-3 thrips/onion leaf (Diaz-Montano et al. 2011) economic threshold levels in field conditions. However, a lot of variations in thrips counts were observed during the field trial. Environmental factors like rainfall had a significant effect on the population densities of thrips by washing or dislodging them from the plants. Results also showed that thrips populations were reduced at insecticidal-treated plots, which is in agreement with Ghelani et al. (2014) findings. Although EPF formulations were efficient in reducing the thrips numbers, they caused moderate population reduction as compared to insecticides. The highest efficacy of B. bassiana against thrips is in accordance with Boopathi et al. (2011) who stated that among different EPF B. bassiana gave better results in reducing the population of thrips. For maximum benefits, therefore, this approach should be integrated with other thrips management strategies, such as the use of resistant varieties, polythene mulches, proper sanitation, sticky traps, and botanicides (Maniania et al. 2003).
T. tabaci population on vegetables may be controlled well with entomopathogens at concentration of 1 × 10 11 conidia/ha in field crops. But there is one limitation in the use of these EPF that they are relatively less persistent (Inglis et al. 1997). Results showed that these EPF were able to persist for 10 days under the field conditions. The present results showed also that B. bassiana, M. anisopliae, and bifenthrin had great potentials for use as important component in developing integrated insect pest management packages against thrips on onion (Nyasani et al. 2015). Further studies are required to standardize concentrations of these EPF at different stages of onion thrips infestations under field conditions.

Conclusion
Entomopathogenic fungi B. bassiana and M. anisopliae significantly reduced thrips population build up in onion crop after 7-10 days post applications. The fungal species used against T. tabaci varied in their ability to reduce its populations. B. bassiana as EPF was much against the onion thrips than the M. anisopliae. Use of EPF to control thrips populations could reduce the application of