Background

Coconut is commonly known as “Kalpavruksha” in Sanskrit language, and to describe its importance, almost all the parts of the palm are economically important. Coconut oil finds extensive use in food and industrial sectors because of its unique characteristics. The saturated fats in coconut oil have antimicrobial properties. Coconut oil also helps in the absorption of vitamins, minerals and amino acids. In India, coconut is grown in an area of 1.89 million hectares covering 18 states/union territories; the southern states including Kerala, Tamil Nadu, Karnataka and Andhra Pradesh account for 89.7% of total area. The productivity of coconut in these four states vary between 5193 (Karnataka) to 13,771 nuts/ha (Tamil Nadu). Biotic stresses are the major constraints in coconut cultivation. Rhinoceros beetle is a serious pest in coconut causing severe economic losses (Chandran et al. 2017). Rhinoceros beetle causes economic loss of 299.3 million USD in coconut plantation, (Abidin et al. 2014) in tropical regions of the world. They are endemic in coconut-growing regions of South and South East Asia (Bedford 1980) and it has also been reported in Guan and Saipan (Moore 2007). Integrated pest management (IPM) is considered to be best management practice to manage rhinoceros beetle. Biocontrol agents are found to have significant role in IPM in the era of organic agriculture. Management of rhinoceros beetle by entomopathogenic bacteria (Bacillus thuringiensis) (Bt) and entomopathogenic fungi (EPF) (Metarhizium robertsii (Metchnikoff) Sorokin. and Metarhizium majus Johnst., Bisch., Rehner and Humber) will be of great significance looking upon advantages of adoption, availability, proven virulence and environment friendliness. Commercial products containing Bt acting against coleopteran pests are rare; however, certain Bt strains toxic to beetles have been characterized (Yamaguchi et al. 2008). The Bt isolate NBAIR-BTAN4 (KY451835.1) was originally isolated from Andaman and was found to be toxic to the beetles in initial laboratory studies. Among EPF, Metarhizium spp. are widely used against coleopteran pests. M. anisopliae is the most studied; however, studies on M. robertsii and M. majus are very limited, and hence, they were included in the present study. Also, very limited data are available on combined use of Bt and EPF in pest management and very few studies have shown that Bt and EPF are compatible (Bahmani et al. 2020). Hence, the present study was carried out to study the biocontrol potential of bio-formulations of Bt and EPF both individually and in combination against Oryctes rhinoceros L. under both potted and field conditions.

Methods

Isolation and mass multiplication of Bacillus thuringiensis strain NBAIR-BTAN4

Bacillus thuringiensis strain NBAIR-BTAN4 (KY451835) was isolated from soils obtained from Andaman Islands as per protocols described by Travers et al. (1987) at ICAR-NBAIR, Bengaluru. The culture was stored and maintained on Luria–Bertani agar medium (HiMedia), and T3 sporulation medium was used for mass multiplication. The BTAN4 strain was inoculated to T3 medium and grown at 37 °C for 72–96 h @ 150 rpm. After complete sporulation, the broth was centrifuged at 8000 rpm for 10 min and the pellet obtained was dissolved in half of the supernatant. This solution was further mixed with surfactants, dispersants and UV protectants to get a liquid-based formulation (Varshney et al. 2020).

Isolation and mass multiplication of Metarhizium robertsii and M. majus

Two promising indigenous EPF, viz. M. robertsii (ArMz6W) and M. majus (VjMz1W), were originally isolated as per the protocol described by Velavan et al. (2021). These efficient strains of M. robertsii (KU983797) and M. majus (KU983771) were chosen for field studies based on earlier research (Velavan et al. 2017). The selected EPF were grown on potato dextrose agar (PDA) media and incubated at 25 ± 1 °C for 10 days to confirm their viability before being used (Bischoff et al. 2009). For mass multiplication, the strains of EPF were inoculated to the Sabauraud’s broth medium and kept for shaking at 150 rpm for 7–8 days at 27 °C. Initial count (colony-forming unit) of M. robertsii and M. majus in talc formulation was calculated and maintained as described by Easwaramoorthy (2002). The fully grown fungal culture was mixed with talc at 1:2 ratio and kept for drying to obtain up to 12% moisture content. The moisture of the talc was analyzed using moisture analyzer (Denver). The talc-based formulation was mixed with water @ 5 g/l for field trials. For each palm, in both the methods of application, individual and combination, the total volume of formulation was kept constant as one liter.

Collection, culturing and maintenance of Oryctes rhinoceros larvae

For field studies, rhinoceros beetle larvae were collected from a single coconut farm situated at Thanjavur District, Tamil Nadu. Collected rhinoceros larvae were sorted based on size and body mass, and larvae were cultured and mass-multiplied in large containers having organically decomposed FYM, decayed coconut wood and root debris for at least one week before used in the experiment for acclimatization.

Soil drench application of bio-formulations in the field

Soil drench experiments were conducted for three bio-formulations (NBAIR-BTAN4-liquid formulation and powder-based formulations of M. robertsii and M. majus) in seven different treatments including control comprising of T1: NBAIR-BTAN4 (liquid formulation), T2: M. robertsii (powder-based formulation), T3: M. majus (powder-based formulation), T4: NBAIR-BTAN4 + M. robertsii (liquid + powder), T5: NBAIR-BTAN4 + M. majus (liquid + powder), T6: NBAIR-BTAN4 + M. robertsii + M. majus (liquid + powder + powder), and T7: control (water + binding solution). Coconut orchards having one- to two-year-old plants were chosen for soil drench experiment. Optimum drainage and moisture were maintained during entire period of experiment. For each plant pit, 5 larvae of similar size and body weight were released, and similar experiment conditions maintained for all the treatments. Five replications (five seedlings) were maintained for each treatment. Twenty-five larvae were used for feeding in soil drench experiments per treatment in each replication. Larval mortality was recorded during 1st to 5th week in soil drench method. Dead larva was gently collected, washed with water, surface-sterilized with sodium hypochloride (0.1%) and 70% alcohol, further washed with sterile water and incubated in Petri plate for spore-forming EPF or bacterial growth to prove the Koch postulates.

Topical spray of entomopathogenic fungi and bacteria bio-formulations against rhinoceros beetles in field

For the topical spray experiment, treatments were similar to soil drench method. Coconut orchard having one- to two-year-old plants was chosen for spaying. For each coconut seedling, five larvae of similar size and body mass were released at different young whorls, after spraying of bio-formulations. For each treatment, five replications were maintained, per each replication five seedlings were taken, and hence, total larvae used per treatment were 25 larvae for one treatment. Mortality of larvae was recorded every week; dead larvae were brought to laboratory to test the Koch postulates.

Statistical analysis

Data recorded were analyzed by three-factor analysis using SAS 9.3 version considering three factors, viz. microbial agents, method of application and duration by post hoc analysis. Significance of mortality of rhinoceros beetles was calculated using Tukey HSD.

Results

Liquid-based formulation of the Bt NBAIR-BTAN4 and powder-based formulations of Metarhizium robertsii and M. majus were tested by soil drench application and topical spray method against rhinoceros beetles in coconut orchards.

Effect of soil drench application of bio-formulations of entomopathogenic bacteria and fungi on mortality of rhinoceros beetle larvae in coconut orchards

Application of Bt strain NBAIR-BTAN4 liquid formulation individually by soil drench method showed 5.6% mortality in the first week, 16% in 3rd week and 24.8% in 5th week. Application of M. robertsii powder formulation individually by soil drench method showed 1.6, 12 and 23.2% mortality in 1st, 3rd and 5th week, respectively. The application of M. majus showed 3.2, 16 and 24% mortality in 1st, 3rd and 5th week, respectively. Combined application of NBAIR-BTAN4 liquid formulation + M. robertsii incited 8.8, 24 and 40.8% mortality in 1st, 3rd and 5th week, respectively. In the other combined application of NBAIR-BTAN4 + M. majus, % mortality observed was 7.2, 25.6 and 44.8 during 1st, 3rd and 5th week, respectively. However, when all three biocontrol agents were applied in combination, the larval mortality recorded was 12, 32 and 52.8% in 1st, 3rd and 5th week, respectively. In control, mortality recorded was 0, 2.4 and 4.8% in 1st, 3rd and 5th week, respectively. Hence, the results indicated that combined soil drench application of all three biocontrol agents was the most effective with more than 50% larval deaths in 5th week.

Effect of topical spray method on larval mortality

Application of NBAIR-BTAN4 liquid formulation individually by topical spray method caused 1.6, 12.8 and 24% mortality in 1st, 3rd and 5th week, respectively. Application of M. robertsii powder formulation individually by topical spray incited 1.6, 13.6 and 23.2% mortality in 1st, 3rd and 5th week, respectively. Application of M. majus showed 2.4, 12.2 and 20.8% larval death in 1st, 3rd and 5th week, respectively. Combined application in topical spray was also found to be effective as was observed in soil application. Combination of NBAIR-BTAN4 + M. robertsii recorded 5.6, 24.8 and 44% larval death in 1st, 3rd and 5th week, respectively. When NBAIR-BTAN4 was combined with M. majus, the larval mortality recorded was 4, 20.8 and 40.8% in 1st, 3rd and 5th week, respectively. Again, the combination of all three bio-agents was highly effective with recorded mortality of 9.6, 32.8 and 57.6% in 1st, 3rd and 5th week, respectively. By analyzing mortality data from all the treatments in topical spray, combination of NBAIR- BTAN4 + M. robertsii + M. majus showed maximum mortality of 57.6% by 5th week and could indicate a synergistic effect.

Results of means of the three-factor analysis (microbials, method of application and duration) for topical showed that T6 (combination of all three bio-agents) gave the highest mortality of 31.52 (18.61) % at 5 weeks which was significantly different to all other treatments (Table 1A). Combination of Bt with Metarhizium (T4 and T5) was on par recording 24 to 25% mortality. Individual treatments of NBAIR-BTAN4-liquid formulation (T1), powder-based formulation (PBF) of M. robertsii (T2) and PBF of M. majus (T3) did not vary significantly to each other and recorded 12 to 15% mortality (Table 1A). However, all treatments with biocontrol agents (T1 to T6) recorded mortality that was significantly higher than control. Three-factor analyses for soil application method gave similar results (Table 1B). The results indicated that combining Bt with Metarhizium was effective in controlling the coconut rhinoceros beetle larvae.

Table 1 (A) Mortality of Oryctes rhinoceros in treatments as topical spray. (B) Mortality of Oryctes rhinoceros in treatments as soil drench method
Full size table

In order to confirm the above observations, two-way analysis of means of mortality recorded in the two treatment methods (soil application and topical spray) with duration (weeks) was carried out. The results again showed that T6 (combination of Bt with two Metarhizium spp.) gave maximum a mortality of 32.4 (19.23) %, which was significantly high to all other treatments (Table 2). Treatments of Bt combined with either M. robertsii or M. majus (T4 and T5) recorded mortality of 24 to 25% and were on par, but were significantly high to individual treatments (T1, T2 and T3). Mortality of O. rhinoceros larvae with Bt alone (T2) was significantly higher than individual treatments of Metarhizium. All biocontrol interventions (T1 to T6) gave significantly higher mortality when compared to control. The results establish that combination of Bt with Metarhizium can be successfully used in the biological control of O. rhinoceros.

Table 2 Combined mortality of Oryctes rhinoceros in field
Full size table

Discussion

Coconut is one of the most important plantation crops in South and South East Asia. Biotic stresses are known to cause significant economic loss to coconut orchards. The present emphasis is on the reduction in the use of chemical pesticides. Studies on the effect of combinations of biological control agents in pest control are very limited, especially on interactions between Bt and EPF (Mwamburi et al. 2009). Hence, in the present study formulations of Bt and Metarhizium spp. were selected and evaluated against O. rhinoceros individually as well as in combination based on their proven efficacy against coleopteran pests. This study clearly indicates that there was improved pest control with possible synergistic interaction between Bt and Metarhizium as significant differences in mortality of larvae were observed. Individual treatments did record larval mortality, but there were twofold increases when Bt and Metarhizium were combined. The two-way analysis for topical spray and soil drench showed that individual treatments did not vary significantly, but combined treatments gave significantly high mortality rates. Mixed infections lead to diverse interactions among natural enemies (Pozo et al. 2021) and microbial consortia can provide diverse modes of action, thereby achieving better pest or disease control than single microorganisms (Sarma et al. 2015). In studies targeting other insect pests, synergistic interactions have been observed from certain combinations of B. bassiana and Bt (Wraight and Ramos 2005), independent (Lewis and Bing 1991) or antagonistic (Ma et al. 2008). Combining Bt with the two Metarhizium spp. gave significantly higher mortality rates when compared to all other treatments. Application of mixed formulations of biocontrol agents shows better pest control (Oestergaard et al. 2006) or may be used as a pesticide resistance management strategy (Corbel et al. 2002). It was carried out comprehensive three-way and two-way analyses of the present results and all results indicated that combination of Bt with Metarhizium spp. was highly efficient.

The Bt (NBAIR-BTAN4) expresses Cry1IA and Cry2Ab (as per genome analysis, results not shown) which could be playing a role in coleopteran toxicity. The two strains of Metarhizium, viz. M. robertsii and M. majus, were previously studied against banana stem weevil and found effective (Velavan et al. 2022). Presently Bt is mainly utilized for biological control of lepidopteran pests and very limited reports are available on coleopteran toxic Bt (Palma et al. 2014). In the present study, we could isolate coleopteran toxic Bt against O. rhinoceros, and when applied in combination with Metarhizium spp., there was an increased larval death indicating synergistic effect.

Conclusion

In the present study, biocontrol potential of coleopteran toxic Bt, Metarhizium robertsii and M. majus individually and in combination against rhinoceros beetle was demonstrated. The results indicated that combined application of Bt with Metarhizium spp. significantly increased the mortality rate of larvae. It was also observed that soil drench application was a more promising strategy than the topical spray method. Thus, synergistic effect of EPF and bacterial consortium in controlling O. rhinoceros in coconut orchards was confirmed by this study. Development of efficient formulations using both Bt and fungi can be an effective and long-term solution for rhinoceros beetle management in coconut orchards. Therefore, in bio-intensive management of rhinoceros beetle, application of Bt and M. robertsii and M. majus consortium is recommended.