Compatibility of Entomopathogenic Fungi with Neonicotinoids for the Management of Brown Plant Hopper of Rice, Nilaparvata lugens stal. (Delphacidae: Hemiptera)

Rice is the staple food of Indians and it is cultivated in an area of 43.39 million hectares in India with an annual production of 104.32 million tonnes and with 2.4 tonnes per hectare average yield (MOSPI, 2018). Insect pests are one of the major problems in rice production, wherein more than 100 species of insects attack and damage the crop. Among all, brown planthopper (BPH), Nilaparvatalugens (Stal) (Hemiptera: Delphacidae) is one of the important sucking insects, which can cause appreciable damage by sap sucking and transmitting the viruses such as ragged stunt and grassy stunt (Jena et al., 2006). Due to infestation plants turn yellow and dry up rapidly. At early infestation, round and yellow patches appear, which soon turn brownish due to drying up of the plants which is called as ‘hopper burn’ and could result in yield loss ranging from 10-75% (Park et al., 2008). Insecticides are the major means of managing the BPH. However, continuous use of insecticides causes health hazards and environmental pollution, besides development of insecticide resistance among the populations of BPH (Jhansi Lakshmi et al., 2010). Hence, it is very difficult to manage the insects by insecticides alone, but on the other hand for this for effective management of insects, combined use of chemical pesticides with bio pesticides such as bacteria, fungi and viruses would bring down the incidence of BPH effectively. Keeping this view in mind, the present investigation has been planned with combined use of fungal formulations and commonly used neonicotinoid insecticides like thiamethoxam 25 SG and dinotefuran 20 SG for managing BPH.

Experiment was carried out to evaluate neonicotinoid insecticides and entomopathogenic fungi (Beauveria bassiana, Metarhiziumanisopliae and Lecanicilliumlecanii) both in the laboratory and glasshouse at the Indian Institute of Rice Research, Hyderabad.
 
Inhibitory studies in the laboratory (Poison food technique)
 
Standard poison food technique was followed to assess compatibility of the entomopathogenic fungi with various insecticides (Moorhouse et al., 1992). Desired quantity of insecticides (i.e. thiamethoxam 25 SG and Dinotefuran 20 SG) based on field application rate (recommended concentration, half recommended concentration and 1.5 fold of recommended concentration) were added to the PDA medium (200 ml), subsequently it was autoclaved at 121^oC (15 Psi) for 15-20 minutes in the conical flask before solidification (at 48^oC) to get desired concentration and later they were mixed thoroughly. The medium was then poured equally into the petriplates. Each treatment was replicated four times. Small disc (5 mm dia.) of young fungal mycelium was cut with sterile cork borer and placed aseptically in the centre of plate containing the poisoned medium. Petri plates were incubated at 27±1^oC, 80±5% relative humidity. Suitable check without poison was kept for comparison under the same conditions. Diameter of the fungal colony was measured at 2, 4, 6, 8 and 10 days after inoculation (DAI) and compared with standard check. The data were expressed as percentage growth inhibition of fungi by insecticide treated PDA (Hokkanen and Kotiluoto, 1992) and calculated by the following formula
   
Where
X, Y, Z stand for percentage growth inhibition, radial growth of the fungus in untreated check and radial growth of the fungus in poisoned medium, respectively. The pesticides were further classified in evaluation categories of 1- 4 scoring index. 1 = harmless (<20% reduction), 2 = slightly harmful (20-35% reduction), 3 = moderately harmful (35-50% reduction), 4 = harmful (>50% reduction) in toxicity tests in vitro according to Hassan’s classification scheme (Hassan, 1989).
 
Inhibitory studies under glasshouse conditions 
 
The recommended dose of neonicotinoid insecticides were mixed with the recommended dose of effective fungal commercial formulations (5g/l) and sprayed on to the rice plants. Individuals of BPH were released on the sprayed plants and mortality was recorded at 24 hrs interval up to five days after spraying. The results were compared with those of neonicotinoid insecticides and fungal pathogens alone by recording data on % mortality.

Effect of Thiamethoxam 25 SG on growth of entomopatho- -genic fungi

Thiamethoxam showed moderately harmful effect on entomopathogenic fungi where radial growth of fungi was affected with increase in insecticide concentration (Table 1). In the present finding, thiamethoxam caused 19.19 to 39.15% reduction over control. Among the three fungi, L. lecanii was the most affected recording highest reduction of radial growth (39.15%) at 1.5 RC, 24.87% and 35.45% reduction at 0.5 RC and RC, respectively. According to Hassan’s (1989) classification, thiamethoxam was slightly harmful to L. lecanii at 0.5 RC and moderately harmful to L. lecanii at both RC and 1.5 RC. With regard to M. anisopliae, Thiamethoxamwas slightly harmful at all three doses i.e. 0.5 RC, RC and 1.5 RC have significantly inhibited growth of M. anisopliae to an extent of 23.26 to 34.50% over control. Similar results were found with B. bassiana where significant inhibition of the radial growth was observed recording 19.19, 27.78 and 36.36% reduction over control at 0.5 RC, RC and 1.5 RC, respectively indicating thiamethoxam being harmless at 0.5 RC, slightly harmful at RC and moderately harmful at 1.5 RC. Baraiya (2003) also observed B. bassiana as highly compatible with thiamethoxam (0.005%) and imidacloprid (0.005%) recording less than 25% growth inhibition.
 

 
Effect of Dinotefuran 20 SG on growth of entomopatho- -genic fungi
 
Mycelial growth of the fungi was inhibited concomitantly with increase in insecticide concentration. The vegetative growth of fungi was nominally affected by dinotefuran recording 17.99% - 34.72% reduction over control. Among the three fungi, M. anisopliae was most affected with 34.72% reduction in growth over control at 1.5 RC. Present findings (Table 2) clearly indicated that dinotefuran was harmless at 0.5 RC (18.87% reduction) and slightly harmful at RC (24.15% reduction) and 1.5 RC (34.72% reduction) to M. anisopliae. Similarly, dinotefuran was found slightly harmful to B. bassiana at all concentrations by recording 26.32, 27.27 and 27.75% reduction in the radial growth of the fungus over control at 0.5 RC, RC and 1.5 RC, respectively. Among three fungi, L. lecanii was least affected by dinotefuran which inhibited the radial growth of fungus up to 22.75% over control even at 1.5 RC. It indicates its safety at 0.5 RC (17.99% reduction) and RC (19.58% reduction) and slightly harmful effect at 1.5 RC (22.75%). This is further confirming the results of Khan et al., (2012) who stated that neonicotinoids (acetameprid, imidacloprid and thiamethoxam) were compatible and safer to B. bassiana and M. anisopliae.
 
 
       
The present results indicated that at recommended concentration of dinotefuran 20 SG, it was recorded 27.27% inhibited the growth of B. bassiana and 24.15% inhibited the growth of M. anisopliae and 19.58% inhibited the growth L. lecanii. Thiamethoxam 25 SG was recorded 27.78% inhibited the growth of B. bassiana, 30.23% inhibited the growth of M. anisopliae and 35.45% inhibited the growth of L. lecanii.
 
Effect of thiamethoxam 25 SG + entomopathogenic fungi on BPH
 
Results presented in the Table 3 indicated that thiamethoxam alone could cause 65.00% mortality of BPH under glasshouse conditions. Mortality by thiamethoxam was enhanced when combined with entomopathogenic fungi including B. bassiana, M. anisopliae and L. lecanii. It was clear from the studies that first two days after spraying, there was no significant difference in the mortality of BPH between thiamethoxam alone and thiamethoxam in combination with entomopathogenic fungi. However, from third day on wards, initiation of mycosis occurred and mortality was observed in entomopathogenic fungi combined treatments and later mycosis was increased. At three days after spraying, fungal mycosis was observed in thiamethoxam + entomopathogenic fungi combination treatments i.e. thiamethoxam + B. bassiana recording 76.25% mortality, followed by thiamethoxam + M. anisopliae recording 73.75% that were on par with each other and significantly superior over other treatments i.e. thiamethoxam and thiamethoxam + L. lecanii that have recorded 60.00% mortality. At 4 DAS, the % mortality increased in combination treatments, where thiamethoxam + B. bassiana resulted in maximum % mortality of 83.75%, thiamethoxam + M. anisopliae recorded 78.75% mortality which were on par with each other followed by thiamethoxam + L. lecanii recording 66.25% mortality that remained on par with thiamethoxam + M. anisopliae (78.75%). Thiamethoxam alone has recorded low 61.25% mortality, which was on par with thiamethoxam + L. lecanii. However, the treatments were significantly superior over control. Similar trend was observed at 5 DAS where thiamethoxam + B. bassiana, thiamethoxam + M. anisopliae, thiamethoxam + L. lecaniiand thiamethoxam alone which resulted in 86.25, 81.25, 68.75 and 65.00 % mortality respectively and were significantly superior over control (2.50% mortality), B. bassianawith 8.75% mortality, M. anisopliaewith 10.00% mortality and L. lecanii with 12.50% mortality (Reddy et al., 2017).
 

       
The present results indicated that thiamethoxam at recommended dose was slightly harmful with 20-35% reduction in fungal growth (Table 4), when combined with fungi for management of BPH under glasshouse conditions, it could result in additive effect.
 

 
Effect of dinotefuran 20 SG with entomopathogenic fungi on BPH
 
Dinotefuran is a neonicotinyl insecticide belonging to neonicotinoid group and was evaluated for its efficacy against BPH alone as well as in combination with entomopathogenic fungi under glasshouse conditions. The findings indicated that (Table 4) dinotefuran alone caused complete mortality of BPH at three days after spraying. Dinotefuran was proved safe for entomopathogenic fungi like B. bassiana, M. anisopliae, L. lecanii without affecting growth and sporulation under laboratory conditions (Table 2). Even though complete mortality of BPH was observed with dinotefuran alone, mycosis was observed in combination treatments which means dinotefuran could not adversely affect the growth and sporulation of the fungi. Results indicated that there was no significant difference in mortality among dinotefuran alone and combination with entomopathogenic fungi. At 4 days after spraying and 5 days after spraying there was 98.75% mortality with dinotefuran alone and in combination with entomopathogenic fungi. All these treatments were on par with each other and significantly superior over control which has recorded 11.25% mortality at 5 days after spraying.
       
Present studies revealed that there is no need of mixing of any entomopathogenic fungi with dinotefuran as the insecticide itself could cause maximum (98.75%) mortality of BPH. However, if BPH develop resistance to dinotefuran in future, it can be used in combination with entomopathogenic fungi like L. lecanii, B. bassiana and M. anisopliae.
 

The first author expresses his gratitude to Department of Science and Technology, Ministry of Science and Technology, Government of India, for providing INSPIRE fellowship. The authors thank the Director, Indian Institute of Rice Research for providing facilities to take up the present investigation.