INTRODUCTION

Cassava is the third largest source of carbohydrates for human food in the world after rice and maize (Fauquet and Fargette, 1990). Worldwide, 600 million people depend on cassava as their primary staple food (Anonymous, 2015). In India, cassava is mainly grown in three southern states–Tamil Nadu, Kerala and Andhra Pradesh. In addition, it is sparsely cultivated in several other parts of India with a total production of 5.04 mt (Anonymous, 2020). Cassava roots are rich in carbohydrates but poor in vitamins and protein, while cassava leaves are an excellent source of protein and vitamins (Montagnac et al., 2009).

Whiteflies (Hemiptera: Aleyrodidae) are sap-sucking insects and are frequently found in thick crowds on the underside of leaves. Whiteflies are globally significant agricultural pests and virus vectors causing direct and indirect damage to crops with approximate losses totalling billions of dollars (US) annually worldwide (Abd-Rabou and Simmons, 2010). The whitefly, Bemisia tabaci is one of the most economically and agriculturally important insect pests worldwide. It is a well–known vector of cassava mosaic disease (CMD). CMD affected cassava plants produce few or no tubers, depending on the severity of the disease and the age of the plant at the time of infection (Alabi et al., 2011).Another important whitefly infesting cassava is spiralling white fly, Aleurodicus dispersus. It is a polyphagous whitefly species of tropical or Neotropical origin (Russell, 1965, Martin, 1987) and in India it was first reported by Palaniswami et al. (1995). 

Associations between insects and endosymbionts are quite common in nature. It has been estimated that at least 15−20% of all insect species live in symbiotic relationships with bacteria (Douglas, 1998, Gosalbes et al., 2010). As a phloem feeding homopteran, whiteflies harbour various endosymbionts for its nutritional requirement and functional fit. Endosymbionts can be categorized as either primary or secondary endosymbionts according to their physiological roles. The primary endosymbiont Portiera aleyrodidarum is harbored within bacteriocytes and supplements B. tabaci with essential amino acids for growth and development (Santos–Garcia et al., 2012). Although secondary endosymbionts are not necessary for host survival, they may play important roles in their host’s physiology, ecology, and evolution (Zchori–Fein and Brown, 2002, Chiel et al., 2007). Important genera of secondary endosymbiotic bacteria associated with whiteflies include, Hamiltonella (Zchori–Fein and Brown, 2002), Rickettsia (Gottlieb et al., 2006), Wolbachia (Skaljac et al., 2010), Arsenophonus (Baumann et al., 2004), Cardinium (Zchori–Fein and Perlman, 2004) and Fritschea (Everett et al., 2005). All these bacteria possess the ability to manipulate the physiological characteristics of their hosts. Specifically, Wolbachia, Arsenophonus, Cardinium and Rickettsia can manipulate host reproduction; Hamiltonella can induce virus resistance in pea aphid (Oliver et al., 2006) and Rickettsia increases thermotolerance (Brumin et al., 2011). They have diverse relationships with the host from being harmful/lethal (parasitism/ pathogenicity) to beneficial (mutualism or symbiosis) (Kikuchi, 2009).

Insecticides are commonly used to manage whiteflies in cassava, but frequent use can cause these pests to become resistant to insecticides (Naveen et al., 2017; Patra and Hath, 2022). According Kikuchi et al. (2012), endosymbionts present in whiteflies can provide resistance to the host. Among the total endosymbionts present in the insect host, roughly about 1% only are culturable now and to detect others we need costly NGS (Next–Generation Sequencing) platform now.  Diagnostic PCR is a unique technique in the sense that they can bypass NGS to identify unculturable bacterial endosymbionts by using specific bacterial primers (Subramanian et al., 2019). The present study focuses on the identification of specific whitefly endosymbionts in cassava using diagnostic PCR and Sanger sequencing and detection of resistant bacterial endosymbionts in whiteflies against major insecticides applied to control whitefly.

MATERIALS AND METHODS

Large (Aleurodicus dispersus)and small (Bemisia tabaci) whiteflies were collected from different cassava fields of Thiruvananthapuram, Kerala (8.5241° N, 76.9366° E) during March–December 2021. Selected whiteflies were transferred to sterile Eppendorf tube (2ml) containing 0.01 % streptomycin as surface sterilizer. After one–minute whiteflies were taken from antibiotic containing tube and then transferred to other tube containing sterile distilled water. Washing procedure was repeated up to 2 times. After surface sterilization, whiteflies were transferred to tube containing 2ml nutrient broth. White flies were crushed with micro pestle and incubated at room temperature for overnight in rotating shaker. After one overnight incubation, one ml of culture from each sample were taken and serial dilution was performed. Initial dilution was prepared by transferring one ml of sample into 9 ml of sterile distilled water. This gave 10^–1 dilution. With a sterile pipette, one ml of diluted sample was transferred from 10^-1 to the next tube containing 9 ml sterile distilled water. This will give 10^-2 dilution. The above steps were repeated until 10^-6 dilution was obtained.

From the field study on the effectiveness of insecticides on whitefly population (Table 1), one commonly used systemic and contact insecticides (Imidacloprid and Chlorpyrifos respectively) were used in the subsequent study. Nutrient agar media was prepared by dissolving 28 grams of nutrient agar in 1000 ml of distilled water. The medium was autoclaved at 121°C temperature and 15 lbs pressure for 20 minutes and under ear bearable temperature. Insecticides Imidacloprid 17.80% SL and Chlorpyrifos 20% EC, 0.3 ml l^-1 and 2 ml l^-1 were respectively added.Each sample was plated on labelled nutrient agar media. After sufficient incubation, the plates were observed for bacterial colonies. Bacterial culture took minimum 24 hours for growth. Different colonies were streaked separately into new agar plates. The pure cultures were maintained in nutrient agar slants. Colony morphology of bacteria of each plate were observed under stereo microscope. The gram staining was performed using Hi–Media kit (Hi–Media Laboratories Pvt. Ltd., India) according to the manufacture’s protocol. The results were observed using Leica DMLB compound microscope (100x).

Table 1: Field study on the effectiveness of insecticides on cassava whitefly population

For molecular identification of promising–sub cultured bacteria, 1.5 ml of the overnight grown bacterial cultures were taken in centrifuge tubes, centrifuged the contents at 12000 rpm for 10 minutes. Discarded the supernatant. Suspended the pellets in 400 µl TE buffer. Vortexed, added 50 µl 10% SDS, mixed the contents by using pipette. Added 500 µl phenol: chloroform: iso–amyl alcohol (25:24:1), mixed well. Centrifuged the contents at 12000 rpm for 10 minutes and carefully collected supernatant into a fresh tube. Added 500 µl chloroform, mixed well and centrifuged at 12000 rpm for 10 minutes. DNA is precipitated after adding 25 µl of 5M NaCl and 1 ml of 95% Ethanol and incubated overnight at -20°C. Next day, centrifuged for 10 minutes at 12000 rpm, discarded the supernatant and washed pellets with 70% ethanol. Centrifuged the contents for 10 minutes at 12000 rpm. Air dried the pellets at 37°C for 30 minutes, dissolved in water (30 µl) and kept in refrigerator at –20°C for storage.

Agarose gel electrophoresis was performed based on the method described by Sambrook et al. (1989) to check the quality of DNA. Also, the purity of DNA was checked using Nano Drop spectrophotometer (model–NanoDrop–1000, Thermo Scientific^TM). PCR was done using 25 µl reaction mixture which contains, Thermo–scientific master mix– 12.5µl, distilled PCR water–9.5 µl, forward primer (16 S F)–0.5 µl, reverse primer (16S R)–0.5 µl and DNA template- 2 µl. Conditions for PCR were set as following: initial denaturation –92°C for 2 min10 sec, 35 cycles of denaturation 94°C for 1 min 10 sec, annealing temperature of primer 49°C for 30 sec, extension 72°C for 2 min, final extension 72°C for 10 min and holding temperature 4°C. PCR products were gel–purified and sent for sequencing. Sequencing was carried out in an automated sequencer (Anonymous) using universal primers in both directions. The nucleotide sequence obtained was processed to remove low quality reads and transformed into consensus sequences with Geneious Pro software version 5.6. The resulted high–quality sequences were analyzed with BLASTn (Anonymous; http://www.ncbi.nlm.nih.gov) to confirm the authenticity of the isolate and the sequences obtained were submitted to NCBI database.

For diagnostic PCR for the presence of bacterial endosymbionts in whitefly samples, at first DNA were isolated from whiteflies using DNeasy blood and tissue kit (Qiagen®), followed manufacturer’s protocol and obtained good quality DNA. DNA of whitefly populations were diagnosed for the presence of different bacterial endosymbionts Portiera, Wolbachia, Rickettsia, Arsenophonus and Cardinium. Specific bacterial primers were used for amplification of 16S rRNA bacterial gene (Subramanian et al., 2019). For each bacterial endosymbiont, PCR Master mix RTU 12.5µl (EmeraldAmp Max PCR Mix), forward and reverse primers (1 µl each), DNA template (5 µl) and the final volume of 25 µl were prepared with nuclease free water. PCR reaction include: denaturation at 94°C for 30 seconds. Annealing was carried out at different temperatures specific for each bacterial endosymbionts (Portiera 58°C, Wolbachia 52°C, Arsenophonus 52°C, Rickettsia 55°C, Cardinium 50°C) for 30 seconds. Extension was carried out at 72°C for 40 seconds with the final extension for 5 minutes at same temperature. The total number of cycles for PCR reaction was 45. Both positive and negative controls were used for each reaction. The plasmids containing 16S rRNA gene of different bacterial endosymbiont were used as positive controls while the reaction without any DNA were used as negative control. The PCR products were then checked on 1.8% agarose gel and the PCR products for different bacteria exhibited bands of different size.

RESULTS AND DISCUSSION

3.1.  Field study on the effectiveness of insecticides on whitefly population

The study conducted using most commonly used insecticides against cassava whiteflies at their recommended concentrations. The observations were taken up to 14 days after treatment and it was observed that the percent reduction in number of insects visiting cassava leaves were maximum in case of imidacloprid 17.8 SL @ 0.3 ml l^–1 and minimum in case of chlorpyrifos 50 EC @ 1.0 ml l^–1 . These two were selected for the study on growth inhibition of whitefly endosymbionts.

3.2.  Isolation of bacteria from whiteflies

From cassava whiteflies (large and small), endosymbiotic bacteria were isolated after treating nutrient agar media with selected insecticides at required concentrations. Based on colony morphology, distinct bacterial colonies were sub–cultured and selected (Table 2) for further molecular identification procedures. Similarly, detailed works on bacterial colony morphology were carried out earlier by Qamer et al. (2003) and Sousa et al. (2013).

Table 2: Colony morphology of endosymbiotic bacteria

3.3.  Molecular identification of bacteria

The quantity of DNA from different samples varied from 517 ng µl^-1 to 4133 ng µl^-1 and the A260/A280 ratio of DNA samples (quality) ranged from 1.76 to 2.26 indicating sufficient purity of isolated DNA for further PCR amplification. PCR analysis was carried out for bacteria using 16S rRNA primer, with an annealing temperature of 49°C and yielded fragments at 1500 bp (Figure 1).

Based on sequencing report the bacteria were identified using NCBI–BLAST. The endosymbiotic bacteria identified from control population of B. tabaci was Clostridium senegalense and from those treated with Chlorpyrifos 20% EC–2 ml l^-1 was Providencia sp. Acinetobacter sp. was identified from control populations of A. dispersus, whereas, Paenibacillus alvei was isolated from Imidacloprid 17.80% SL–0.3 ml l^-1 treated ones and Chlorpyrifos 20% EC–2 ml l^-1 treated ones. The % identity with reference samples for these bacteria were 96.09%, 94.01%, 93.66% and 92.32% respectively. The sequences were submitted to NCBI and the accession numbers obtained were OP303253, OP303256, OP303254 and OP295124 (Table 3).

Figure 1: PCR analysis of bacterial isolates using 16S primers (Lane 1: 1Kb plus ladder; 2 and 4–7: bacterial endosymbionts of whiteflies)

Table 3: Endosymbiotic bacteria of whiteflies

The endosymbiont Clostridium and Acinetobacter are already reported to be present in insect gut (Colman et al., 2012, Engel and Moran., 2013). Providencia reported from Nasonia vitripennis (Wang and Brucker, 2019) and major olive pests in Tunisia (Ksentini et al., 2019), whereas, Paenibacillus alvei is common in honeybee larvae (Anderson et al., 2011). The development of resistance to insecticides in insect pests is an important and increasing problem. As insecticide use increases in tropical areas, the numbers of resistant insect strains will increase.  From the present study, we can see that whitefly endosymbionts Providencia sp. and Paenibacillus alvei are resistant against insecticides Chlorpyrifos and Imidacloprid respectively and possibly, they may even contribute to insects’ insecticidal resistance/ detoxification capability. Similarly, Kikuchi et al. (2012) reported symbiont–mediated detoxification of Riptortus pedestris against the insecticide fenitrothion. Barman et al. (2021) conducted works on the action of neonicotinoid insecticides on whitefly endosymbionts and Raina et al. (2015), studied the effect of selective elimination of whitefly endosymbiont Arsenophonus using antibiotic treatment.

3.4. Diagnostic PCR for the identification of bacterial endosymbionts in whitefly samples

The quantity of DNA from B. tabaci and A. dispersus were 18.34 ng µl^–1 and 30.32 ng µl^–1 respectively and were with A260/A280 ratio of 1.85 and 1.93 (Figure 2).

Figure 2: Diagnostic PCR for presence of bacterial endosymbionts in whitefly sample (L: 1Kb plus ladder; P: Portiera, W: Wolbachia; A: Arsenophonus; R: Rickettsia; C: Cardinium; B: Blank)

In B. tabaci, presence of primary endosymbiont Portiera was confirmed at fragment length of 1000 bp and secondary endosymbiont Wolbachia at 650 bp. In A. dispersus, secondary endosymbiont Rickettsia presence was noticed at 800 bp (second band obtained is a non–specific banding at 650 bp). A primary endosymbiotic bacterium called Portiera aleyrodidarum, which is fixed in populations and confined to bacteriocyte cells in all whitefly individuals. These endosymbionts are obligate and have mutualistic relationship with B. tabaci. This bacterium is essential for host survival, development and has a long co-evolutionary history with all members of the subfamily Aleyrodidae (Thao and Baumann, 2004, Santos-Garcia et al., 2012). Presence of secondary endosymbionts Rickettsia (Gottlieb et al., 2006) and Wolbachia (Skaljac et al., 2010) are already reported in whiteflies and other insects and they probably help in resistance development of the host (Liu and Guo, 2019). According to Brumin et al. (2011), Rickettsia influences thermotolerance in B. tabaci, whereas, Kontsedalov et al. (2008) states, its presence is associated with increased susceptibility to insecticides. Rickettsia even found to influence body colour of aphids (Tsuchida et al., 2010). Wolbachia reported to cause cytoplasmic incompatibility in the parasitic wasp Encarsia inaron (White et al., 2009).

CONCLUSION

Presence of insecticidal resistant endosymbiotic bacteria in cassava whitefly populations, probably contributing to their success. As diagnostic PCR technique is able to detect unculturable and important endosymbionts, in near future it could effectively replace the costly NGS (Next–Generation Sequencing) platform, in many cases.

ACKNOWLEDGEMENT

Authors extend sincere gratitude to ICAR–Central Tuber Crops Research Institute, Thiruvananthapuram for technical and financial support.

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