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. 2024 Mar 5;14(4):100. doi: 10.1007/s13205-024-03949-0

Diversity and functional characteristics of culturable bacterial endosymbionts from cassava whitefly biotype Asia II-5, Bemisia tabaci

Venkatesh Kumar 1, Jeyarani Subramanian 1,✉, Murugan Marimuthu 1, Mohankumar Subbarayalu 2, Venkatachalam Ramasamy 3, Karthikeyan Gandhi 4, Manikandan Ariyan 5
PMCID: PMC10914660  PMID: 38456084

Abstract

Whitefly Bemisia tabaci, a carrier of cassava mosaic disease (CMD), poses a significant threat to cassava crops. Investigating culturable bacteria and their impact on whiteflies is crucial due to their vital role in whitefly fitness and survival. The whitefly biotype associated with cassava and transmitting CMD in India has been identified as Asia II 5 through partial mitochondrial cytochrome oxidase I gene sequencing. In this study, bacteria associated with adult B. tabaci feeding on cassava were extracted using seven different media. Nutrient Agar (NA), Soyabean Casein Digest Medium (SCDM), Luria Bertani agar (LBA), and Reasoner’s 2A agar (R2A) media resulted in 19, 6, 4, and 4 isolates, respectively, producing a total of 33 distinct bacterial isolates. Species identification through 16SrRNA gene sequencing revealed that all isolates belonged to the Bacillota and Pseudomonadota phyla, encompassing 11 genera: Bacillus, Cytobacillus, Exiguobacterium, Terribacillus, Brevibacillus, Enterococcus, Staphylococcus, Brucella, Novosphingobium, Lysobacter, and Pseudomonas. All bacterial isolates were tested for chitinase, protease, siderophore activity, and antibiotic sensitivity. Nine isolates exhibited chitinase activity, 28 showed protease activity, and 23 displayed siderophore activity. Most isolates were sensitive to antibiotics such as Vancomycin, Streptomycin, Erythromycin, Kanamycin, Doxycycline, Tetracycline, and Ciprofloxacin, while they demonstrated resistance to Bacitracin and Colistin. Understanding the culturable bacteria associated with cassava whitefly and their functional significance could contribute to developing effective cassava whitefly and CMD control in agriculture.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-03949-0.

Keywords: Cassava whitefly, Culturable endosymbionts, Chitinase, Protease, Siderophore, Antibiotics

Introduction

Cassava is an essential source of nutrition for over a billion people in 105 countries, with annual output reaching 302 million tons (Latif and Müller 2015; FAOSTAT 2022). Tamil Nadu accounted for 83.75% of total output in India in 2021–22 (APEDA 2022). Globally, Cassava mosaic disease is the biggest problem to cassava production, which is spread by whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). In India in the early 1940s, the Cassava mosaic virus (CMV) was discovered to be a severe threat to cassava (Manihot esculenta Crantz) (Abraham 1956). The Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV), two Gemini viruses transmitted by whiteflies, are widely prevalent in the Indian subcontinent, infecting cassava (Varma et al. 2011). Following the identification of Cassava mosaic disease (CMD) in Southeast Asia, an initial assessment was conducted to evaluate the impact of SLCMV on cassava root production in Vietnam. The results revealed that utilizing SLCMV-infected cuttings at planting led to a significant decrease in both cassava root yield and starch content, with reductions ranging from 16 to 33% and 22 to 38%, respectively, when compared to the use of disease-free, healthy cuttings (Uke et al. 2022).

Whitefly, B. tabaci has become a worldwide issue due to its quick proliferation across the tropical and subtropical regions of the world. This insect physically harm plants by consuming their phloem sap and acts as a vector for many viral infections (Brown and Czosnek 2002; Basu 2019). B. tabaci transmits non-persistent Potyvirus, semi-persistent Closterovirus and Carlavirus, and the most crucial persistent Begomovirus. Despite similar morphological resemblances, B. tabaci belongs to over 40 genetically distinct groupings (Kanakala and Ghanim 2019). These cryptic species were identified using the nucleotide (3.5% variance) between mitochondrial cytochrome oxidase I gene sequences (Dinsdale et al. 2010; De Barro et al. 2011). A study of the specificity of Begomovirus transmission by several cryptic species of the B. tabaci complex was published by Fiallo-Olivé et al. (2020). The diversity of whitefly biotypes associated with cassava has been studied in different countries such as Central African Republic (Tocko-Marabena et al. 2017), South Sudan (Misaka et al. 2020), Africa (Chen et al. 2019), East Africa (Mugerwa et al. 2012), Zambia (Chiza Chikoti et al. 2020), and Lao PDR (Leiva et al. 2022).

Numerous commensal, pathogenic, and mutualistic species comprise the bacterial community linked to B. tabaci (Dillon and Dillon 2004). Pseudomonadota, Bacillota, Actinomycetota, Bacteroidota, Spirochaetota, and Acidobacteriota were the bacterial phyla studied from the insect gut environment (Banerjee et al. 2022). Microbial symbionts are crucial for insect hosts, as they supply necessary nutrients; control development, reproduction, metabolism, immunity, defense against antagonists; detoxify harmful substances; and help hosts adapt to their specific environments (Hansen and Moran 2014; Gao et al. 2020). Whitefly, B. tabaci harbor several primary and secondary endosymbionts. Portiera aleyrodidarum, a primary endosymbiont in whiteflies, resides in bacteriocytes and is essential for supplying necessary amino acids and carotenoids that are lacking in phloem sap (Thao and Baumann 2004; Sloan and Moran 2012). In addition, whitefly species host the following seven facultative secondary endosymbionts: Arsenophonus, Cardinium, Fritschea, Hamiltonella, Hemipteriphilus, Rickettsia, and Wolbachia (Zchori-Fein and Brown 2002; Nirgianaki et al. 2003; Weeks et al. 2003; Everett et al. 2005; Gottlieb et al. 2006; Bing et al. 2013). The primary symbionts are in the role of maintaining balance (Baumann 2005), while other symbionts provide host specificity (Chen et al. 2016), adaptability in whitefly biology, virus transmission effectiveness (Ghosh et al. 2018; Wu et al. 2022), resilience to temperature, contribution to the morphogenesis, production of pheromones, and immune responses to their hosts (Dillon and Dillon 2004; Genta et al. 2006; Engel and Moran 2013). Some whitefly-associated bacteria can potentially be entomopathogens and biocontrol agents (Roopa et al. 2014). Data on 16S rRNA gene sequencing from a greater variety of insect species found indications of change in the bacterial community dependent on the host diet and taxonomic structure (Colman et al. 2012).

The role of bacterial symbionts in persistence virus transmission in insect is very crucial. Gut commensal microbiome may regulate host defenses against viral infections of gut epithelial cells (Yin et al. 2020). Kliot et al. (2019) found that Rickettsia infections downregulated immune system genes in whiteflies, enhancing their acquisition, retention, and transmission of the Tomato yellow leaf curl virus (TYLCV). Lei et al. (2021) revealed that the interaction between the rickettsial secretory protein, BtR242, and the protein coat of the Cotton leaf curl Multan virus (CLCuMuV) benefited its transmission by whiteflies. They also observed that vitellogenin (Vg) levels in Rickettsia-infected whiteflies were over twofold higher than that in non-infected whiteflies, with Vg facilitating TYLCV movement across the midgut barrier in the whitefly vector (He et al. 2021). The GroEL protein produced by symbiotic bacteria is involved in preserving the stability of Geminivirus and Luteovirus particles within the whitefly’s hemolymph (Hogenhout et al. 1998; Morin et al. 1999). Simultaneously, the obligatory symbiotic bacteria Sulcia and Nasuia play a role in facilitating the transmission of Rice dwarf virus to the succeeding generation (Jia et al. 2017; Wu et al. 2019b). Wu et al. (2019a) found that the gut bacterium Serratia marcescens promotes mosquito susceptibility to arboviruses. This is achieved through a secreted protein called SmEnhancin, which digests mucins in the mosquito gut, disrupting the gut barrier and enhancing arboviral infection. In the silkworm, the intestinal bacterium Bacillus pumilus SW41 secretes a potent antiviral lipase against Bombyx mori NPV (Liu et al. 2018). Meanwhile, certain bacteria from Aedes albopictus, such as Pseudomonas rhodesiae, Enterobacter ludwigii, and Vagococcus salmoninarium, produce antiviral compounds to inhibit La Crosse encephalitis virus infection (Joyce et al. 2011). On the contrary, Serratia odorifera, a gut commensal bacterium in Aedes aegypti mosquitoes, increases mosquito susceptibility to DENV-2 by releasing bacterial polypeptides that disrupt the binding of mosquito prohibition to DENV-2 virions. In summary, these resident symbiotic microorganisms can influence virus infections by regulating gut immune responses, affecting physical barriers, or producing specific antiviral compounds (Apte-Deshpande et al. 2012).

Chitin is the second most abundant natural polymer, present in the cell walls of many fungi and algae, as well as in the exoskeletons of crustaceans, insects, and arthropods (Moussian 2019). Chitinase-producing bacteria, including various types such as Aeromonas, Streptomyces, Vibrio, Serratia, Bacillus, Enterobacter, Burkholderia gladioli, Aeromonas hydrophila, and A. punctata, play a crucial role in controlling the spread of fungus and insect pests by breaking down chitin (Cody 1989; Bhattacharya et al. 2007; Kuddus and Ahmad 2013; Veliz et al. 2017). Proteases, produced by a range of organisms, account for a significant portion of the global market for industrial enzymes and are also known to have insecticidal activity from insect-associated microbes (Harrison and Bonning 2010). Insects may harbor proteolytic bacteria in their digestive systems to aid in protein digestion, particularly to counteract dietary protease inhibitors (Zhu-Salzman and Zeng 2015). Microorganisms produce siderophores, iron-binding molecules, for acquiring iron from host insects and protecting themselves against entomopathogens (Ciche et al. 2003; Indiragandhi et al. 2007). Bacteria also produce siderophores to scavenge iron (Guerinot 1994; Lee et al. 2012). Antibiotics are used to explore the role of symbionts associated with insect hosts (Zhao et al. 2022). Zhang et al. (2015b) showed that antibiotic treatment with rifampicin led to a rapid decrease in the secondary symbiont, while the primary symbiont remained stable until later in the host’s life. These effects also affected the offspring’s fitness, with a greater impact on female hosts.

Understanding the bacterial community associated with insect hosts is pivotal, given the crucial role bacteria play in the insect–plant connection (Gueguen et al. 2010; Casteel and Hansen 2014; Gupta and Nair 2020; Wu et al. 2022). With no previous exploration of culturable endosymbionts from cassava whiteflies, and considering the significant impact of endosymbionts on the life of whiteflies, this study focuses on culturable bacteria associated with the cassava-specific whitefly, B. tabaci. The investigation aims to assess their functional contributions through biochemical analyses, including chitinase, protease, siderophore production, and antibiotic resistance. This research strives to offer valuable insights for potential management strategies in future studies.

Materials and methods

Collection and mass rearing of whitefly

B. tabaci adults were collected from cassava fields at Tapioca and Castor Research Station (TCRS), Tamil Nadu Agricultural University (TNAU), in Yethapur, Salem, Tamil Nadu, India (11.65299° N, 78.46858° E), and released into potted (41 cm dia) cassava variety MVD 1 (commonly grown variety) that were placed inside a nylon net cages (60 cm × 60 cm × 90 cm; mesh size 120 micron) maintained at Insectary, Department of Agricultural Entomology, TNAU, Coimbatore, with conditions of 28 ± 2 °C, 60–80% RH under the natural light condition.

Whitefly biotype confirmation by mitochondrial DNA amplification and analysis

Ten whiteflies from lab culture were taken after three generations, and DNA was extracted using a Qiagen blood and tissue kit according to the manufacturer’s procedure. DNA was stored into − 20 °C until further use. Partial mitochondrial cytochrome oxidase I (COX1) sequences were produced using the primers C1-J-2195 (5′-TTGATTTTGGTCATCCAGAAGT-3′) and L2-N-3014 (5′-TCCAATGCACTAATCTGCCATATTA-3′). It is used to categorize the biotypes of whiteflies. The PCR was carried out in a final volume of 25 μL, which included 2 × Emerald Amp GT PCR master mix (12.5 μl), 20 μmol/L of each primer, and 10 ng of insect DNA extract, as well as required volume of nuclease- and protease-free molecular biology-grade water (Hi-media, Mumbai). The PCR conditions for amplification were as follows: initial denaturation at 94 °C for 2 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 30 s, and final elongation at 72 °C for 10 min (Akintola et al. 2020). The PCR product was sequenced (Biokart India Pvt. Ltd., Bangalore), and MEGA version 11 was used to perform multiple sequence alignment and phylogenetic analysis on identified sequences (Tamura et al. 2021).

Isolation of bacteria associated with cassava whitefly

Because the virus is transmitted most efficiently during the adult stage of the whitefly, the adult stage was employed for bacterial isolation. One hundred adult whiteflies from the cassava colony were gathered using an aspirator and starved for 24 h to remove the transitory bacteria from the host plant’s sap. Before homogenizing in 0.1 M phosphate buffer (pH 7.0), the adults were cleaned to remove surface microbes with 70% ethanol and 5% sodium hypochlorite for 1 min and three to five rinses with sterile distilled water. Insect homogenates were serially diluted with sterile water, and then 0.1 mL solution was spread on Petri plates containing various bacterial growth media, including Luria Bertani agar (LBA); nutrient agar (NA); Soyabean Casein Digest Medium (SCDM); Endo Agar (EA); Reasoner’s 2A agar (R2A); MacConkey agar (MCA); and de Man, Rogosa, and Sharpe agar (MRSA) (M/s.Himedia Laboratories, Mumbai, India) (de Vries et al. 2001; Saranya et al. 2022a) and incubated for 48 h at 28 ± 2 °C. As a control to verify the absence of epiphytic bacteria, the water obtained during the final rinsing step was introduced into the bacterial growth medium. Bacterial colonies with varied colony morphologies were streaked four to six times to establish a pure culture. A microscope was used to determine their purity. Purified bacterial isolates were stored at − 80 °C in 60% glycerol (Indiragandhi et al. 2007; Saranya et al. 2022b).

Molecular characterization of bacterial isolates

The DNA was extracted from 24 h established cultures using a Qiagen blood and tissue kit following the manufacturer’s directions. The PCR was carried out in a final volume of 25 μL using 2 × Emerald Amp GT PCR master mix (12.5 μL), 10 pmol of each primer (universal 16S forward primer 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ and reverse primer 1492R- 5′-GGTTACCTTGTTACGACTT-3′), and 10 ng of bacterial DNA extract and nuclease- and protease-free molecular biology-grade water (Hi-media). Initial denaturation at 95 °C for 5 min was followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 30 s, extension at 72 °C for 1 min, final extension at 72 °C for 10 min, and hold at 4 °C in the thermocycler (Veriti™ 96 WELL Fast Thermocycler, Applied Biosystems™). Amplified DNA was observed and recorded using gel documentation and analysis equipment (Medicare Gelstan 1312 CHEMI imaging system). The Bio Basic INC. EZ-10 Spin Column DNA Gel Extraction Kit was used to purify the PCR results. Biokart India Pvt. Ltd. performed Sanger sequencing in Bengaluru, India. MEGA version 11 performed multiple sequence alignment and phylogenetic analysis on the resultant sequences (Tamura et al. 2021).

Protease activity

The bacterial isolates were resuspended in nutritional broth and incubated at 28 ± 2 °C for 24 h to perform the protease experiment. Skim milk agar plates were used to identify isolates of bacteria that produce protease (Cattelan et al. 1999). A 24-h-old bacterial culture (1 × 108 cfu/mL) was spotted on 10 L of skim milk agar plates and then left to incubate for 24 h at 28 ± 2 °C. The perimeter of the cleared area was calculated. The protease activity has been calculated using the following formula:

Proteaseactivity(%)=DiameteroftheclearzoneDiameterofthecolony×100

Chitinase activity

Preparation of colloidal chitin

Five grams of crab shell chitin powder (C7170, Sigma-Aldrich Co., USA) was vigorously mixed with 60 ml of concentrated HCl (Merck S.A.) for 1 h at room temperature. Then, it was filtered with glass wool, and the filtrate was added to 200 ml of 50% ethanol and vigorously agitated throughout the operation. The precipitate was transferred to a glass funnel lined with 80 g/m filter paper, and the colloidal chitin was washed with sterile distilled water until it became neutral (pH 7.0). The remaining colloidal chitin on the filter paper was scraped with a spatula, weighed, and stored in the dark at 4 °C (Skujins et al. 1965; Souza et al. 2009).

Preparation of a minimal medium containing the colloidal chitin

Following Furukawa et al. (1978), a minimal medium (MM) with the following concentrations of salts (g l−1): (NH4)2SO4, 1.0; KH2PO4, 0.2; K2HPO4, 1.6; MgSO4.7H2O, 0.2; NaCl, 0.1; FeSO4.7H2O, 0.01; and CaCl2.2H2O, 0.02 was prepared. Colloidal chitin was added to the medium at a concentration of 1.2% (w/v), and the final pH was adjusted to 7.0 ± 0.5. It was autoclaved for 15 min at 121 °C to eliminate any microorganisms. To detect chitin hydrolysis, 24-h pure broth cultures were streaked over a medium and kept at 27 ± 2 °C for 96 h.

Siderophore production assay

The ChromAzurol S (CAS) agar plate method was developed to detect siderophore-producing bacterial isolates (Milagres et al. 1999). The autoclaved basal agar medium and the CAS indicator solution were cooled to 50 °C. The 50% glucose solution was prepared and autoclaved. A 2 mL cooled 50% glucose solution was added to the autoclaved basal agar medium. Then, 10 mL of the CAS indicator solution was gradually applied along the walls while constantly agitated. After mixing, the CAS agar medium (100 mL) was placed into sterile plates. The plates were then spotted with 10 µL of culture (1 × 107 cfu mL−1, 24 h old) and incubated for 48 h at 28 ± 2 °C. Around the spotted colonies, a zone with an orange halo formed, indicating siderophore production. The siderophore production was calculated using the following formula:

Siderophoreproduction(%)=DiameteroftheorangehalozoneDiameterofthecolony×100

Antibiotic susceptibility test

The antibiotic susceptibility of cassava whitefly gut bacteria was tested using the Kirby–Bauer disc diffusion technique (Hudzicki 2009; Sharma 2021). Vancomycin VA 30 µg, erythromycin E 15 µg, streptomycin S 10 µg, kanamycin K 30 µg, doxycycline DO 30 µg, bacitracin B 10 µg, chloramphenicol C 30 µg, colistin CL 10 µg, tetracycline TE 30 µg, and ciprofloxacin CIP 5 µg were the antibiotics investigated. Bacteria were cultivated in a nutrient broth (HIMEDIA M002-100G) for 24 h at 28 ± 2 °C. Bacterial isolates were equally disseminated for 5 min on nutrient agar plates before being incubated at 28 ± 2 °C for 24 h with antibiotic discs (Hi-Media, India) put on top of the agar with sterile forceps. The diameter of the inhibition zone was measured and compared with the diameter of the inhibition zone previously published by CLSI to determine the antibiotic’s sensitivity (Saranya et al. 2022a).

Results

Cassava-associated whitefly biotype

DNA sequences that confirmed the species were found using the GenBank (http://www.ncbi.nlm.nih.gov) BLASTn program, and they were then submitted with the accession number OQ848452. The classified biotype sequences provided by Dinsdale et al. (2010) were obtained from the NCBI nucleotide database, and the Asia II-5 biotype of the whitefly in our lab culture was confirmed (Fig. 1).

Fig. 1.

Fig. 1

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Phylogenetic relationships of cassava whitefly biotype with available biotypes using partial Mitochondrial Cytochrome Oxidase I (COX1) gene sequence. Our cassava associated biotype represented in bold font. The evolutionary distances were computed using the Maximum Composite Likelihood method in MEGA11 software. HKY + G model were used. 1000 bootstrap were maintained

Bacterial isolates and their molecular characterization

Four of the seven different bacterial growth mediums, including NA, LBA, SCDM, and R2A, but not MCA, EA, and MRSA, supported the bacterial growth, resulting in the isolation and characterization of a total of 33 bacteria associated with the adult cassava whitefly. The NA medium produced the most bacterial isolates, featuring 19 bacterial isolates of Bacillus cereus CWF6 & CWF16, Bacillus subtilis CWF4, CWF10, & CWF32, Lysobacter firmicutimachus CWF11, Exiguobacterium homiense CWF13, Staphylococcus epidermidis CWF14, Exiguobacterium aurantiacum CWF15, Exiguobacterium profundum CWF28, Bacillus halotolerant CWF18, Terribacillus saccharophilus CWF19, Brucella pseudogrignonensis CWF20, Enterococcus sp. CWF23, Bacillus pumilus CWF25, Brevibacillus agri CWF26 & CWF29, Pseudomonas stutzeri CWF31, and Bacillus sonorensis CWF33. SCDM recorded six isolates, namely Bacillus tropicus CWF7, Staphylococcus pasteuri CWF1 & CWF9, Bacillus velezensis CWF17, and Staphylococcus epidermidis CWF2 & CWF27, while R2A (Bacillus sp. CFW21., Staphylococcus aureus CWF8, Cytobacillus horneckiae CWF5, and Staphylococcus pasteuri CWF3) and LB medium (Bacillus cereus CWF24 & CWF30, Novosphingobium panipatense CWF12, and Bacillus licheniformis CWF22) recorded four isolates each (Table 1). Most bacterial isolates belonged to the phylum Bacillota (class Bacilli), with a few from the phylum Pseudomonadota (class Alphaproteobacteria and Gammaproteobacteria). Bacteria identified belonged to the genus of Bacillus, Exiguobacterium, Terribacillus, Brevibacillus, Enterococcus, Staphylococcus, Brucella, Novosphingobium, Lysobacter, and Pseudomonas (Supplementary file 1). The isolates were subjected to phylogenetic analysis, the results of which are shown in Fig. 2.

Table 1.

Molecular characterization of bacterial isolates of cassava whitefly

S. No Isolate designation Closest match Similarity (%) Size of the sequence NCBI accession number Media
1 CWF1 Staphylococcus pasteuri MT539733 98.91 1379 OQ674479 SCDM
2 CWF2 Staphylococcus epidermidis MZ350515 99.50 1330 OQ674503 SCDM
3 CWF3 Staphylococcus pasteuri ON761772 99.13 1260 OQ674505 R2A
4 CWF4 Bacillus subtilis OP009945 98.82 1389 OQ674514 NA
5 CWF5 Cytobacillus horneckiae MT487623 98.95 1330 OQ674515 R2A
6 CWF6 Bacillus cereus MK967017 98.95 1330 OQ674794 NA
7 CWF7 Bacillus tropicus MT835152 98.98 1376 OQ674805 SCDM
8 CWF8 Staphylococcus aureus MW644610 99.26 812 OQ674810 R2A
9 CWF9 Staphylococcus pasteuri MT539733 99.19 1364 OQ674823 SCDM
10 CWF10 Bacillus subtilis MF037830 99.19 1361 OQ675140 NA
11 CWF11 Lysobacter firmicutimachus MW897735.1 99.03 1340 OQ675153 NA
12 CWF12 Novosphingobium panipatense MG996842 99.27 1376 OQ675157 LB
13 CWF13 Exiguobacterium homiense MG733598 99.08 1306 OQ675162 NA
14 CWF14 Staphylococcus epidermidis MT585538 97.93 1462 OQ675263 NA
15 CWF15 Exiguobacterium aurantiacum MG705832.1 99.23 1292 OQ675541 NA
16 CWF16 Bacillus cereus MW774443 98.87 1236 OQ675558 NA
17 CWF17 Bacillus velezensis OQ586464 99.56 1369 OQ675615 SCDM
18 CWF18 Bacillus halotolerans MN186794 99.48 962 OQ676121 NA
19 CWF19 Terribacillus saccharophilus MN704802 99.18 1216 OQ676149 NA
20 CWF20 Brucella pseudogrignonensis MK517588 98.28 1334 OQ676216 NA
21 CWF21 Bacillus sp. MF346110 98.73 1418 OQ676405 R2A
22 CWF22 Bacillus licheniformis MG705593 99.47 1330 OQ676461 LB
23 CWF23 Enterococcus sp. KC819131 98.82 1361 OQ676546 NA
24 CWF24 Bacillus cereus MW440681 99.22 1411 OQ676573 LB
25 CWF25 Bacillus pumilus MH260994 99.53 1065 OQ676638 NA
26 CWF26 Brevibacillus agri KP284437 99.11 1355 OQ676842 NA
27 CWF27 Staphylococcus epidermidis MT604781.1 98.98 1280 OQ676881 SCDM
28 CWF28 Exiguobacterium profundum ON261410.1 98.70 1380 OQ676884 NA
29 CWF29 Brevibacillus agri MT422060.1 99.28 1246 OQ676885 NA
30 CWF30 Bacillus cereus MT605291.1 99.40 1333 OQ676886 LB
31 CWF31 Pseudomonas stutzeri MT386141.1 99.18 1342 OQ676920 NA
32 Cwf32 Bacillus subtilis KY806228.1 99.28 1384 OQ676926 NA
33 Cwf33 Bacillus sonorensis MT314051.1 98.98 1371 OQ676949 NA
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Fig. 2.

Fig. 2

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Phylogenetic relationship between known bacterial 16S rRNA sequences and bacterial isolates associated with Whitefly, Bemisia tabaci. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura 3-parameter model in MEGA 11 software. The bootstrap consensus tree inferred from 1000 replicates. Our isolates are represented with bold font

Biochemical characterization of bacterial isolates

Chitinase activity

After incubation, bacterial cultures grown on chitin-amended media produced a hydrolysis zone, demonstrating the existence of chitinase activity (Supplementary. 2). Chitinase activity was detected in nine of the 33 bacterial isolates examined. Those isolates were Bacillus cereus CWF6, Bacillus subtilis CWF10, Lysobacter firmicutimachus CWF11, Exiguobacterium homiense CWF13, Brucella pseudogrignonensis CWF20, Bacillus sp., CWF21, Bacillus licheniformis CWF22, Bacillus cereus CWF24, and Bacillus cereus CWF30 (Table 2).

Table 2.

Biochemical characterization of bacterial isolates of cassava whitefly

S. No Isolate designation Chitinase activity (+)/ (−) Protease activity Siderophore
1 CWF1 − 30.16 ± 0.28defg 37.36 ± 0.61bc
2 CWF2 − 28.54 ± 0.44efg NDk
3 CWF3 − NDi 8.37 ± 0.07ijk
4 CWF4 − 52.31 ± 0.97a 33.23 ± 0.34bcde
5 CWF5 − 25.71 ± 0.74fgh 11.20 ± 0.13hij
6 CWF6 +  34.44 ± 0.56cdef NDk
7 CWF7 − 15.08 ± 0.15ghi 24.52 ± 0.48efg
8 CWF8 − NDi 34.92 ± 0.16bc
9 CWF9 − 48.74 ± 0.75abc NDab
10 CWF10 +  36.30 ± 0.60bcdef NDk
11 CWF11 +  22.54 ± 1.18fgh 28.55 ± 0.39bcdef
12 CWF12 − 35.26 ± 0.70cdef 8.12 ± 0.05jk
13 CWF13 +  11.43 ± 0.31hi 43.56 ± 0.29ab
14 CWF14 − 38.33 ± 0.57abcdef NDk
15 CWF15 − 15.87 ± 0.18ghi 10.37 ± 0.08hij
16 CWF16 − 11.13 ± 0.43hi 37.99 ± 0.40bc
17 CWF17 − 38.52 ± 0.65abcde 28.03 ± 0.99bcdef
18 CWF18 − 11.20 ± 0.18hi NDk
19 CWF19 − 18.10 ± 1.04gh 21.30 ± 0.63fgh
20 CWF20 +  10.32 ± 0.59hi 42.76 ± 0.62ab
21 CWF21 +  46.07 ± 0.83abcd NDk
22 CWF22 +  34.52 ± 1.20cdef 11.57 ± 0.13hij
23 CWF23 − NDi 24.44 ± 0.67efg
24 CWF24 +  46.19 ± 0.99abc NDk
25 CWF25 − 51.27 ± 1.03ab 12.04 ± 0.15ab
26 CWF26 − 45.15 ± 1.02abcd 26.87 ± 1.36def
27 CWF27 − NDi NDa
28 CWF28 − NDi 18.65 ± 1.33fghi
29 CWF29 − 39.29 ± 0.80abcde 33.46 ± 1.28bcde
30 CWF30 +  16.67 ± 1.67gh 52.31 ± 0.66a
31 CWF31 − 27.78 ± 2.31 fg 15.87 ± 1.20fghi
32 CWF32 − 34.70 ± 1.95cdef 26.28 ± 1.63def
33 CWF33 − 30.20 ± 1.75defg NDk
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Serial 1–33 (CWF1-CWF33) indicates whitefly, Bemisia tabaci Asia II-5 bacterial isolates from cassava. Columns with the same letter are not significantly different at 0.05 levels (Turkey’s HSD test). Values in each column are the mean of three replications ± standard error (SE). ND not detected

Protease activity

On skim milk agar plates, bacteria that generated proteases were recognized by the clear zone formed after 24 h of inoculation (Supplementary. 2). Twenty-eight of the 33 strains tested positive for protease activity at varying levels. Bacillus subtilis CWF4 (52.31%), Bacillus pumilus CWF25 (51.27%), Staphylococcus pasteuri CWF9 (48.74%), Bacillus cereus CWF24 (46.19%), Bacillus sp. CWF21 (46.07%), and Brevibacillus agri CWF26 (45.15%) all demonstrated more than 40% activity. Brevibacillus agri CWF29 (39.29%), Bacillus velezensis CWF17 (38.52%), Bacillus subtilis CWF10 (36.30%), Novosphingobium panipatense CWF12 (35.26%), Bacillus subtilis CWF32 (34.70%), Bacillus licheniformis CWF22 (34.52%), Bacillus cerus CWF6 (34.44%), Staphylococcus epidermidis CWF14 (33.38%), Bacillus sonorensis CWF33 (30.20%), and Staphylococcus pasteuri CWF1 (30.16%) showed more than 30% protease activity. Staphylococcus pasteuri CWF3, Staphylococcus aureus CWF8, Enterococcus sp. CWF23, Staphylococcus epidermidis CWF27, and Exiguobacterium profundum CWF28 were discovered to lack detectable protease activity (Table 2).

Siderophore production

Bacterial isolates generating siderophores producing an orange halo around an inoculation culture in the ChromAzurol S (CAS) agar plate are shown in Supplementary 2. Bacillus cereus CWF 30 (52.31%), Exiguobacterium homiense CWF13 (43.56%), and Brucella pseudogrignonensis CWF20 (42.76%) were found to have more than 40% siderophore activity. Bacillus cereus CWF16 (37.99%), Staphylococcus pasteuri CWF1 (37.36%), Staphylococcus aureus CWF8 (34.92%), Brevibacillus agri CWF29 (33.46%), and Bacillus subtilis CWF4 (33.23%) showed more than 30% activity. Staphylococcus epidermidis CWF2, Bacillus cerus CWF6, Staphylococcus pasteuri CWF9, Bacillus subtilis CWF10, Staphylococcus epidermidis CWF14, Bacillus halotolerans CWF18, Bacillus sp. CWF21, Bacillus cereus CWF24, Staphylococcus epidermidis CWF27, and Bacillus sonorensis CWF33 lacked detectable siderophore activity (Table 2).

Antibiotic sensitivity

Bacterial isolates were exposed to ten antibiotics with varied modes of action, including those that decrease cell wall production, permeability, DNA gyrase, topoisomerase IV activity, and several locations on protein synthesis. The zone of inhibition of bacterial culture surrounding the antibiotic disc was evaluated to categorize the degree of sensitivity of bacterial strains (Supplementary. 3). The majority of the cultures were highly sensitive to antibiotics, such as vancomycin VA30, erythromycin E15, streptomycin S10, kanamycin K30, doxycycline DO30, tetracycline TE30, and ciprofloxacin CIP5, intermediately sensitive to chloramphenicol C30, and resistant to bacitracin B10 and colistin CL10. Bacterial isolate Bacillus cereus CWF30 was resistant to most of the antibiotics with minimal inhibition zone, followed by Lysobacter firmicutimachus CWF11, Novosphingobium panipatense CWF12, Staphylococcus epidermidis CWF27, Exiguobacterium profundum CWF28, Brevibacillus agri CWF29, and Bacillus sonorensis CWF33. Many antibiotics were effective against the bacterial isolates Staphylococcus epidermidis CWF2, Cytobacillus horneckiae CWF5, Bacillus subtilis CWF10, Exiguobacterium homiense CWF13, Terribacillus saccharophilus CWF19, Enterococcus sp. CWF23, and Bacillus cereus CWF24 (Supplementary files. 4 and 5).

Discussion

The critical need to identify the specific B. tabaci biotype associated with CMV transmission arises due to variations in their efficiency in spreading the virus. Chi et al.'s (2020) research prominently emphasized the Asia II 1 biotype as the most efficient CMV transmitter among the three biotypes investigated. Moreover, within the Indian setting, the Asia II 5 biotype, which is exclusive to CMV, has been observed to be involved in the transmission of the Tomato leaf curl virus (TYLCV) (Chowda-Reddy et al. 2012) and its existence in cotton fields (Ashfaq et al. 2014; Ashwathappa et al. 2020). Our study results emphasize the importance of giving priority to the Asia II 5 biotype for the successful management of CMD.

The diversity of gut flora in insects is affected by the host plants they feed on, as demonstrated in prior studies (Zhao et al. 2022; Saranya et al. 2022a). In our research, we discovered unique bacterial isolates in cassava whiteflies. In their study of B and Q whitefly biotypes, Indiragandhi et al. 2010 identified Actinobacteria (Actinomycetota), Alpha, Beta, and Gamma Proteobacteria (Pseudomonadota), as well as Firmicutes (Bacillota) classes of bacteria. In our study, we found similar classes of bacteria, with the exception of Actinobacteria and beta Proteobacteria. Several bacterial species, including Staphylococcus epidermis, Staphylococcus pasteuri, Bacillus cereus, Bacillus pumilus, Bacillus licheniformis, and Bacillus subtilis, identified in our research, have been previously documented in studies involving various whitefly biotypes (Ateyyat et al. 2010; Indiragandhi et al. 2010). The presence of these bacterial species in different whitefly biotypes hints at their potential importance in whitefly biology, possibly affecting development, nutrition, or resistance to pathogens. Further investigation is required to determine their specific roles and potential applications in pest management and crop protection.

The choice of growth medium for isolating bacteria from insects can significantly impact the diversity and abundance of bacterial phylotypes, as demonstrated by Davidson et al. (2000). In our study, the bacterial isolates from cassava whiteflies were successfully established in SCDM, R2A, NA, and LB mediums, which is similar to a study on the cultivable bacteria in rugose spiraling whiteflies by Saranya et al. (2022a). This highlights the significance of selecting appropriate growth media to effectively isolate and study bacterial communities associated with insects.

Chitin is an essential component of arthropods, chitons, yeasts, fungi, diatoms, corals, and sponges (Muzzarelli 2011), and the role of bacteria in chitin breakdown and its mechanism is clearly detailed by Beier and Bertilsson (2013) and these chitinase-producing bacteria have been used to control fungal and insect species (Dahiya et al. 2006; Subbanna et al. 2018). Chitinase-producing bacteria, such as Bacillus cereus, Bacillus subtilis, Bacillus licheniformis, Brucella pseudogrignonensis, Exiguobacterium homiense, and Lysobacter firmicutimachus, were identified in our study. These bacterial species have previously been recognized for their chitinase production and employed in the management of insects and fungi in numerous research studies (Karunya et al. 2011; Chandrasekaran et al. 2012; Lee et al. 2012; Chakraborty et al. 2019; Drewnowska et al. 2020; Essghaier et al. 2021; Lv et al. 2022). Chitinase-producing bacteria can play a dual role in assisting insect survival by weakening fungal cell walls to protect against fungal infections while also posing a potential risk by compromising insect cell walls. To better understand their specific functions, further research involving individual cultures is necessary.

Bacteria from various insect digestive tracts, particularly Bacillota and Pseudomonadota, produce proteases, crucial for digestion (Rao et al. 1998; Akman Gündüz and Douglas 2009; Banerjee et al. 2022). This phenomenon spans multiple insect orders and plays a role in insect adaptation to dietary protease inhibitors, affecting their ecological success (Zhu-Salzman and Zeng 2015). Microbiotas may facilitate whitefly adaptation to cassava, possibly due to the plant’s cyanogenic glycosides (Ravindran 1993; Hue et al. 2012). However, bacterial proteases can harm insect cuticles, hemocoel, and midguts, making them susceptible to pesticides (Harrison and Bonning 2010). Gut bacteria producing chitinase and proteases can affect the peritrophic membrane, potentially causing nutritional imbalances and insect mortality (Okongo et al. 2019; Krishnamoorthy et al. 2020). Evaluating individual protease-producing bacteria is essential for a comprehensive understanding of their impact on insect health and adaptation.

Bacteria produce siderophores to scavenge iron (Lee et al. 2012). Siderophores, with a high affinity for iron binding, aid in acquiring essential Fe3+ for bacterial growth (Guerinot 1994). The grasshopper Sathrophyllia femorata’s digestive tract hosted siderophore-producing bacteria from 37 species across 19 genera, primarily from Bacillota and Pseudomonadota (Sonawane et al. 2018). In our study, we found that most Pseudomonadota and Bacillota isolates produced siderophores. Notably, Pseudomonas sp. strain PRGB06’s siderophore offers protection against entomopathogenic fungal infections (Indiragandhi et al. 2007). For future studies, evaluating the specific functions of these bacterial cultures is crucial.

Antibiotics with distinct modes of action can alter the whitefly endosymbiont population, potentially impacting the host insect B. tabaci (Zhang et al. 2015a; Shan et al. 2016). While most examined bacterial isolates showed resistance to Bacitracin and Colistin, which affect cell wall production and permeability, they were susceptible to several other antibiotics. These antibiotics, including chloramphenicol, streptomycin, erythromycin, kanamycin, doxycycline, tetracycline, vancomycin, and ciprofloxacin, target various bacterial processes involved in protein synthesis, peptidoglycan synthesis, and DNA gyrase and topoisomerase IV (Spotts and Stanier 1961; Nishimura et al. 1962; Zhang et al. 2018; Patel et al. 2022).

Saranya et al. (2022a) investigated bacterial endosymbionts in rugose spiraling whiteflies and their response to antibiotics. Feeding rugose spiraling whiteflies Ceftriaxone, Carbenicillin, and Ciprofloxacin led to reduced hatching rates and overall egg production. Their findings also included bacterial endosymbionts’ resistance to colistin, matching our results. Raina et al.'s (2015) research has shown that eliminating endosymbionts from B. tabaci results in improved fitness. Visôtto et al. (2009) isolated bacteria from the digestive tracts of fifth-instar velvet bean caterpillars and assessed their antibiotic sensitivity. Tetracycline had the most significant impact on the bacterial isolates, followed by chloramphenicol. Our cultures exhibited similar sensitivity, displaying a greater response to tetracycline and a moderate response to chloramphenicol. Our findings suggest that antibiotics with a substantial impact on our bacterial isolates warrant further exploration to assess their consequences on whitefly fitness and adaptability in changing environments. This is crucial for gaining comprehensive insights into effective pest management strategies.

A global study on whitefly biotypes has suggested a potential connection between whitefly genetic groupings and bacterial populations. These symbionts play complex roles in regulating insect vector virus permissiveness (Gnankine et al. 2013; Yin et al. 2020; Ma et al. 2021; Wu et al. 2022). Notably, the presence of Rickettsia infections enhances the whitefly’s ability to acquire, retain, and transmit TYLCV with a downregulation of immune system gene expressions in the infected population (Kliot et al. 2019; Lei et al. 2021). Furthermore, GroEL, a heat shock protein produced abundantly by bacterial endosymbionts in insects, plays a vital role in the transmission of Geminivirus to host plants (Morin et al. 1999; Pinheiro et al. 2015). Given the multifaceted roles that endosymbionts play in virus transmission, it becomes imperative to thoroughly study these interactions for effective strategies in combatting cassava mosaic disease.

Conclusion

In this study, the presence of the Asia II-5 whitefly biotype in cassava cultivation areas was confirmed. We successfully cultured whitefly-associated bacteria on four out of seven tested growth media: NA, LBA, SCDM, and R2A. A total of 33 bacterial isolates were obtained, falling into the Bacilli (Bacillota) and Alpha and Gamma Proteobacteria (Pseudomonadota) classes. The majority of these isolates exhibited chitinase, protease, and siderophore activity, indicating their potential functional significance. Notably, antibiotic resistance levels varied among these bacterial isolates. The study emphasizes the importance of targeting whitefly endosymbionts for effective whitefly species management due to their diverse roles, including biotype specificity, host adaptation, and viral transmission. Furthermore, our findings hint at the possibility of utilizing these newly acquired bacteria as efficient biocontrol agents. Looking forward, future research should focus on unraveling the functional implications of these bacterial isolates, expanding our understanding of their roles and potential applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 5738 KB) (5.6MB, docx)

Acknowledgements

KV acknowledges the Department of Biotechnology, Government of India, New Delhi, India, for the Junior Research Fellowship (DBT-JRF) financial support during the doctoral program under grant DBT/2021-22/TNAU/1691. The Department of Agricultural Entomology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India, is acknowledged for providing research facilities and funding. All authors accepted the final version of manuscript submitted to the journal.

Author contributions

KV conducted all the laboratory experiments and manuscript drafting. SJ and MM conceived the hypothesis, designed the experiment, and corrected the manuscript. SM, RV, GK, and AM assisted in laboratory experiments and drafted the manuscript. All authors have seen and approved the manuscript and its contents, and that they are aware of the responsibilities connected to the authorship.

Data availability

The datasets generated during and/or analysed during the current study are already included in this article.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. On behalf of all authors, the corresponding author states that there is no conflict of interest.

Human or animals rights

All authors declare that this work did not involve the use of humans or animals, and that they all approved the final manuscript before it was submitted to the journal.

Informed consent

Not applicable.

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Associated Data

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Supplementary Materials

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Data Availability Statement

The datasets generated during and/or analysed during the current study are already included in this article.

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