Introduction

Eggplant (Solanum melongena L.) also known as brinjal is an important vegetable crop mainly grown in tropical and sub-tropical countries. It is prone to attack by several pathogens, among them brinjal little leaf (BLL), a phytoplasma associated disease is considered as the most important as it causes significant yield losses up to 40% (Mitra et al. 1993). BLL disease was reported first time in India by Thomas and Krishnaswamy (1939). So far, six groups and nine subgroups of phytoplasmas (16SrI-B, 16Sr II-D, 16Sr III-B, −J and –U, 16Sr VI-A, 16Sr VI-D, 16SrIX-C and 16SrXII-A) have been reported to be associated with BLL disease in different countries (Rao and Kumar 2017). Under epidemic condition yield loss up to 100% was reported in affected eggplants (Rao et al. 2018). This disease could be artificially transmitted from infected to healthy plants through the parasitic plant dodder (Cuscuta spp.) or through vegetative grafting. Several leafhopper species are reported as potential vectors for transmission of BLL phytoplasmas under natural conditions (Kumar et al. 2015). Besides, several weeds such as Datura inoxia, D. stramonium, Cannabis sativa subsp. sativa, Portulaca oleracea and P. grandiflora have also been reported as natural and putative alternative hosts of BLL associated phytoplasma strains in India. They play an important role in the natural spread of the BLL disease through leafhopper vectors (Rao and Kumar 2017).

Farmers in India are repeatedly using broad spectrum insecticides to manage pests and diseases in the eggplant crop which results in higher production costs, resistance to pesticides, harmful pesticide residues having adverse implications towards consumer health. Hence, there is an urgent need to find out alternative and safer methods including host plant resistance for the management of BLL disease. Identification of durable resistance sources through screening of available eggplant cultivars, lines, and wild species of Solanum against BLL disease is an effective prerequisite to develop a resistant variety. Little attempt has been made, so far, for identifying the resistance sources in eggplant for BLL. Das and Mitra (2000) screened eggplant varieties against the BLL disease and germplasms under natural field conditions and identified eight resistant and 14 moderately resistant lines to BLL. Chakrabarti and Choudhury (1975) were also screened eggplant genotypes against the BLL disease and identified two wild species of eggplant viz., Solanum integrifolium and S. gilo as resistant to BLL. In addition, they also developed S.212–1 (cross between S. integrifolium and S. gilo), which was graded as resistant to BLL. Afterwards, no attempts have been made towards screening of potential cultivated eggplant genotypes in India for the resistance potential against BLL.

In addition to phenotypic characteristics, the biochemical parameters also play a key role in the selection of resistance sources. Biochemical compounds like, phenols, polyphenol oxidase and peroxidase enzymes help in protecting the host plant against several biotic stresses (Jabeen et al. 2009; Kumar et al. 2010). Therefore, analyses of biochemical parameters are also important to exploit host plant resistance potential in eggplant varieties against insect vector and phytoplasma disease.

Thus, the aim of the present study was: (a) to characterize the phytoplasma strains associated with brinjal little leaf disease in three major eggplant growing districts of Uttar Pradesh; (b) to record the major leafhopper vector responsible for natural spread of BLL; (c) to screen popular cultivated eggplant varieties/genotypes and wild Solanum species to identify resistant sources against BLL disease; and (d) to study the effect of phytoplasma infection on biochemical properties in different eggplant genotypes.

Materials and Methods

Survey and collection of eggplant little leaf samples

A survey was conducted during the years 2015 and 2016 to record the incidence and severity of BLL disease and presence of leafhopper species in eggplant fields of three districts (Varanasi, Mirzapur and Jaunpur) of Uttar Pradesh state, India. The incidence of BLL disease (percentage of plants with little leaf symptoms) was recorded visually in each field (40 fields in Varanasi, 13 in Mirzapur and five in Jaunpur) by counting the number of symptomatic over asymptomatic plants by randomly selecting 10×10 m plots at each location during October 2015 to November 2016 between vegetative and reproductive stage of the crop. Per cent Disease Index (PDI) was calculated by the following procedure.

$$\mathrm{PDI}=\frac{\mathrm{Number}\;\mathrm{of}\;\mathrm{infected}\;\mathrm{plants}}{\mathrm{Total}\;\mathrm{number}\;\mathrm{of}\;\mathrm{plants}\;\mathrm{observed}}\times100$$

Fifty-eight samples showing the typical phytoplasma-like symptoms (little leaf and bunchy top) were collected from Varanasi (40), Mirzapur (13) and Jaunpur (5) along with two asymptomatic plant samples from each location (Table 1, Fig. 1). The collected eggplant samples were kept in separate polythene bags after labelling and brought to Plant Pathology laboratory of ICAR-Indian Institute of Vegetable Research (IIVR), Varanasi for further processing through PCR assays.

Table 1 Survey and incidence of little leaf disease of eggplant in different places of Uttar Pradesh state of India along with GenBank submission and phytoplasma identification in brinjal and insect vector
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Fig. 1
figure 1

Eggplant displayed different types of symptoms under field conditions (a) witches’ broom at Varanasi; (b) Bunchy top symptoms at Mirzapur; (c) little leaves turned into brown and fell down at Uttar Pradesh; (d) Upper leaves turn pale yellow in colour at Varanasi; (e) growing buds are converted into little leaf symptoms at Janupur; (f) eggplant showing the big bud symptoms at Varanasi

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Insect vector sampling, identification, and dynamics

During the field survey, different leafhopper species feeding on brinjal plants were collected using a sweeping net method during vegetative and reproductive stage of the crop. The collected insects were kept in plastic vials containing 70% ethanol and stored at 4 °C. They were identified at Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi. The identified leafhoppers were analyzed for phytoplasma indexing (Table 1). To monitor the insect vector population, yellow sticky trap cards @ 1–2 traps per 50 m2 (placed at East, West, South and North side of the field, 2 m inside the border row at a height of 40 cm near to the crop canopy) were fixed when the crop (eggplant cv. Punjab Barsati) was 4 weeks old at ICAR-Indian Institute of Vegetable Research, Varanasi (Fig. 3a). Leaf hopper population was counted at weekly intervals from September to March months during two consecutive years (2015 and 2016) and correlated with the incidence of BLL disease in the field (Un Nabi et al. 2015).

Phytoplasma source and maintenance

The BLL samples collected from the ICAR-IIVR experimental plots were maintained in the polyhouse under insect-proof cage through graft transmission on eggplant cv. Punjab Barsati (Acc. No. KC478607). This highly susceptible cultivar was used as standard check for screening resistant in different eggplant germplasms and the wild Solanum species (Fig. 2).

Fig. 2
figure 2

Test egplant cv. Punjab barsati showing (a) pale yellowing of leaves and malformed buds, (b) Little leaf with witches’ broom symptoms by graft transmission.

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Molecular detection and characterization of phytoplasma in eggplant

DNA isolation and PCR assay

Total genomic DNA was isolated from 100 mg of leaf midrib tissue from 58 symptomatic eggplant samples (collected from different surveyed locations) using CTAB method. The DNA extracted from asymptomatic samples collected from healthy brinjal fields was used as negative control. Before PCR assays, the quality of the DNA was checked with Nanodrop. The DNA extracted from the susceptible standard check maintained at ICAR-IIVR was used as positive control in the study. Approximately, 50 ng/μl DNA was used in the PCR for the detection of phytoplasma using phytoplasma-specific 16S ribosomal DNA (rDNA) primers P1/P7 followed by R16F2n/R2 nested primer pairs (Deng and Hiruki 1991; Gundersen and Lee 1996). The samples were collected from fields at distances of 10 km and used for 16S rDNA gene sequencing as described by Ashwathappa et al. (2020). Similarly, the total genomic DNA extracted from leafhoppers (five each from different surveyed locations as well as from eggplant experimental plot at ICAR-IIVR) was used as template for PCR assays (De Barro and Driver 1997).

Pair wise sequence comparison of 16Sr RNA gene and phylogenetic analysis

The 16S rRNA gene sequences derived from the eggplant and insect samples were assembled and edited using ClustalX2 software. They were subjected to BLAST, NCBI to search for similar sequences in the database. Query sequences with close and similar matches to 16S rRNA gene sequences of the members of phytoplasma group/subgroup representative’s available in the GenBank database were retrieved (Table 2). The sequence identity matrices for the BLL strains together with phytoplasma group/subgroup available in the database were generated using Bioedit Sequence Alignment Editor (version 5.0.9) and a phylogenetic tree was constructed by MEGA 7 software using the neighbor-joining method with 1000 bootstrap replications (Kumar et al. 2016). The 16S rRNA gene sequence (Acc. No. U14905) of Acholeplasma laidlawii was used as outgroup to root the tree.

Table 2 List of the 16Sr DNA sequences of phytoplasmas employed in the phylogenetic analysis.
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Virtual RFLP analysis

The 16S rRNA gene sequences of BLL strains corresponding to the R16F2n/R16R2 fragments were subjected to in silico RFLP analysis using the iPhyClassifier online tool and compared with published RFLP patterns of representative sequences of phytoplasma strains of 16SrVI-D (Ac. No. X83431) and 16SrVI-A (Ac. No. AY390261) subgroups (Zhao et al. 2009).

Screening of eggplant varieties and wild Solanum spp. against BLL disease

The seeds of 55 eggplant varieties and 17 wild Solanum spp. were obtained from the Crop Improvement Division, ICAR-Indian Institute of Vegetable Research (IIVR), Varanasi for screening against BLL disease. The seeds were sown in the raised seed bed (10 m × 5 m) with potting soil in an insect proof greenhouse at ICAR-IIVR. The seedlings were transplanted in an open field 5 weeks after sowing and also in plastic pots for natural and artificial screening by grafting technique, respectively.

Open field screening

A separate field experiment was conducted using 55 cultivated eggplant genotypes and 17 wild Solanum spp. at the research farm of ICAR- Indian Institute of Vegetable Research, Varanasi (82.52 E longitude; 68 25.10 N latitude), Uttar Pradesh, India for the two consecutive seasons during 2015 and 2016 in a randomized block design with three replications. The 100 seedlings of each eggplant genotypes and wild Solanum spp. accessions were transplanted and raised in plots of size 5 m × 5 m with a spacing of 75 × 75 cm by following the recommended standard agronomic practices. Based on preliminary studies, eggplant cultivar Punjab Barsati was used as a standard susceptible check for this experiment. The transplanted eggplant genotypes and wild Solanum spp. accessions were exposed to natural infestation of leafhoppers. The incidence of the BLL disease was recorded at weekly intervals after 5 weeks of transplanting till the flowering and fruiting stage of the crop. The mean percentage of disease incidence was calculated based on diseased over healthy plants from all the replications. Based on per cent disease incidence, the eggplant genotypes were classified into five categories: (i) immune (0%), (ii) resistant (0.1–10%), (iii) moderately resistant (10.1–20%), (iv) susceptible (20.1–50%), and (v) highly susceptible (> 50%) (Memane et al. 1987; Devi et al. 1995). Further, the infected and healthy plant samples collected from the 55 eggplant genotypes and 17 wild Solanum spp. accessions were subjected to molecular DNA-based screening (PCR assay) using the primer pairs R16F2n/ R16R2 through nested PCR assay as described above.

Artificial Screening by grafting

The 55 accessions of eggplant genotypes and 17 wild Solanum spp. were also subjected to further screening through graft transmission technique (Venkataravanappa et al. 2018a). Ten seedlings from each of different accessions of eggplant genotypes were transplanted in plastic pot with dimension of 30 cm × 15 cm filled with a potting mixture. Grafting was made on the 35 days old eggplant as described earlier using scion from the phytoplasma positive eggplant maintained under polyhouse conditions (Venkataravanappa et al. 2018a). The grafted plants were maintained and observed for symptom expression up to 95 days after grafting followed by PCR indexing.

Study of biochemical parameters

The leaf samples of cultivated eggplant genotypes infected with phytoplasma were collected during peak incidence of the disease including susceptible cultivar (Punjab Barsati) after 45 days after planting for the estimation of defence enzyme activities such as peroxidase, polyphenol oxidase and total phenol content.

Peroxidase enzyme

Peroxidase enzyme activity was analyzed by following the method of Shannon et al. (1996). One gram of fresh leaf tissue from each cultivar was macerated with help of sterilized pestle and mortar by adding Tris-HCl buffer (pH 7.6). The extract was centrifuged for 30 min at 15000 × g at 4 °C. The chilled supernatant (0.1 ml) was mixed with 2.8 ml of reaction mixture containing 0.5% o-dianisidine dissolved in methanol, 0.28 ml sodium acetate buffer and 2.4 ml water. Hydrogen peroxide (30%) was added (0.1 ml) to reaction mixture to start the reaction. Changes in absorbance values were recorded at an interval of one minute at 430 nm up to 3 min.

Polyphenol oxidase enzyme

For estimation of polyphenol oxidase activity, one-gram fresh leaf tissue from each cultivar was macerated with phosphate buffer (pH 6.0) at 4 °C in a sterilized pestle and mortar. The extract was centrifuged at 15000 × g for 30 min at 4 °C. To the extract (0.5 ml), 2 ml phosphate buffer and 0.5 ml (0.01 M) catechol was added. Changes in absorbance values were recorded at 410 nm up to 3 min at an interval of one minute (Matto and Diamond 1963).

Total phenol content estimation

Total phenol content of the eggplant cultivars was estimated by grinding 1 g leaf sample with 10 ml of 95% ethanol in a pestle and mortar, subsequently boiling in a water bath for 30 min. Then, the extract was centrifuged for 20 min at 10000 rpm. Each sample was extracted three times and the supernatants were pooled followed by evaporation of ethanol at 80 °C in a hot water bath. The residue was dissolved in 50 ml of water, 3 ml of extract was taken and 0.5 ml of Folin-Ciocalteau reagent and 4 ml of Na2CO3 (20%) was added. The mixture was kept in boiling water bath for 1 min and cooled. The phenol content was estimated using a catechol standard by recording the absorbance at 650 nm.

Results

Survey, incidence, and symptomatology

Under natural conditions, the major symptoms recorded on eggplant varieties were pale green-coloured little leaf leaves, phyllody, big bud, witches’ broom, and excessive axillary shoot proliferation (Fig.1a–f). The floral buds were malformed into leaf like structures and infected plants failed to set fruits. Infected plants could be easily identified from a distance because of their little leaf and bushy appearance. The incidence and severity of the BLL in various fields of Varanasi, Mirzapur and Janupur was recorded from 8 to 30%.

Leafhopper identification and population studies

The major leafhopper species collected from the eggplant fields at surveyed locations and experimental plots were identified as Amrasca biguttula (Ishida), Empoasca prima (Distant) and Hishimonus phycitis (Distant). Out of these, H. phycitis, was identified as predominant species present in brinjal fields at all the surveyed locations as well as at the experimental plots of ICAR-IIVR, Varanasi (Fig. 3b-d). The highest count of insect vector (H. phycitis) was recorded during second fortnight of September (29 insects/trap) and first fortnight of October (26 numbers/trap) (Table 1). The high populations of H. phycitis in experimental eggplant plots were found to be positively correlated with the incidence of BLL disease in later months. The percent little leaf disease in the experimental plots ranged from 10.21 to 52.48% (Table 1).

Fig. 3
figure 3

(a) Installation of yellow sticky trap under field condition, (b) Insect vector (Hishimonus phycitis) on yellow sticky trap, (c) Lateral and (d) dorsal view of Hishimonus phycitis

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Detection of phytoplasma

Phytoplasma indexing in 58 symptomatic eggplant samples (collected from different surveyed locations) along with standard susceptible check as a positive control were confirmed through amplification of ~1.8 kb products in the first round PCR (P1/P7 primers) and ~1.2 kb products in nested PCR assays with primer pair R16F2n/R2, respectively (Table 2). No amplifications were noticed in any of the asymptomatic samples collected from different surveyed locations.

Three leafhopper species, Amrasca biguttula, Empoasca prima and Hishimonus phycitis, were counted as major dominant species in eggplant fields based on populations trapped during the 2 years. For phytoplasma indexing (19 samples of three species of leaf hopper collected from surveyed locations of 10 m × 10 m plot size at Varanasi, Mirzapur and Jaunpur and five samples from experimental plots at ICAR-IIVR) were subjected to PCR assay using primer pairs, P1/P7 and R16F2n/R2. Only four H. phycitis samples (two each from the experimental plots at Varanasi and Mirzapur) were detected positive for phytoplasma with the amplification product of 1.25 kb in nested PCR assay (data not shown). However, no phytoplasma amplification was achieved in any of other two leafhopper species (A. biguttula, E. prima) collected from Varanasi, Mirzapur and Jaunpur districts of Uttar Pradesh in the present study.

Since most of the samples from similar places have 16S rRNA sequence identity of 100%, representative 15 BLL samples of which six were from Varanasi (BLL-1, BLL-14, BLL-15, BLL-16, BLL-22, BLL-23), six from Mirzapur (BLL-24, BLL-25, BLL-26, BLL-27, BLL-28, BLL-29), three from Jaunpur districts (BLL-30, BLL-31, BLL-32) and four leaf hopper samples of H. phycitis (Hp1 to Hp4) (two from the experimental plot and two from Varanasi and Mirzapur) (data not shown) were sequenced in both directions and the consensus sequences of the isolates were deposited in the NCBI database (Acc. Nos KC478607, MW273752-MW273769) (Table 1).

Pair wise comparison of 16S rRNA gene sequences of BLL and leafhopper phytoplasma strains

Pair wise comparison of 16S rRNA gene sequences from 15 eggplant phytoplasma isolates (BLL-1, BLL-14, BLL-15, BLL-16, BLL-22, BLL-23, BLL-24, BLL-25, BLL-26, BLL-27, BLL-28, BLL-29, BLL-30, BLL-31, BLL-32) and four leafhopper isolates (Hp-1 to Hp-4) with different phytoplasma group/subgroup representatives available in NCBI database showed maximum 16S rRNA gene sequence similarity (99.3 to 100%) of BLL and Hp isolates with Ca. P. trifoli (16SrVI group) related strains (Table 2).

Phylogenetic analysis of 16S rRNA gene sequences

The phylogenetic tree was generated by comparing the 19 (15 eggplants and four from insect vector) 16S rRNA gene sequences of BLL phytoplasma and Hp isolates identified in the present study with other selected phytoplasma of different groups and subgroups retrieved from GenBank database. The phylogenetic tree analysis of 16SrDNA sequences of BLL and Hp phytoplasma isolates showed clustering of BLL and Hp isolates into two subgroups strains,16SrVI-D (KC478607) and 16SrVI-A (AY390261), which belonged to Ca. P. trifoli (16SrVI group) related strains (Kumar et al. 2015; Venkataravanappa et al. 2018a) (Fig. 4).

Fig. 4
figure 4

Phylogenetic tree based on sequences of 16SrRNA gene from little leaf phytoplasma and Hishimonus phycitis with other phytoplasma strains using Neighbor-joining algorithm. Horizontal distances are proportional to sequence distances; vertical distances are arbitrary. The trees are unrooted. A bootstrap analysis with 1000 replicates was performed and the bootstrap percent values more than 50 are numbered along branches.

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The restriction RFLP profiles of the three BLL phytoplasma isolates (BLL23, BLL24, and BLL25) showed 100% similarity to the restriction profiles of 16SrVI-D subgroup representative strain (Acc No. X83431) with a similarity coefficient of 1.00 and restriction profiles of the other three BLL isolates (BLL16, BLL27 and BLL30) were found similar with 16SrVI-A subgroup representative strain (Acc. No. AY390261) with a similarity coefficient of 1.00. However, the BfaI endonuclease restriction profile of thirteen BLL isolates (BLL1, BLL14, BLL15, BLL22, BLL26, BLL28, BLL29, BLL31, BLL32, Hp1, Hp2, Hp3 and Hp4) of the present study was matched different from all previously reported 16SrVI subgroup strains with a similarity coefficient ranging from 0.86 to 0.97 (Fig. 5).

Fig. 5
figure 5

Virtual RFLP patterns derived from in silico digestions, using iPhyClassifier for R16F2n/R16R2 fragment of 16S rRNA gene from strains of BLL and insect vector (BLL1, BLL22, BLL28, BLL31, Hp1, Hp2, Hp3 and Hp4). The virtual RFLP patterns strains of BLL (BfaI) distinguish the strain from those in a number of subgroups in group 16SrVI. The restriction fragments were resolved through 3% virtual agarose gel. M: Molecular Ladder phiX174 DNA HaeIII digest.

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Screening of eggplant genotypes/lines and wild Solanum sp. against BLL disease

Open field screening

Responses of 55 eggplant genotypes and 17 wild Solanum species to phytoplasma infection was observed and their levels of resistance was recorded (Table 3). Out of these, only one cultivated eggplant variety Uttara and 17 wild Solanum species were found to be immune to the BLL disease, as no visual symptoms were observed during the entire course of study and were tested negative for phytoplasma DNA amplification in nested PCR assays (Fig. 6a-b). Whereas Pusa Ankur variety was found resistant with disease incidence of 6.5% and other 12 eggplant genotypes viz., KKM 01, Ajad Eggplant 4, CHBR 1, Rajendra Eggplant 9, BH 2, CHBR 2, Utkal Madhuri, Bhagyamati, Shobha, Arka Nidhi, CH 215 and Arka Keshav were recorded as moderately resistant with disease incidence ranging between 10.1–20%. The remaining 41 eggplant genotypes were graded as susceptible and highly susceptible to BLL disease (Table 3). Further, total genomic DNA extracted from all the 55 eggplant genotypes/lines and 17 wild Solanum species were subjected to PCR analysis using phytoplasma specific universal primers. Further, immunity in Uttara and 17 wild Solanum species was confirmed with no amplification of phytoplasma DNA in PCR indexing (Fig. 7a & b).

Table 3 Reaction of different eggplant varieties/lines and wild Solanum spp. to phytoplasma (Little leaf disease)
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Fig. 6
figure 6

Response of Uttara variety along with a susceptible check (Punjab barasati) to phytoplasma disease under field conditions (a) Punjab barasati, (b) Uttara variety

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Fig. 7
figure 7

PCR amplification of 16S rRNA gene of little leaf phytoplasma infecting (a) 55 eggplant varieties/lines and (b) 17 wild Solanum spp.

(a) Marker 1 kb, Lane 1 to 55: Punjab Barasati, KKM 01, Kashi Prakash, IVBL-22, Ajad Brinjal-3, Ajad Brinjal-1, Ajad Brinjal-2, Ajad Brinjal-4, PR-5, CHBR 1, Rajendra Eggplant-9, Pusa Ankur, JB-9, JB-8, Arka neelkanth, Co-2, Arka kranti, Jb 69, IBH-3, BH 2, DBL-24, CHBR-2, Aruna, Uttakal Tarini, Br14, Pusa Shymel, RS 356, Uttra, Utkal Madhuri, Bhagyamati, Pusa Uphar, JB-6, KS-339, B. Devariya, ABSR-2, RCMBL-02, Shobha, Adm-190, Pant Rituraj, RM Jaint, DBR-8, DBR-31, Arka Nidhi, Jawahar Brinjal, CH-215, SLW, Punjab sadabahar, Gulabi, S. pratibha, JB- 67, Arka keshav, Co-11, JB-2, Pusa Purple Long, RCMBL 04–04,

(b) Marker 1 kb, Lane 1 to 17: Solanum torvum, S. incanum, S. gilo (ADM-117) S. kashianum (ADM-183), S. laciniatum (EC 790351), S. sisymbifolium (EC 790352), S. aethiopicum (EC 790353), S. macrocarpum (EC 790354), S. anguivi (EC 790358), S. aethiopicum (EC 790360), S. aethiopicum (EC 790361), S. viarum (EC 790363), S. xathocarpum (EC 790365), S. sundatum (EC 790349), S. anguivi (EC 790359), S. aethiopicum (EC 790357)

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Artificial screening by grafting

All the 55 cultivated eggplant genotypes and 17 wild Solanum species screened under field conditions were also used for artificially screening against BLL disease through grafting technique. Among them, 54 cultivated eggplant genotypes expressed the typical little leaf and phyllody symptoms after incubation of 20–25 days; whereas, the variety Uttara did not show any symptoms even after grafting for one month. Similarly, 17 wild Solanum species did not show any trace of phytoplasma symptoms after incubation of 20–25 days (Table 4). Further, total genomic DNA extracted from all the 72 eggplant genotypes and wild Solanum species were subjected to PCR analysis using phytoplasma universal primers. PCR amplicons of ~1.2 kb were detected in all genotypes of eggplant except DNA isolated from the variety Uttara and 17 wild Solanum species (Table 4).

Table 4 Artificial Screening (grafting) different eggplant varieties/lines against phytoplasma (Little leaf disease)
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Evaluation of eggplant varieties for defence enzyme activities

The eggplant genotypes were evaluated in two consecutive years under natural disease pressure, showed varying degree of susceptibility to BLL disease. Therefore, to know the status of defence enzymes activity in eggplant genotypes infected with BLL disease, the healthy and diseased leaf samples of 55 eggplant genotypes were collected during peak severity and incidence of the disease at 60 days after sowing from the field for the estimation of peroxidase, polyphenol oxidase activities and the total phenol content.

Peroxidase enzyme activity

The peroxidase enzyme activity in terms of changes in absorbance (g/min) was recorded in range from 0.47–2.11 and 0.58–1.81 in healthy and BLL diseased eggplant genotypes, respectively. The highest activity was recorded in Uttara, an immune variety followed by PR-5, Shobha, ABSR-2, Pusa Barsati, KKM01, and Rajendra Brinjal-9. The lowest enzyme activity was observed in the highly susceptible genotypes JB6, Co11, Pusa purple long, Akra Nidhi and Pusa Uphar. Overall, the healthy samples of the eggplant varieties/lines recorded maximum peroxidase enzyme activity than the phytoplasma associated identified diseased varieties/lines (Fig. 8a).

Fig. 8
figure 8

(a) Peroxidase, (b) Polyphenoloxidase activity and (c) Phenol content in the 616 healthy and phytoplasma infested 55 eggplant varieties/lines

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Polyphenol oxidase enzyme activity

The polyphenol oxidase enzyme activity profile in terms of changes in absorbance (g/min) ranged from 0.50–0.94 and 0.41–0.83 in healthy and BLL infected eggplant genotypes. The genotypes with maximum polyphenol oxidase activity showed resistance reaction to phytoplasma infection. The eggplant genotypes such as Azad Kranti, SLW, Pusa Shymel, CO2, JB6, CHBR1 and KKM01 recorded highest enzyme activity along with the immune variety Uttara. The susceptible eggplant genotypes such as Utkal Tarini, Aruna, BH2, Punjab Sadabahar and ABSR-2 were recorded with lowest enzyme activity (Fig. 8b).

Total phenol content in eggplant varieties

The total phenol content (mg/100 g) ranged from 133.72–785.12 and 349.2–567.21 in healthy and BLL infected eggplant genotypes, respectively. The moderately resistant varieties like Arka Nidhi, Rajendra Brinjal 9 and Azad Brinjal 4 recorded a higher phenol content. The lowest phenol content was observed in susceptible genotypes such as DBL24, DBR8, Pant Rituraj, CHBR2, JB2, RM_Giant, BH2, DBR31 and other susceptible varieties/lines (Fig. 8c).

Discussion

Phytoplasma diseases are major constraints for production of many economically important field crops (Rao et al. 2018). The diseases caused by phytoplasmas are constantly increasing over the years with an uncertain etiology and diverse geographic distribution (Bertaccini and Duduk, 2009; Rao et al. 2018). Accurate detection of phytoplasma is a prerequisite for the management of the disease. The nucleic acid-based diagnostic technique such as polymerase chain reaction is routinely used for authentic detection of phytoplasmas. In the present study, initial diagnosis was done based on symptoms appearance in different eggplant samples collected from three eggplant growing districts of Uttar Pradesh, India. This was subsequently confirmed by PCR and nested-PCR assays (Kumar et al. 2015; Rao and Kumar 2017; Venkataravanappa et al. 2018a).

Phytoplasma specific primers designed based on conserved 16S rRNA gene are being used for detection of a wide range of phytoplasmas associated with different plants and insect vectors (Namba et al. 1993). However, nested-PCR assay is performed for preliminary amplification of universal primer pair followed by use of a second set of internal sized primer capable of detection of phytoplasmas in the infected tissues. It also enhances the sensitivity and specificity for detection of phytoplasmas present even in low titres in the plant tissue (Ashwathappa et al. 2019,2020; Hemmati et al. 2020).

So far, phytoplasmas belonging to 16Sr II-D and 16Sr VI-D were reported to infect eggplant crop in India (Kumar et al. 2017; Rao 2021; Venkataravanappa et al. 2018a). In the present study, 16SrVI-A and 16SrVI-D subgroups have been identified and characterized in eggplant samples collected from three districts (Varanasi, Mirzapur and Jaunpur) of Uttar Pradesh, India based on 16S rRNA gene sequence comparison and restriction profiles in virtual RFLP analysis. Further the virtual RFLP analysis 16S rRNA gene of thirteen BLL isolates (BLL1, BLL14, BLL15, BLL22, BLL26, BLL28, BLL29, BLL31, BLL32, Hp1, Hp2, Hp3 and Hp4) revealed that they could be tentatively classified into a new variant under 16SrVI phytoplasma group, because of different restriction pattern of Bfa, which needs further investigations with real RFLP analysis and other multilocus gene mapping. Available literature also suggests that 16SrVI group infects a wide range of plants including weeds, ornamentals, vegetables, fruits and trees in India (Rao 2021).

In the present study, the leaf hopper H. phycitis has been identified as a putative vector that transmits BLL disease under natural conditions. It was confirmed based on the presence of phytoplasma in leafhoppers collected from infected eggplant fields in PCR assays. The high population densities of vector, H. phycitis was recorded in eggplant fields from March to May and Sept to Nov during 2015 and 2016 followed by high incidence of the BLL disease in eggplant fields at Varanasi. The H. phycitis was reported as a natural and putative vector for BLL phytoplasma strain associated with 16SrVI-D subgroup in different states of India (Kumar et al. 2017; Dutta et al. 2020). Similarly, H. phycitis is also reported as natural vector of sesame phyllody in India (Un Nabi et al. 2015), lime witches’ broom in Iran (Siampour et al. 2006; Salehi et al. 2007) and several phytoplasma strains associated with ornamentals in India (Rao 2021).

Different defense mechanisms function in host plants to tolerate/overcome different biotic/abiotic stresses. The mechanism of host plant resistance in response to biotic stress consists of a series of changes in biochemical events such as emergence of free radicals, damage of cellular biomolecules, and subsequently malfunctioning of immune system (Bendich 1996). The antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) play major roles in the detoxification of ROS and maintain adequate level of antioxidants in the cells (Baker and Orlandi, 1995). In the present study, the peroxidase (PO), polyphenol oxidase (PPO) enzyme activity and total phenols content in the eggplant was studied in BLL resistant and susceptible eggplant genotypes. An increased activity of defense enzymes such as PO and PPO were observed in resistant genotypes of eggplant vis-à-vis susceptible ones. The literature survey showed that, defense enzymes like PO and PPO play a major role in the defense mechanism of plants (Shivalingaiah and Umesha 2016). PPO carries out oxidation of phenols to quinones. The level of resistance to pathogens varies with different plants and varieties of same plant (Bingham et al. 2009). Enhanced PO and PPO enzyme activities confer resistance against pathogen infection (Melo et al. 2006). Similarly, the total phenol content and antioxidative enzyme levels were compared in tomato leaf curl Palampur virus in infected and healthy pumpkin plants revealed that, there was substantial increase in the total phenol content and antioxidative enzymes (SOD, GPX, APX and CAT) levels in leaves (72%) and fruits (300%) in response to virus infection of pumpkin plants (Namrata et al. 2013). The increased activity of polyphenol oxidase has been well documented in many plants subjected to various biotic and abiotic stresses (Kumar et al. 2010). Phenols also play an important role in plant defense, host pathogen interaction and disease development in infected plants (Jabeen et al. 2009; Kumar et al. 2010). The increased quantity of phenolics in phytoplasma-infected eggplants may also contribute towards the resistance against infection.

The major limitation in the production of eggplant is its susceptibility to different biotic stresses. Among them, little leaf, is an important disease of eggplant which is caused by phytoplasma and transmitted by leaf hoppers (Rao and Kumar 2017). At present, there is a lack of information about resistant sources and reliable screening techniques to identify resistance source in India for effective phytoplasma management. Also, there was often a confusion between resistance of the plant to the phytoplasma and resistance to the insect vector. In the present study, we have identified one immune variety (Uttara), 17 immune wild Solanum species, one resistant (Pusa Ankur) and 12 moderately resistant eggplant varieties against BLL phytoplasma. These resistant accessions identified in the present study could be used as pre-breeding materials to breed resistant cultivars and map BLL resistance genes in eggplant. We have also confirmed the presence of new tentative 16SrVI subgroup variant strains of phytoplasma associated with BLL disease in north eastern plain region (Varanasi, Mirzapur and Jaunpur) of India, which needs further investigation for finer classification and nomenclature of the subgroups associated. Further studies on survey, detection, identification, and characterization of hitherto unknown phytoplasma strains are required to understand the clear picture of the diversity and geographical distribution phytoplasmas associated with BLL to develop a suitable and effective management strategies to manage the BLL incidence in India.