Utilization of 16Sr RNA and secA genes for molecular discernment of ‘Candidatus Phytoplasma australasiaticum’ strain associated with linseed germplasm in India

Abstract

The growing prevalence of phytoplasma associated symptoms on linseed or flax (Linum usitatissimum L.) germplasm at Indian Council of Agricultural Research- National Bureau of Plant Genetic Resources (ICAR-NBPGR) fields was noticed during the 2019–22 growing seasons. The characteristic phytoplasma symptoms of phyllody, stem fasciation, stunting, along with floral and capsule malformations were observed in 41 linseed accessions grown at experimental fields of ICAR-NBPGR, Delhi. During 3 years, the presence of phytoplasma in symptomatic linseed accessions was confirmed by nested-PCR assays utilizing 16S rRNA and secA gene-specific primers. The 16S rRNA and secA gene sequences of linseed phytoplasma strains from the representative symptomatic 41 linseed accessions exhibited 100% sequence identity among themselves and 99.93% and 99.82% sequence homology with reference strain, ‘Candidatus Phytoplasma australasiaticum’ (GenBank Accession: Y10097). Phylogenetic analysis of 16S rRNA and secA gene sequences clustered the linseed isolates with the peanut witches’ broom group belonging to ‘Ca. P. australasiaticum’ strains. The virtual RFLP analysis of 16S rRNA F2nR2 fragment (~1.2 kb) of linseed phytoplasma strains further classified it into 16Sr group II, subgroup D. Our results suggested confirmation of the association of ‘Ca. P. australasiaticum’ strain (16SrII-D) in the linseed germplasm accessions from North India, which is the first report from India. The phytoplasma infection also reduced the growth and yield parameters of two linseed accessions (IC0498748 and EC0718851).

Introduction

Linum usitatissimum L., commonly known as linseed or flax, is one of the key oil and fiber yielding crop species having prodigious applications in the food, textile, paint and pharmaceutical sectors (Żuk et al. 2015; Kaur et al. 2023b). Its cultivation can be traced back > 8000 years (van Zeist and Bakker-Heeres 1975). Canada is the leading producer and exporter of linseed worldwide, while China is the largest importer accounting for 26.8% of total global flax import in the year 2020 valuing 31,108 M US$. India ranks third in terms of the area under cultivation and 7th in production (FAOSTAT 2022; Yadav et al. 2022). Besides being an important dual-purpose commercial crop for oil and fiber extraction, linseed has rapidly emerged as an ideal nutritional supplement and functional food owing to very dense nutritional composition constituted by high alpha-linolenic acid (ALA; 55–57%), proteins (up to 18.29%), fibers (27.3%) and lignans, particularly secoisolariciresinol diglucoside (SDG; 294–700 mg/100 g) (Kajla et al. 2015; Langyan et al. 2023). However, numerous biotic stresses are the major impediments to achieving high yield stability and sustainability in linseed production amid the rising global demands (Kaur et al. 2018; 2023b; Ajithkumar et al. 2021; Yadav et al. 2023; Nair et al. 2023).

Phytoplasmas are non-culturable, cell wall-less prokaryotic microorganisms colonizing the phloem tissues of plants. They are known to infect over 800 plant species and cause devastating losses in agronomically important crops worldwide. The threat of phytoplasma diseases in the world is increasing with a high impact on crop yield, quality and economic loss (Rao et al. 2018; Bertaccini 2022; Tiwari et al. 2023). In India, recent shreds of evidence showed that phytoplasma associated with plants including vegetables, fruits, ornamentals, oil crops, sugarcane, palms, and weeds are increasing at an alarming rate with significant yield losses (Rao 2021; Tiwari et al. 2023).

Flax phyllody was first documented in Canada in the year 1957, where it caused an epidemic and resulted in significant yield losses (Saharan and Mehta 2005). The causal agent of the disease was initially thought to be a virus, but it was later determined to be a phytoplasma. Akhtar et al., (2013) identified ‘Candidatus P. aurantifolia’ (subgroup 16SrII-D) strain associated with flax phyllody and stem fasciation from Pakistan. In India, ‘Ca. P. asteris (16SrI-B) and Clover proliferation (16Sr-VI) groups were reported associated with phyllody and stem fasciation of linseed from West Bengal (Biswas et al. 2014). Further, Ajithkumar et al., (2021), reported linseed to be a new host for ‘Ca. P. cynodontis’ (16SrXIV-A) strain associated with stem fasciation and phyllody from Karnataka, India. Since then, there has been no report available on the occurrence of phytoplasma strains from other parts of India and abroad. Therefore, the present investigation was planned and executed to characterize phytoplasma associated with linseed germplasm accessions by using molecular markers employing 16S rRNA and secA gene-specific primers.

Materials and methods

Plant materials

The whole set of linseed germplasm collection (2800 accessions) conserved at the National Genebank at the Indian Council of Agricultural Research- National Bureau of Plant Genetic Resources (ICAR-NBPGR) was grown in the field for three consecutive years (2019–20, 2020–21 and 2021–22) for agro-morphologic characterization and natural disease incidence. The sowing was done in the last week of October at ICAR-NBPGR, experimental field, IARI, New Delhi (28° 38′ 53.7″ N, 77° 09′ 05.4″ E and 218 m above mean sea level) as per the methodology and experimental details mentioned by Kaur et al., (2023a). The other important objective of the study was to evaluate 2800 linseed germplasm accessions (2515 representing India and 285 exotic collections primarily from Canada, Argentina, Australia, Russia and the USA) for observing phytoplasma disease incidence under field conditions. After the appearance of the first incidence of phyllody symptoms in linseed accessions in the experimental field during the cropping season of the year 2019–20, the number of symptomatic accessions were counted and incidence was expressed on percent basis by dividing the number of accessions showing symptoms by the total number of germplasm (2800 accessions) grown in the field. Various symptoms expressed on different linseed germplasms were also recorded under natural field conditions during all 3 years of cropping seasons. Percent disease incidence for each plot of symptomatic accession was recorded by counting the number of symptomatic plants/total number of plants in the plot × 100.

Agro-morphologic data

The agro-morphologic data on plant height (cm) and yield parameters (seed yield/plant (g), number of capsules/plant and number of seeds/capsule) were recorded in two representative accessions (one indigenous germplasm: IC0498748 and one exotic germplasm: EC1074585) having severe disease incidence in all the 3 years. The observations were recorded upon attainment of physiologic maturity (when 80% of the plants in the plot showed browning of capsules in three replicates) in symptomatic and healthy plants. Student’s t-test was used to analyze statistical significance difference (*P < 0.05, **P < 0.01 and ***P < 0.001) using the online GraphPad web server https://www.graphpad.com/quickcalcs/ttest1/?format=SD (accessed April 2024).

DNA extraction and PCR-based phytoplasma detection

The molecular confirmation of phytoplasma infection was conducted for 3 years in the representative symptomatic accessions. Initially, the infected leaf and stem tissues were excised from symptomatic accessions in the field during the year 2020–21, while two chosen accessions exhibiting severe symptoms consistently were considered further in the next two years (2021–22 and 2022–23). The tissue samples were excised from the representative plants of symptomatic as well as asymptomatic accessions in three replicates. Total genomic DNA was isolated using 0.5 g of tissue from the samples following the CTAB method (Doyle and Doyle 1987). Qualitative and quantitative analysis of DNA was carried out spectrophotometrically using Nanodrop™ One Microvolume UV–Vis Spectrophotometer (Thermo Scientific™). An amount of 50 ng/µl DNA extracted from symptomatic and asymptomatic linseed plants were subsequently used as a template for the nested-PCR assays.

The 16S rRNA gene was amplified using phytoplasma universal primer combination P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) followed by R16F2n/R16R2 (Gundersen and Lee 1996) using TaKaRa LA Taq® DNA polymerase (Takara Bio). The PCR conditions for the amplification with primers P1/P7 involved an initial denaturation at 94 °C for 3 min, followed by 32 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min and final extension at 72 ℃ for 10 min. The PCR product was diluted 1:30 to be used as template for the nested-PCR cycle with all the reagent concentrations and conditions like first PCR except the primer R16F2n/R16R2 pair were used instead of P1/P7. The PCR product of the first as well as second nested-PCR product of the 16S rRNA was gel eluted and purified using gel extraction kit (QIAGEN, Chatsworth, CA, USA). The phytoplasma positive linseed selected accessions showing DNA amplifications were Sanger sequenced initially and analyzed for homology using NCBI BLASTn programme (http://www.ncbi.nlm.nib.gov/).

To further confirm and delineate the phytoplasma infestation, conserved housekeeping gene secA was amplified using secA gene-specific primers pairs (secA-Forward/secA-Reverse3 and secA-Forward2/secA-Reverse3) (Hodgetts et al. 2008) (Table 1). Five µl of the 16S rRNA and secA genes amplified PCR products were subjected to electrophoresis in a 1.2% (w/v) agarose gel, stained with ethidium bromide and observed under UV-transilluminator. The amplified gel-eluted PCR products were Sanger sequenced and submitted to NCBI GenBank.

Table 1.

List and sequence of primers used in the present study

Primer name	Sequence
P1	5′-AAGAATTTGATCCTGGCTCAGGATT-3′
P7	5′-CGTCCTTCATCGGCTCTT-3′
R16F2n	5′-TGACGGGTGTGTACAAACCCCG-3′
R16R2	5′-GAAACGACTGCTAAGACTGG-3′
secA-F	5′-AGGTGTTATTGGGGATATTTT-3′
secA-R	5′- GCTTGATGCAAACCATCACTG-3′

Phylogenetic analysis and in-silico restriction fragment length polymorphism (RFLP) analysis

To establish the phylogenetic relationship of identified linseed phytoplasma isolates with identified phytoplasma groups, representative 16S rRNA and secA gene sequences were retrieved from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) and assembled using Clustal Omega program (Sievers et al. 2011). The selected sequences represented major phytoplasma species and subgroup lineages infecting different economically important crops and plant species. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) with default values using MEGA11 (Tamura et al. 2021). The phytoplasma sequence corresponding to the 16S rRNA region delineated with R16F2/R16R2 primers were subjected to in-silico RFLP analysis through iPhyClassifier tool (Zhao et al. 2009) and compared with restriction profiles of the representative phytoplasma strains of different ribosomal groups and sub-groups.

Results

Disease symptomology and incidence

The phytoplasma-induced symptoms were observed in two linseed accessions (IC0498748 and EC0718851) out of 2800 germplasm accessions during the first-year (2019–20) cropping season at the experimental farm at ICAR-NBPGR, New Delhi, India. The disease symptoms later progressed to 25–41 accessions in 2nd and 3rd years respectively in the same set of germplasm accessions under similar field conditions. The number of symptomatic plants increased with cropping season beginning with 0.07% recorded in the first year of 2019–20 to ∼1.5% in the year 2021–22 (Table 2). The characteristic phytoplasma-induced symptoms were apparent after 45 days of sowing in the month of December in all 3 years. The major common symptoms recorded in 41 linseed germplasm accessions were little leaf, witches’ broom, fasciated stem, and twisted inflorescence with malformed flowers and capsules at ICAR-NBPGR research farm, New Delhi (Fig. 1). The maximum severity of symptoms was recorded in two genotypes (IC0498748 and EC1074585) in all the 3 years of planting.

Table 2.

Linseed germplasm accessions showing phytoplasma symptoms during three consecutive cropping seasons

Year	National identity	No. of symptomatic accessions	Total no. of accessions grown	Percent incidence (%)
2019–20	IC0498748*, EC0718851	2	2800	0.07
2020–21	IC0621685, IC0199761, IC0118849, IC0096737, IC0118857*, IC0498933, IC0498767, IC0526119, IC0498738, IC0499193, IC0305055, IC0356381, IC0526027, IC0499103, IC0384566*, IC0498748*, IC0499073, IC0499047, IC0526095, IC0384576, EC1074585*, EC0041774-A, IC0053284, IC0623723, EC0541198	25	2800	0.90
2021–22	EC0041672, IC0621686, IC0621685, IC0623723, IC0426928, IC0498530, IC0498679, IC0498866, IC0096485, IC0525924, IC0525924, EC0718851, IC0564628, IC0096648, IC0498748*, IC0499135, EC0718826, EC0041528, IC0499073, IC0267547, IC0199753, IC0499195, IC0054977, IC0096638, EC0541213, IC0526064, IC0498768, IC0096755, IC0498769, EC1074585*, IC0058332, IC0567432, IC0564617, EC0041478, IC0498626, IC0585288, IC0499193, EC0541198, IC0499103, IC0118857*, IC0384566*	41	2800	1.46

IC and EC denote Indigenous and exotic collections, respectively. The symbols * represent the accessions used for Sanger sequencing of 16S rRNA gene

Fig. 1.

Comparative morphotype of a phytoplasma-infested and healthy linseed plant at different developmental stages (A, B) Abnormal green structure, shoot apex fasciation (phyllody, virescence and yellowish-green leaves); (C) Twisted inflorescence axis showing small-sized leaves and closely arranged flowers forming clusters giving bushy broom-like appearance; (D) Malformed flowers; (E, F) Intense proliferation of lateral buds causing the formation of fused capsules with sterile and reduced seeds/capsule; (G–K) Phenotype of a healthy non-symptomatic plant bearing buds, flowers and capsules

Phytoplasma characterization

All the 41 symptomatic linseed samples yielded phytoplasma-specific amplification of the expected size of ∼1.2 kb with P1/P7 and R16F2n/R16R2 primer pairs (data not shown). The amplified nested-PCR products (∼1.2 kb) (Fig. 2) were purified and sequenced from both ends.

Fig. 2.

Nested PCR confirmation of symptomatic plants using different sets of universal 16 s rRNA primer combinations followed by secA gene. Left panel: Amplicon of size 1.8 kb using P1/P7 primer combinations; Middle panel: Amplicon of size 1.24 kb using R16F2n/R16R2 primer combinations. Right panel: PCR amplification of secA gene (partial sequence) exhibiting amplicon size of ∼559 bp. M1 and M2 indicate 1 kb DNA marker, while M3 indicates 100 bp DNA marker; NC: Non-symptomatic healthy plant DNA used as negative control; Lanes 1–6: Isolates from different linseed accessions namely EC1074585, IC0498748, IC0096737, IC0526119, IC0356381 and IC0498767, respectively

Consequent sequence analysis of all the 41 PCR products was found to be 100% identical among themselves. Henceforth, the four-sequence data of two of the linseed phytoplasma isolates were deposited in the NCBI GenBank (GenBank accession number: OR178021 for exotic accession EC1074585 and GenBank accession number: OR178439, PP838570, and PP838573 for Indian accession IC0498748, IC0118857, and IC0384566, respectively). The 16S rRNA gene sequence analysis of four linseed phytoplasma isolates examined exhibited the highest sequence identity of 99.93% with ‘Ca. Phytoplasma australasiaticum’ (formerly Ca. P. australasia) 16SrII-D reference strain (GenBank accession number Y10097).

Further, 480 bp amplicon of the secA gene was also amplified in all the symptomatic 41 linseed accessions with secA gene-specific primers (data not shown). The resulting PCR amplicon of ∼559 bp (secA-F and secA-R) from partially amplified secA gene (Fig. 2) was sequenced and deposited in the NCBI database with the GenBank accession number: PP027973 for exotic accession namely EC1074585 and accession number: PP027974 for Indian accession IC0498748. No amplification was obtained in any of the asymptomatic linseed germplasm sample (negative control) collected from the same field during all 3 years (data not shown). The results obtained indicated the presence of phytoplasma in 41 symptomatic linseed germplasm accessions (Table 2).

The comparative virtual RFLP profiling analysis with 17 restriction enzymes using iPhyClassifier was carried out with the query 16S rRNA F2nR2 ~1.2 kb fragment with each other and with the reference strain of 16Sr group II, subgroup D (GenBank accession number: Y10097), which assigned the detected linseed phytoplasma isolates in 16SrII-D subgroup (Fig. 3).

Fig. 3.

Comparative analysis of virtual RFLP using 17 different restriction enzymes (A) Restriction pattern of reference strain (Y10097) showing maximum similarity with the query sequence (B) ‘Ca. P. australasiaticum’ (OR178021)

Phylogenetic characterization

Phylogenetic analysis was conducted based on 16S rRNA and secA gene nucleotide sequences of four linseed phytoplasma strains with the representative strains of the different ‘Ca. Phytoplasma species and sub-groups. The identified linseed phytoplasma strains associated with linseed clustered with the identified members of the 16SrII group representing ‘Ca. Phytoplasma australasiaticum’ into a distinct branch in both phylogeny trees constructed for 16S rRNA (Fig. 4A) and secA gene sequences (Fig. 4B), respectively.

Fig. 4.

Phylogenetic clustering of representative ‘Candidatus Phytoplasmas’ species and sub-groups based on (A);16S rRNA and (B) secA gene sequences. The associated taxa clustered in the bootstrap test (1000 iterations) are shown next to the branches. Red color dots depict the sequence of isolates identified in the present study (Red shaded). A. laidlawii and A. oculi sequences (yellow-shaded) were used as outliers for 16S rRNA and secA genes respectively. GenBank accession numbers for sequences are given in parentheses

Effect of phytoplasma infection on growth and yield parameters

Healthy plants of linseed accessions IC0498748 and EC1074585 were recorded with a mean plant height of 70.6 cm and 69 1 cm, respectively, while the corresponding phytoplasma-infected plants recorded with a mean height of 28.61 cm and 27.6 cm, respectively accounting for a reduction of around 40.2% in the infected plants as compared to healthy ones (Fig. 5A). Total number of capsules per plant were reduced from 209.6 to 62.6 in IC0498748 and 118.4 to 33 in EC1074585 owing to structural malformation (Fig. 5B). Likewise, a lower number of seeds were formed per capsule (2–4) compared to healthy plants (7–8) (Fig. 5C), which may be attributed to poor seed setting in the phytoplasma-infected accessions. All these factors negatively contributed to the overall yield reduction by 68.9 and 61.8% in the accessions IC0498748 and EC1074585, respectively in comparison to the healthy plants (Fig. 5D).

Fig. 5.

Comparative analysis of various agro-morphologic traits in healthy and infected plants (A) Plant height (cm); (B) Total number of capsules per plant; (C) Number of seeds per capsule; (D) Seed yield per plant (g)

Discussion and conclusion

Phytoplasma disease is the major constraint for production and yield losses of many economically important field crops including oil-yielding crops in India and abroad (Rao et al. 2017a, b; Rao 2021; Vemana et al. 2023). The diseases associated with phytoplasmas have consistently increased over the years with diverse geographic distributions (Bertaccini 2022; Tiwari et al. 2023). Accurate detection of phytoplasma infection is a prerequisite for appropriate management of these diseases. In the present study, the detection of phytoplasma strain ‘Ca. P. australasiaticum’ has been confirmed utilizing 16S rRNA and secA gene sequence comparison analysis on 41 linseed germplasm accessions. So far, very few reports are available on phytoplasma infecting linseed crop all over the world. Phytoplasma belonging to the 16SrII-D subgroup has been reported on linseed from Pakistan (Akhtar et al. 2013). However, 16SrI, 16SrVI and 16SrXIV group-associated phytoplasma strains have been reported on linseed earlier from India (Biswas et al. 2014; Ajithkumar et al. 2021).

There is no earlier report available on ‘Ca. P. australasiaticum’ related strain 16SrII-D association with linseed from India. Thus, the presence of ‘Ca. P. australasiaticum’ 16SrII-D related strain associated with linseed is the first report from India. The ‘revised guidelines’ followed proposed amendments and emphasis on the utility of RFLP-based grouping of phytoplasma strains (Wei and Zhao 2022) and whole genome sequences (OGRI values) (Kirdat et al. 2023; Jardim et al. 2023). In the present study, the sequence analysis and phylogenetic classification of linseed phytoplasma strains revealed that identified linseed phytoplasma isolates shared 99.93% sequence homology with ‘Ca. P. australasia’ strain Y10097. However, Oren et al., (2020) have suggested previously, the name Ca. P. australasia violates the international code of nomenclature of Prokaryotes and its orthography appendix. Keeping in view the linguistic accuracy suggested by Oren (2017), it was proposed by Jardim et al., (2023) that ‘Ca. P. australasia’ should not be used in further classification for the 16SrII-D subgroup strain and should be replaced as ‘Ca. P. australasiaticum’. Therefore, the strain examined in the present study has been identified and classified as ‘Ca. P. australasiaticum’ within the 16SrII-D subgroup (Figs. 3, 4).

The growing prevalence of phyllody and the expansion of the geographicdistribution of phytoplasma in linseed is an alarming threat amid climatic fluctuations globally. The present study highlights the incidence of phytoplasma infestation of a phytoplasma strain of ‘Ca P. australasiaticum’ strain 16SrII-D subgroup on the new host, linseed in the northern part of India. This study provides fresh insight into genetic diversity, geographicdistribution, and host range of phytoplasma ‘Ca P. australasiaticum’ strain.

Phytoplasma belonging to the 16SrII-D subgroup is the most widespread phytoplasma strain infecting several vegetable, ornamental, fruit, legume, and oil crops in India (Rao 2021). Besides the 16SrII-D subgroup phytoplasma strains have also been reported widely in several weed species in India and many leafhopper vectors have been identified as natural vectors for these phytoplasma strains from similar locations (Rao 2021). Very recently, the 16SrII-D subgroup strain of phytoplasma was identified in sesame germplasm accessions in 2020–21 from the same location as of present study (Ranebennur et al. 2022a, b). Since, we have again identified a similar strain of phytoplasma associated with several linseed germplasm accessions from the same geographic location, where sesame was reported as a natural host of 16SrII-D strain in previous years, suggests possible transmission of this phytoplasma strain by insect vectors, which needs to be confirmed. The increasing spread of the 16SrII-D phytoplasma strain to new hosts in the northern part of India alarms to study the spread sources and other epidemiologic factors involved in natural spread during previous years. The widespread occurrence of phytoplasma strain ‘Ca. P. autralasiaticum’ (16SrII-D strain) in diverse crops at Delhi including sesame, ornamentals, vegetables and weeds in similar fields may be a major source of natural reservoirs of 16SrII-D subgroup strain. Several leafhoppers are reported as natural vectors for transmission of ‘Ca. P. autralasiaticum’ strains at the same geographiclocation (Rao 2021) which might facilitate the transmission of phytoplasma strain associated with linseed in the present study. The preliminary evidence of ‘Ca. P. autralasiaticum’ presence in several linseed germplasm accessions alarms scientific fraternity and farmers to take important initiatives in developing appropriate management strategies.

Breeders might consider the asymptomatic linseed germplasms identified in the study during the 3 years as a source of resistance in breeding programs or may directly be prompted after confirming their desirable yield and market traits. The germplasm accessions without any phytoplasma suspected symptoms in all 3 years of the experiment (Table 2) could be used as pre-breeding materials to breed resistant cultivars and map phytoplasma resistance genes in linseed. In future, there is a need to continue the search for resistance sources in linseed germplasm. At present, there is no information available for grading resistance in linseed germplasm for effective phytoplasma management. Since, in the present study, losses up to ~60% in growth and yield were recorded in two linseed germplasms, it would be important to develop a resistance scale for screening of linseed germplasm in India and abroad. Further studies on survey, detection, identification and characterization of novel phytoplasma strains in linseed from other geographiclocations in India are required to understand the clear picture of the genetic diversity of phytoplasmas associated with linseed to develop a suitable and effective management strategy.

Author contribution

SKY conducted molecular studies and wrote the manuscript; DS and BJ — provided performance of the plant experiments and assessment of plant characteristics; Field survey and data-record study; LP, GPR and MSS manuscript review; VK — provided chemicals, laboratory facilities, overall supervision and manuscript review. All the authors have read the manuscript and agreed for publication.

Funding

This work was supported by funding for the Linseed Network Project (No. BT/Ag/Network/Linseed/2019–20) from the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India.

Data availability

The original contributions presented in the study are included in the article/supplementary material. The sequence data of the isolates from the present study have been submitted in NCBI database (GenBank IDs: OR178021, OR178439, PP838570, PP838573, PP027973 and PP027974).

Declarations

Conflict of interest

The author(s) declare no conflict of interest.