Identification Of Phytoplasmas Research Articles

Diagnosis of Phytoplasma Associated with the Sandalwood Spike Disease

Sandalwood spike disease (SSD) is a serious disease of Indian sandal tree (Santalum album). The disease is caused by a Candidatus Phytoplasma asteris strain of subgroup 16SrI-B. The disease is naturally spread through leafhopper and root-grafting. The infected plants take long time to express disease symptoms and eventually die. The diagnostic disease symptom of SSD is crowded small chlorotic leaves on stiff twigs that has spike like appearance. There are various biological, histopathological, electron microscopic and molecular methods available for the detection and identification of phytoplasma. Biological and histopathological tests are time consuming and ambiguous. Serological test like ELISA and molecular test like PCR are more versatile and accurate for the detection of phytoplasma. Application of various molecular techniques like nested PCR, RFLP and sequence analysis of 16S ribosomal RNA led to identify the SSD phytoplasma isolates as a member of 16SrI-B and 16SrXI-B phytoplasma subgroups. In this chapter, various diagnostic methods utilised for the detection and identification of SSD phytoplasma are summarised.

First report of Flavescence dorée phytoplasma identification and characterization in three species of leafhoppers

Phytoplasma strains were sporadically found in specimens of Hishimonus hamatus, Japananus hyalinus and Graphocephala fennahi caught during an entomological survey conducted in Southern Switzerland on the edges and woods surrounding cultivated vineyards in 2017. The subsequent molecular characterization of the pathogens allowed to identify them as strains strictly associated to Flavescence dorée, a serious European grapevine disease, transmitted from vine to vine by the leafhopper Scaphoideus titanus. In particular, M12 and M50 genotypes were found in H. hamatus and J. hyalinus, respectively, while G. fennahi harboured mixed phytoplasma genotypes with at least two different Flavescence dorée isolates. The potential importance of these findings in the frame of the Flavescence dorée epidemics in Europe is discussed.

Identification of 16SrII-V Phytoplasma Associated with Mungbean Phyllody Disease in Taiwan.

Mungbean (Vigna radiata (L.) R. Wilczek), an important legume crop in Asia, is primarily cultivated in the central-southern region of western Taiwan. In 2020, mungbean exhibiting typical phytoplasma-induced disease symptoms such as witches' broom, phyllody, virescence, and proliferation was observed in Yunlin County, Taiwan. Moreover, the seed harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbean demonstrated that the occlusion of sieve tubes resulted from the accumulation of phytoplasma-like bodies in sieve elements along with filament-like structures in sieve pores. The association of phytoplasma in symptomatic mungbean was confirmed by PCR analyses of the 16S ribosomal RNA (rRNA) and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma-associated mungbean phyllody disease identified in this study belongs to the 16SrII-V subgroup. BLAST analysis and the phylogenetic analysis indicated that the SAP11-like protein identified in mungbean phyllody disease is identical to peanut witches' broom phytoplasma SAP11, which explains the witches' broom phenotype observed in symptomatic mungbean. The results described in this report confirm that the 16SrII-V phytoplasma, a widely distributed phytoplasma associated with peanut witches' broom disease in Taiwan, has also infected mungbean. This is not only the first instance of mungbean phyllody disease found in Taiwan but also the first instance of mungbean phyllody disease caused by 16SrII-V subgroup phytoplasma.

Improvement in molecular detection of phytoplasma associated with rose by selection of suitable primers and development of a multiplex PCR assay.

Sensitive and effective phytoplasma DNA amplification in symptomatic rose cultivars is a long unresolved problem. In the present study, improvement in standardization for PCR assay for phytoplasma detection was established with rose samples by selection of various combinations of nested primer pairs of 16S ribosomal gene and secA gene. CTAB DNA extraction method was slightly modified by adding 2% polyvinyl pyrrolidone and increased the isopropanol volume which yieldedbetter quality DNA. Best amplification results were achieved in nested PCR assay employing P1/P7, R16mF2/R16mR2 and R16F2n/R16R2, P1/P7 and R16mF2/R16mR2, and R16mF2/R16mR2 and fU5/rU3 primer pairs. Besides, a multiplex PCR assay was also developed and optimized for consistent identification of phytoplasma in rose samples by employing primer pairs of 16S rRNA and secA genes together in a single PCR reaction by optimizing annealing temperature at 55°C.

SecA gene suitability for fast and easy identification of Phytoplasmas by RFLP analysis

In this study we investigated the secA gene as possible marker to supplement the classification scheme based on 16S rRNA sequences. We cloned and sequenced secA gene and its flanking region of ‘Ca. P. asteris’ CPh strain phytoplasma, as well as 1997 bp length secA gene fragment of Canada peach X-disease CX and 1327 bp fragment of ‘Ca. P. ziziphi’ JWB. This helped us to select a primer pair for a single step PCR that can amplify ~1200 kb length fragment of secA gene from different phytoplasma groups. We propose that this fragment could be used in RFLP analysis to quickly identify and distinguish 16SrI-A, I-B, I-C, I-D, III-A, III-B, III-E, III-F, III-H, V-B, XII-B and XXI-A subgroup phytoplasmas using just two restriction endonucleases.

Hishimonus phycitis: Possible Vector of 16SrI-B Subgroup Phytoplama

Witches-broom symptoms of Catharanthus roseus are caused by ‘Candidatus phytoplasma is a terrible disease in the Uttar Pradesh State of India. The disease has been transmitted to infected C. roseus seedlings by grafting and cutting of stems. Heavy infestation of Hishimonus phycitis was recorded in ornamental garden during rainy season with witches- broom and phyllody disease of C. roseus at surveyed location of Shahjahanpur, Uttar Pradesh. To confirm the phytoplasma in C. roseus and the identification of phytoplasma causing insect vector, DNA were isolated from both of the infected plants and suspected H. phycitis. Isolated DNA of both insects and plants were performed to PCR assay with the help of phytoplasma specific universal primer P1/P7 and nested PCR with R16F2n/R2 which yielded about 1.2 kb amplicon in both the H. phycitis and witches-broom symptomatic samples. Both suspected amplified DNA samples from C. roseus and H. phycitis were sequenced. BLASTn and RFLP analysis were performed for the sequence alignment and subgroup assignment. BLASTn analyses were produces a result of 100% sequence similarity among themselves and 99% similarity with strain of 16SrI group whereas RFLP produces 16SrI-B subgroup. The study indicated that H. phycitis may play role as a vector in the transmission of these phytoplasma in C. roseus crops.

Molecular identification of phytoplasmas associated with little leaf disease of brinjal in Andhra Pradesh and Telangana states of India

Brinjal little leaf (BLL) is a very important phytoplasma disease in India that causes severe economic losses every year. During 2020, symptoms of little leaf and witches’ broom were observed in brinjal plants grown in the farmer fields at Kadapa, Andhra Pradesh and Mahabubnagar district, Telangana states. Four symptomatic and two symptomless plants collected from the surveyed places of both states were subjected to DNA extraction and used as template in nested PCR assays with universal phytoplasma 16S rRNA primers. Amplification of about 1.25 kb product with primers R16F2n/R2 was obtained only from symptomatic BLL plants and the positive control, but not from asymptomatic plants. Pair wise sequence comparison and phylogenetic analyses of 16S rRNA gene sequences of these phytoplasma strains confirmed that the phytoplasmas from Mahabubnagar district in Telangana were ‘Candidatus Phytoplasma trifolii’-related strains, while those from Kadapa, Andhra Pradesh, were related to Ca. P australasia’. Further virtual RFLP analysis of 16S rDNA sequences allowed the classification of these strains into 16SrVI-D and 16SrII-D subgroups, respectively.

Molecular detection of phytoplasmas in potato psyllids in ecuador

Since the potato purple top appeared and the potato psyllid was reported in Ecuador, crop losses have been significant. So far, phytoplasmas enclosed in two ribosomal groups (16SrI-F, 16SrII) and Candidatus Liberibacter solanacearum’ were molecularly identified in symptomatic potato plants. Later, Bactericera cockerelli, a potato psyllid, was also first reported and correlated to the potato purple top disease. Molecular identification of the pathogens involved in this disease has been quite elusive. Samples of potato psyllids were analyzed in this study resulting in the identification of phytoplasmas enclosed in group 16SrXI, and others related to Candidatus Phytoplasma prunorum’ and Ca. P. solani’. Potato purple top in Ecuador is a complex disease that constitutes a severe threat for the potato production in the region.

Genetic diversity of phytoplasma strains infecting chrysanthemum varieties in India and their possible natural reservoirs

Symptoms typical of phytoplasma infection such as phyllody, virescence, witches’ broom and yellowing were observed in 12 varieties of Chrysanthemum morifolium in floral nurseries and experimental fields at New Delhi, Karnataka, Maharashtra and Andhra Pradesh, India, during surveys made from 2015 to 2017. Disease incidence ranged from 15 to 30%. Phytoplasma presence was confirmed in all symptomatic chrysanthemum varieties by molecular identification assays. Sequence comparison, phylogenetic and in silico RFLP analyses of 16S rDNA sequences allowed the identification of the chrysanthemum infecting phytoplasma strains into different ribosomal groups and subgroups, namely 16SrI, 16SrII-D, 16SrVI-D and 16SrXIV. Detection of phytoplasma strains of 16SrII-D subgroup were also confirmed in symptomatic Chenopodium album and Parthenium hysterophorus plants grown in and around the surveyed chrysanthemum fields at New Delhi, whereas 16SrVI-D phytoplasma strains were detected in symptomatic Cannabis sativa weed and leafhopper Hishimonus phycitis individuals collected from the symptomatic chrysanthemum fields at New Delhi. This is the first report on the presence of 16SrVI and 16SrXIV groups of phytoplasmas in chrysanthemum plants. Studies on genetic diversity of phytoplasmas infecting the major chrysanthemum varieties in India and their epidemiological aspects had previously not been reported. The detection and identification of phytoplasmas in different chrysanthemum varieties could contribute to increase the awareness among farmers in the management of these diseases.

Detection and Identification of Phytoplasmas in the 16SrIV-A, -B, and -D Subgroups in Palms in Tabasco, Mexico.

The 16SrIV-A phytoplasmas are associated with the devastating disease lethal yellowing (LY) of palms. In Tabasco (Mexico), the death of Cocos nucifera, Adonidia merrillii, and Attalea butyracea palms have been suspected to be associated with LY based on symptomatology. Samples from the trunk of both symptomatic and nonsymptomatic palms were collected in three different environments: two species of palms within a rural zone and the other within an urban zone. DNA was extracted to perform a nested PCR with phytoplasma primers P1/P7-LY16SF/R16R2. A 1,345-bp fragment was amplified from the DNA extracted from each of the 29 LY-symptomatic palms sampled. Phytoplasma identification was achieved by amplicon sequencing and virtual restriction fragment length polymorphism analyses. Three 16SrIV phytoplasma subgroups were detected: 16SrIV-A in C. nucifera, 16SrIV-B in A. merrillii, and 16SrIV-D in C. nucifera, A. merrillii, and A. butyracea. Phylogenetic analysis showed also that the three phytoplasma strains found in the palm species clustered with phytoplasmas reported in the literature in the three subgroups identified. This is the first report of phytoplasmas associated with these palm species in Tabasco.

Evaluating the effect of tuf and secA gene sequence length for discrimination of phytoplasmas

Phytoplasmas are a major liming factor to the growth and production of many plant species worldwide. Most studies have used the conserved 16S rRNA gene for phytoplasma identification and classification. The efficiency of the tuf and secA genes length in the characterization of phytoplasmas was evaluated by designing universal primers to amplify the long and short tuf (740 bp and 414 bp) and secA (1232 bp and 536 bp) genes from the phytoplasma 16SrII-A and -D sub-groups, which are closely related phytoplasma strains. The neighbour joining (NJ) and the maximum likelihood (ML) methods were used to test the accuracy and stability of the phylogenetic trees. The NJ and ML phylogenetic trees from the long and short DNA sequences of the 16S rDNA, tuf, and secA genes produced two major clades (WB and AS). The NJ and ML phylogenetic trees showed high similarity in their differentiation between and within the 16S phytoplasma groups. This study indicates that the long DNA sequences of tuf and secA genes produced phylogenetic trees with higher resolution and separated very closely related phytoplasma groups. The accurate characterization of phytoplasmas is important for epidemiological studies and in disease management.

First Report of Association of a ‘Candidatus Phytoplasma aurantifolia’ 16SrII-D Related Phytoplasma with Geranium (Pelargonium hortorum) Little Leaf Disease in Iran

Geranium, an important ornamental plant belong to the family Geraniaceae, is widely cultivated in urban landscapes and private gardens all over the world. During a survey in spring 2019, a few seed-propagated geranium plants exhibiting typical symptoms induced by phytoplasmas, including pale green and little leaves, phyllody, virescence, and witches’-broom symptoms, were observed in Jiroft, Kerman province of Iran. Total genomic DNA extracted from leaf midribs and petioles of the three symptomatic and two asymptomatic plants was used as a template in a nested polymerase chain reaction (PCR) assay using universal phytoplasma primer pairs P1/P7 followed by R16F2n/ R16R2 (Gundersen and Lee 1996) to amplify a part of the 16S rDNA phytoplasma genome as previously described by Azadvar et al. (2011). The generated 1.8- and 1.25-kb amplicons in the first and second round of the nested PCR assay, respectively, from each of the three symptomatic plant samples revealed phytoplasma infection of the plants. No amplicon was generated in PCR assays by using the DNA templates from symptomless plants. The purified PCR products were directly sequenced (Bioneer, South Korea) in both forward and reverse directions. The consensus sequences were analyzed by the iPhyClassifier tool (Zhao et al. 2009) and pDRAW32 software (AcaClone software, http://www.acaclone.com) for 16Sr group/subgroup phytoplasma identification. The 16S rDNA nucleotide sequences from the three diseased plants were identical (GenBank accession no. MT187639). iPhyClassifier analysis showed that the F2nR2 16S rRNA encoding gene sequence of the phytoplasma associated with geranium little leaf (GrLL) disease in Iran was identical (similarity coefficient 1.00) to those of the reference pattern of 16Sr group II subgroup D (GenBank accession no. Y10097) and had 98.7% similarity with the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession U15442). Virtual restriction fragment length polymorphism (in silico RFLP) analysis of the 16S rDNA F2nR2 sequence with the pDRAW32 program using AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, SspI, and TaqI endonucleases further confirmed that the GrLL phytoplasma belong to subgroup II-D. The 16SrII-D subgroup phytoplasma has been previously reported in association with Calendula officinalis (Esmailzadeh Hosseini et al. 2016), Helianthus annuus (Salehi et al. (2015), and Petunia hybrida (Faghihi et al. 2014) ornamental plants in Iran. We also found many petunia (P. hybrida) plants showing little leaf, yellowing, phyllody, and witches’-broom symptoms next to the GrLL-infected plants, which hosted the same phytoplasma (GenBank accession no. MT187637). A 16SrII group-related phytoplasma has previously been reported associated with Pelargonium capitatum weed plant exhibiting severe stunting and necrosis in Australia (Lee et al. 2010). To our knowledge, this is the first report of a ‘Ca. P. aurantifolia’ 16SrII-D subgroup-related phytoplasma associated with geranium (Pelargonium hortorum) little leaf disease in the world.

Simultaneous Detection and Direct Identification of Phytoplasmas in the Aster Yellows (16SrI), Clover Proliferation (16SrVI) and Stolbur (16SrXII) Groups Using a Multiplex Nested PCR Assay in Potato Plants

Phytoplasmas in the aster yellows (16SrI), clover proliferation (16SrVI) and stolbur (16SrXII) groups have a wide host range with common hosts. In the current study, specific primers based on the 16-23S rRNA spacer region were designed and optimised for the detection and identification of phytoplasmas in these groups in a multiplex nested PCR assay. Fifty-two phytoplasma symptomatic potato plants were tested, and clover proliferation (16SrVI) and stolbur (16SrXII) phytoplasmas detected coinfecting 16 potato plants with single infection of these phytoplasmas in 5 and 21 samples, respectively. ‘Candidatus P. asteris’ (16SrI) was not detected. The specificity of the primers was proven in both single and multiplex PCR assays. Detection of multiple infections can be useful in assisting epidemiological studies and in deciding appropriate disease management strategies.

Identification of 16SrII-D group phytoplasma associated with Setaria verticillata (L.) P. Beauv. in India

Phytoplasma suspected symptoms of little leaf and chlorosis were observed on Setaria verticillata grass at DDU Gorakhpur University Campus, Gorakhpur,Uttar Pradesh, India during 2018. To confirm the presence of phytoplasma, total DNA was extracted followed by PCR assay utilizing phytoplasma primers P1/P7 and R16F2n/R16R2. The ~ 1.2 kb amplicon was obtained by nested PCR assay from symptomatic Setaria samples. The amplified product was directly sequenced, aligned and submitted to GenBank. Comparison of 16S rRNA gene sequence of Setaria grass isolate shared 100% sequence identity with strains of 16SrII-D phytoplasma group (Ca. Phytoplasma aurantifolia). This is the first record of the occurrence of 16SrII-D subgroup phytoplasma in a weed grass, S. verticillata in India.

Identification of phytoplasma in six fruit crops in India

Symptoms of decline, leaf yellowing and reddening, little leaf and malformation were observed in apricot, guava, lychee, mango, pomegranate and grapevine in fruit orchards belonging to three states of India namely Delhi, Maharashtra and Jammu & Kashmir during 2016–2017. Phytoplasmas belonging to three different groups were detected in all the symptomatic fruit tree samples in polymerase chain reaction with phytoplasma specific primer pairs amplifying 16S rRNA and secA genes. Pair wise sequence comparison and phylogenetic analysis confirmed the presence of phytoplasmas. A ‘Candidatus Phytoplasma asteris’ - related strain was detected in apricot showing decline symptoms in Siot (Jammu & Kashmir). ‘Ca. P. australasia’ - related strain was identified in guava, lychee, mango and pomegranate in Rajbag, Sunjwan (Jammu & Kashmir), Pusa (Delhi) and Baramati (Maharashtra), respectively, exhibiting little leaf, leaf yellows and malformation symptoms. Furthermore, a rice yellow dwarf group (16SrXI-B) - related strain was detected in pomegranate and grapevine that showed leaf yellowing and reddening at Pusa (Delhi) and Baramati (Maharashtra), respectively. Subgroup analysis using virtual RFLP of 16S rDNA sequences allowed enclosing these phytoplasma strains into 16SrI-B, 16SrII-D and 16SrXI-B subgroups. In the study, identification of phytoplasma subgroups, 16SrI-B in apricot, 16SrII-D in guava, lychee, mango and pomegranate (Pusa) and 16SrXI-B in pomegranate (Baramati) and grapevine, are the first records globally.

Identification of phytoplasmas and Auchenorryncha in Tunisian vineyards

To verify the risk that plant pathogens such as phytoplasmas could have been introduced with infected propagation material, a preliminary investigation for the presence of phytoplasmas and their insect vectors in the main Tunisian viticultural regions was carried out. To identify phytoplasmas and leafhopper fauna several survey have been carried out on grapevine and spontaneous plant species in the main grapevine growing regions of Tunisia, i.e. Rafraf, Baddar, Mraissa, Belli, Gobba, and Bousalem. A total of 1,109 insect specimens were collected and 30 species were identified, 25 Cicadomorpha and 5 Fulgoromorpha. The 82.58% of the leafhopper species recorded are reported either as vectors of viruses or of phytoplasmas or direct damaging pests, while the 17.49% remaining do not have an important role in field management. In some of the collected grapevine materials aster yellows phytoplasmas (16SrI-B) were identified, confirming previous reports.

Identification of 16SrXI phytoplasmas in sugarcane and alternative hostplants in Karnataka, India

Sugarcane crop is severely affected by diverse phytoplasma-associated diseases worldwide. Symptomatic sugarcane, eggplant and cluster bean together with weeds such as false daisy, Croton sparsiflorum, and Parthenium hysterophorus samples were collected at Khanapur village in Karnataka, India in 2018. The molecular analyses of the samples allowed to identify the presence of 16SrXI phytoplasma group after nested PCR amplification of the 16S rRNA gene followed by RFLP and sequencing analyses. The virtual and real RFLP analysis of the 16S rRNA gene sequence suggested its classification in a new ribosomal subgroup tentatively named 16SrXI-G. Amplification of secA and tuf genes from the sugarcane samples also confirmed the presence of 16SrXI group phytoplasmas in sugarcane and other host plant species in India and indicate their close relationship with sugarcane white leaf phytoplasmas from Sri Lanka and Vietnam. The identification of this phytoplasma strain in sugarcane, brinjal, cluster beans and weeds, in and around sugarcane fields in Karnataka, is an important information that should be employed to help the disease management aimed to reduce the phytoplasma incidence and economic losses in sugarcane.

Identification of phytoplasmas in 16SrI-D subgroup in mango with malformation and witches’ broom disease in Punjab, India

Symptoms of mango malformation and witches’ broom were observed in mango orchards at Ludhiana, Punjab, India during 2020. Phytoplasmas were detected in the symptomatic mango samples by polymerase chain reaction with specific primer pairs amplifying 16S rRNA and secA genes. Pair wise sequence comparison and phylogenetic analysis of 16S rRNA and secA gene sequences of mango phytoplasma strains indicate their relatedness with phytoplasmas in the aster yellows group. In silico RFLP analyses using the iPhy Classifier of the 16S rDNA sequence of the mango phytoplasma allowed its classification into the 16SrI-D subgroup. This is first report of this phytoplasma in mango plants showing a malformation and witches’ broom disease.

Molecular detection and identification of a strain related to ‘Candidatus phytoplasma australasia’ in marigold in India

Marigold plants with suspected symptoms of phytoplasma infection were collected during field surveys from three different districts of Karnataka where the incidence of these symptoms was ranging from 1 to 11%. The presence of phytoplasmas in the samples showing phyllody and virescence symptoms was confirmed by nested PCR assays using P1/P7 and R16F2n/R16R2 phytoplasma specific primer pairs. The nucleotide sequence identity and phylogenetic analyses of the 16S rRNA gene sequence indicated the presence of a ‘Candidatus Phytoplasma australasia’ -related strain in the diseased marigold samples. The phylogenetic analysis of secA gene sequences of this phytoplasma strain showed its clustering with strains enclosed in the 16SrII-D subgroup, confirming the phytoplasma identification. To the best of our knowledge, this constitutes the first report of 16SrII phytoplasmas associated with marigold phyllody from the Karnataka state. As the most widely cultivated commercial flower of Karnataka, marigold has a significant role in the national and international flower trade. Phyllody and associated symptoms have a direct negative impact on the quality and market value of these flowers.

Identification and transmission of phytoplasmas and their impact on essential oil composition in Aerva javanica.

Aerva javanica plants showing symptoms of witches' broom, little leaf, and leaf roll were observed in 50% of the A. javanica plants grown in the deserts of Hormozgan province, Iran. This study was carried out to verify possible phytoplasma association with this disease, insect vector, and modification of the chemical composition of symptomatic A. javanica. Aerial parts were collected at the full flowering stage from symptomatic and asymptomatic plants and samples were examined for phytoplasma presence by nested-PCR assays. PCR and sequencing analysis showed that a phytoplasma was associated with the disease sharing 99% sequence identity with 'Candidatus Phytoplasma aurantifolia'-related strains (16SrII). Furthermore, this phytoplasma was transmitted to healthy periwinkle plants under experimental conditions by the leafhopper Austroagallia sinuata that was then demonstrated to be a vector of this phytoplasma. Analysis of the oils carried out by GC-MS revealed the enrichment of the triterpene hydrocarbons (2.3% in asymptomatic vs. 11.8% in symptomatic samples) and the hydrocarbons (69.3% vs. 85.93%) as compared to the abundance in asymptomatic samples. Conversely, a matching decrease in ketone (3.51% vs. 0.6%) was observed in the symptomatic plants. These results indicate that phytoplasma presence is influencing the secondary metabolites production in infected plants.