Broom Symptoms Research Articles

Increased sodium and fluctuations in minerals in acid limes expressing witches' broom symptoms.

Witches’ broom disease of lime (WBDL), caused by ‘Candidatus Phytoplasma aurantifolia’, is a very serious disease of acid limes. The disease destroyed more than one million lime trees in the Middle East. WBDL results in the production of small, clustered leaves in some branches of lime trees. Branches develop symptoms with time and become unproductive, until the whole tree collapses within 4–8 years of first symptom appearance. This study was conducted to investigate differences in minerals between symptomatic and asymptomatic leaves of infected lime trees. The study included one set of leaves from uninfected trees and two sets of infected leaves: symptomatic leaves and asymptomatic leaves obtained from randomly selected acid lime trees. Nested polymerase chain reaction detected phytoplasma in the symptomatic and asymptomatic leaves from the six infected trees, but not from the uninfected trees. Phylogenetic analysis showed that all phytoplasmas belong to the 16S rRNA group II-B. Mineral analysis revealed that the level of Na significantly increased by four times in the symptomatic leaves compared to the non-symptomatic leaves and to the uninfected leaves. In addition, symptom development resulted in a significant increase in the levels of P and K by 1.6 and 1.5 times, respectively, and a significant decrease in the levels of Ca and B by 1.2 and 1.8 times, respectively. There was no significant effect of WBDL on the levels of N, Cu, Zn, and Fe. The development of witches’ broom disease symptoms was found to be associated with changes in some minerals. The study discusses factors and consequences of changes in the mineral content of acid limes infected by phytoplasma.

Multigene characterization of a new 'Candidatus Phytoplasma rubi'-related strain associated with blackberry witches' broom.

A new phytoplasma was identified in naturally infected blackberry plants exhibiting witches' broom symptoms in Portugal. The 16S rRNA gene sequence revealed that it is related to 'Candidatus Phytoplasma rubi' (16SrV-E ribosomal subgroup) and RFLP analysis revealed a unique profile following MseI endonuclease digestion of R16F2n/R2 amplicons that distinguished it from the strains belonging to previously established 16SrV phytoplasma subgroups. The in silico restriction analyses confirmed that the phytoplasma strain from blackberry is different from all the other strains reported in group 16SrV. Phylogeny of the 16S rRNA gene sequences, sequence analyses of 16S-23S, tuf, rplV-rpsC, rplF-rplR, rplO-SecY-map and uvrB-degV genetic loci, as well as the variability of unique oligonucleotide sequences defined for 'Candidatus Phytoplasma rubi' confirmed the uniqueness of this phytoplasma strain from Portugal for which a novel ribosomal subgroup, 16SrV-I, is proposed. The representative of this new subgroup was named blackPort phytoplasma (Portuguese blackberry phytoplasma).

Identification of new plant hosts of 16SrII group phytoplasmas in Saudi Arabia

Qassim region is one of the main agricultural areas in Saudi Arabia. During 2015, ornamental, landscape and bindweed plants showing virescence; yellowing, phyllody, witches’ broom, proliferation, fasciation, short internode, and dieback symptoms were observed in this region. To confirm the presence of phytoplasmas, DNA extracted from symptomatic and asymptomatic plants was used to amplify 16S rRNA-encoding gene through direct and nested PCR assays using P1/P7, R16F2n/R16R2 and fU5/rU3 primers. Phytoplasma DNA was detected in all symptomatic plants collected. Sequencing and phylogenetic analysis were performed to identify the phytoplasmas detected. BLAST comparison indicated that the phytoplasmas showed more than 99.2% identity with phytoplasmas in 16SrII group. Three phytoplasma strains from Albizia and periwinkle shared 100% identity with ‘Candidatus Phytoplasma aurantifolia’, subgroup 16SrII-B, while phytoplasmas affecting Parkinsonia aculeate, Gazania rigens, Hibiscus rosa-sinensis, Calendula officinalis, Plectranthus scutellarioides, Convolvulus arvensis, Zinnia Magellan, Conocarpus lanceolatus and Catharanthus roseus were identical to papaya yellow crinkle, classified in the 16SrII-D subgroup.

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4.

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches’ broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.

A subgroup 16SrIII-J phytoplasma associated with Aegiphila verticillata, a typical wild plant of Brazilian savanna region, exhibiting witches’ broom symptoms

Aegiphila verticillata (parrot fruit) is a wild plant that commonly grows in Brazilian savanna fields, which are also denominated Cerrado. Trees and shrubs of this species were observed exhibiting profuse shoot proliferation that produced small leaves with mild chlorosis and flowers and fruit of reduced size which fell off prematurely. In the present study, PCR assays demonstrated the association of a phytoplasma with naturally affected plants. Identification performed with group-specific primers indicated this phytoplasma as belonging to the 16SrIII group. Molecular analysis based on virtual RFLP patterns and similarity coefficient calculations allowed to classify the phytoplasma as a representative of the 16SrIII-J subgroup. Phylogenetic analysis confirmed this result and evidenced that this phytoplasma was closely related to the reference strain for the 16SrIII-J subgroup. The disease was denominated Aegiphila witches’ broom (AegWB). In South America, representatives of the 16SrIII-J subgroup are frequently found in association with diseases of phytoplasmal etiology that occur in several wild and cultivated species. This study reveals for the first time that A. verticillata, a typical plant from the Brazilian Cerrado region, is a host of phytoplasmas, specifically of a member of the 16SrIII-J subgroup.

Sunn hemp, a major source-plant of the phytoplasma associated with huanglongbing symptoms of sweet orange in São Paulo State, Brazil

In Sao Paulo State (SPS), sweet orange (Citrus sinensis) trees with huanglongbing (HLB) symptoms are infected with Candidatus (Ca.) Liberibacter (L.) asiaticus (Las) or Ca. L. americanus (Lam). However, in 2007, 3 years after HLB was first reported in SPS, some trees with characteristic HLB symptoms were found free of liberibacters, but infected with a phytoplasma of 16Sr group IX. This phytoplasma was further characterized by PCR amplification of ribosomal protein genes rpsC-rplV-rpsS and amplicon sequencing. A qPCR test to detect the phytoplasma in plants and insects was also developed on the basis of the ribosomal protein genes. The phytoplasma was transmitted from citrus-to-citrus by grafting. The 16Sr group IX phytoplasma associated with HLB symptoms in sweet orange in SPS and characterized by the above techniques was named “HLB-phytoplasma”. Although the HLB-phytoplasma is widely distributed in many municipalities of central, northern, and northwestern SPS, the number of HLB-phytoplasma-infected trees in each municipality is very small. Experiments have been undertaken to identify the origin of the HLB-phytoplasma and the source of inoculum on which a putative insect vector could become infected with the HLB-phytoplasma. In SPS, sunn hemp (Crotalaria juncea L.) is a major, widely distributed cover crop. A 16Sr group IX phytoplasma was detected in sunn hemp plants with witches’ broom and virescence symptoms, and was shown to have 16Sr DNA sequences and ribosomal protein gene sequences with 100% identity to the corresponding sequences of the sweet orange HLB-phytoplasma. Transmission electron microscopy revealed the presence of phytoplasma cells in the phloem sieve tubes of infected C. juncea stalks. These results were taken as evidence that the sunn hemp phytoplasma and the sweet orange HLB-phytoplasma were identical. Scaphytopius marginelineatus, a leafhopper frequently found in sweet orange orchards, was shown to acquire the HLB-phytoplasma efficiently from affected sunn hemp plants, but acquisition from, and transmission rates to, sweet orange were very low. On the whole, these data suggest that (i) sunn hemp is a major source of inoculum of the HLB-phytoplasma, (ii) S. marginelineatus becomes infected on sunn hemp and transmits the phytoplasma to sweet orange, and (iii) transmission from sweet orange to sweet orange occurs only rarely, if at all. 16Sr group IX phytoplasmas, very closely related to the SPS HLB-phytoplasma, have also been detected in citrus in Minas Gerais and Bahia states (Brazil) and Mexico.

Molecular characterization of ‘Candidatus Phytoplasma asteris’ subgroup I-B associated with sesame phyllody disease and identification of its natural vector and weed reservoir in India

An incidence of 12 to 35 % sesame phyllody (SP) disease was observed in sesame fields at Kushinagar and Gorakhpur districts in Uttar Pradesh and Indian Agricultural Research Institute, New Delhi, India during July-October 2013. The association of phytoplasma with sesame phyllody plants at all the three locations and a weed, Sclerocarpus africanus Jacq (SA) showing little leaf and witches’ broom symptoms at Gorakhpur was confirmed by direct and nested PCR amplification of phytoplasma 16S rDNA. Out of four predominant feeding leafhopper species in the symptomatic sesame fields, viz. Empoasca prima (Distant), Exitianus indicus (Distant), Hishimonus phycitis (Distant) (HP) and Cofana unimaculata (Signoret), only H. phycitis was found to be associated with phytoplasma in nested PCR assays with phytoplasma universal primer pair R16F2n/R16R2. Transmission test and population dynamics study further confirmed that H. phycitis was the proven natural and potential vector to transmit the SP phytoplasma from diseased to healthy sesame plants in transmission assays. BLAST analysis of 1.25 kb 16SrDNA partial sequences of nested PCR products obtained from symptomatic SP plants, weed (SA) and the leafhopper (HP) revealed 99 % sequence identities among themselves and 99 % identity with other reported strains of ‘Ca. P. asteris’ (16Sr I group). Phylogenetic analysis also suggested the closest phylogenetic relationship of SP, SA and HP phytoplasmas with those of ‘Ca. P. asteris’ group. RFLP analysis of R16F2n/R16R2 primed 16S rDNA sequences of SP, SA and HP phytoplasma isolates using iPhyClassifier online tool with 17 selected restriction enzymes confirmed the association of ‘Candidatus Phytoplasma asteris’ subgroup B with SP, SA and HP in India. The identity of natural vector and new alternative host of SP phytoplasma in the present study suggested that these may act as potential/natural source for secondary spread of sesame phyllody phytoplasma.

Phytoplasma identification in iberis exhibiting stunting and witches’ broom symptoms

Iberis sempervirens samples from varieties Tahoe and Fish Back showing stunting, yellowing and witches’ broom symptoms were tested to verify possible phytoplasma presence. Detection of 16SrX group phytoplasmas was obtained after nested PCR amplification and RFLP analyses in both varieties. Sequencing of 16Sr DNA allowed verification that phytoplasmas from iberis showed 99% identity with ‘Candidatus Phytoplasma mali’. Moreover, RFLP analyses also indicated the presence of aster yellows phytoplasmas (subgroups 16SrI-B and -A) in mixed infection with 16SrX phytoplasmas in the Fish Back variety. This is the first report of phytoplasma infection in iberis and also one of the few detections of phytoplasmas related to 16SrX-A group from an herbaceous species worldwide.

Detection and multigene characterisation of a ‘CandidatusPhytoplasma prunorum’-related phytoplasma associated with witches’ broom symptoms in pineapple fruit

Pineapple samples showing witches’ broom symptoms in the leaves at the top of the fruit were analysed to verify phytoplasma presence. 16SrX group phytoplasmas were detected after nested PCR amplification with specific primers and RFLP analyses. Sequencing of 16Sr DNA allows verification that phytoplasmas from pineapple show a 99% shared identity with ‘Candidatus Phytoplasma prunorum’. Further characterisation of the phytoplasma on ace, imp and secY genes by RFLP analyses on amplicons obtained in nested PCR assays enable the differentiation of the strain infecting pineapple from those detected in fruit trees in Europe and used as reference strains. This is the first report of phytoplasmas belonging to the 16SrX-B group from a species different from pome and stone fruit worldwide.

Identification ofCannabis sativaL. ssp.sativaas putative alternate host of sesame phyllody phytoplasma belongs to 16SrI group in India

Little leaf and witches’-broom symptoms on Cannabis sativa L. ssp. sativa plants were recorded in sesame phyllody infected fields at Kushinagar district of eastern Uttar Pradesh, India in September 2013. The DNA extracted from symptomatic Cannabis sativa L. ssp. sativa (CSS) plants yielded amplicons of 1.25 kb in nested PCR assays with R16F2/R2n primer pair. BLASTn comparison and phylogenetic analysis of 16SrDNA phytoplasma sequence of CSS phytoplasma isolate revealed association of ‘Ca. Phytoplasma asteris’ (16Sr I group) with symptomatic cannabis plants. Our results suggest that CSS may play an important role in perpetuation of sesame phyllody phytoplasma in nature.

First report of phytoplasma associated withEucalyptus urophyllashowing witches’ broom in Brazil

Eucalyptus production is of great importance to Brazil's economy. Diseases affecting the growth and the architecture of these trees lead to large losses in yield and quality of the wood. The occurrence of eucalyptus trees showing witches’ broom and little leaf symptoms in different areas of Brazil is affecting seedling production. In this work, a phytoplasma was found associated with these symptomatic plants and was identified as belonging to the 16SrII group. This is the first report of a phytoplasma associated with eucalyptus in Brazil.

Phytoplasma diseases on major crops in Vietnam

The nested-PCR technique was applied to detect and identify phytoplasmas associated with major crops in Vietnam including sugarcane, cassava and longan. Phylogenetic analysis confirmed that phytoplasmas associated with the sugarcane grassy shoot and white leaf diseases belong to the rice yellow dwarf group (16SrXI). In cassava showing witches’ broom symptoms a mixed infection of phytoplasmas related to ‘Candidatus Phytoplasma asteris’ (group 16SrI) and ‘Ca. P. aurantifolia’ (group 16SrII) was detected. In samples showing longan witches’ broom disease three phytoplasmas including ‘Ca. P. aurantifolia’ (group 16SrII), elm yellows (group 16SrV) and “stolbur” (group 16SrXII) were identified.

The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia.

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51'49″ N, 20°39'33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII 'Candidatus Phytoplasma solani' group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia.

Rice yellow dwarf phytoplasma (16Sr XI-B subgroup) infecting Jasminum sambac in India

Leaf yellowing and witches’ broom symptoms were recorded on Jasminum sambac (L.) plants in August 2012 at Jaipur, Rajasthan, India. Total DNA was isolated from symptomatic jasmine plants and nested PCR was performed using primers pairs P1/P7 and 3Far/3Rev. Expected size amplicons of ~1.3 kb were obtained in nested PCR from symptomatic jasmine plants which were cloned and sequenced. BLASTn analysis of partial 16S rRNA gene sequence of jasmine leaf yellows and witches’ broom (Jas LY and WB) isolate revealed highest sequence identity (99%) with phytoplasma associated with sugarcane white leaf, sugarcane grassy shoot, coconut root wilt, sorghum grassy shoot and goose grass white leaf, all members of the rice yellow dwarf phytoplasma (16Sr XI) group. Phylogenetic analysis of the Jas LY and WB phytoplasma sequence revealed further its closest relationship with members of the 16Sr XI group. This confirms the association of 16Sr XI group phytoplasma with Jas LY and WB disease. Real and virtual RFLP analysis of 16S rRNA gene of jasmine phytoplasma further confirmed that it belonged to the 16Sr XI-B subgroup. This is the first report of 16SXI-B subgroup phytoplasma infection on Jasminum sambac in the world.

A novel phytoplasma associated with witches’ broom disease of Ligustrum ovalifolium in Turkey

California privet (Ligustrum ovalifolium Hassk.) plants exhibiting leaf yellowing, witches’ broom, dieback and decline symptoms were observed for two years (2010–2011) in three gardens at Adana region (Turkey). DNA isolated from symptomatic and healthy plants was used to amplify 16S rDNA fragments by direct and nested-PCR. Phytoplasmas were detected in 21 symptomatic plants, out of 30 samples collected, whilst no PCR amplifications were obtained from asymptomatic plants. BLAST analysis of the 16S rDNA showed that the phytoplasma found in L. ovalifolium from Turkey, denoted as Turkish Ligustrum witches’ broom phytoplasma (TuLiWB), most closely resembled members of group 16SrII (peanut witches’ broom group) and shared up to 92 % sequence identity. Based on in silico 16S rDNA RFLP analysis and automated calculation of the pattern similarity coefficient, TuLiWB showed molecular characteristics different from all previously described phytoplasma species to represent a new taxon. Similar indication also emerged from the phylogenetic tree which allocated it in a novel discrete subclade within the phytoplasma clade. This is the first report on the presence of a phytoplasma affecting L. ovalifolium and whether this novel phytoplasma is the same agent reported as a mycoplasma-like organism (MLO) and associated with witches’ broom disease of Ligustrum in Korea (1989) is yet to be determined.

‘CandidatusPhytoplasma asteris’ association with leaf yellows and witches’ broom symptoms ofBrachycomespecies in India

During a survey in flower nurseries at IARI campus, New Delhi carried out in 2012–13, Brachycome species plants grown in pots and flower beds showed leaf yellows (LY) and witches’ broom (WB) symptoms. Samples were collected from these plants and analyzed for phytoplasma presence through PCR assays. DNA from four symptomatic plants was extracted and produced amplicons of 1.2 kb in nested PCR assays. Symptomless plants did not produce amplicons. The sequencing and BLASTn analysis revealed 99% identity with strains related to ‘Candidatus P. asteris’. Phylogenetic analysis further confirmed the clustering of Brachycome phytoplasma with ‘Ca. P. asteris’ strains. This is the first report of 16SrI group phytoplasma association with Brachycome LY and WB disease in the world.

Sweet potato little leaf strain V4 phytoplasma associated with snake bean in the Northern Territory, Australia

Sweet potato little leaf strain V4 phytoplasma (SPLL-V4) was found to be associated with 12-week old snake bean plants (Vigna unguiculata ssp. sesquipedalis) from a block with 890 plants in Lambells Lagoon, Northern Territory. Symptoms included witches’ broom, big bud, phyllody and little leaf. There was a 19.9 % incidence of symptoms on 366 plants assessed. The same phytoplasma was also detected from a single plant of Phyllanthus amarus with witches’ broom symptoms growing near the snake bean plants. The SPLL-V4 phytoplasma was identified based upon the 16S ribosomal RNA gene and intergenic spacer region nucleotide sequence and restriction fragment length polymorphism.

Morphological, anatomical and molecular investigation into witches’ broom disease of mamejvo (Enicostemma axillare)

Mamejvo (Enicostemma axillare Raynal) is a perennial herb with diversified health benefits. It was found to be affected by a witches’ broom disease under the field conditions at Anand, Gujarat, India. Affected plants were 53.33% reduced in height due to shortened internodes, leading to typical witches’ broom symptoms. Flower size was reduced significantly in the affected plants compared with the healthy ones and their petal color gradually turned green (virescence). Affected plants produced 2.5 times more dry herbage than the normal plants but with less active ingredient (swertiamarin). Symptomatic plants died early while root suckers from such plants failed to establish after transplanting. DAPI stained transverse sections of affected shoot tips showed the presence of discrete greenish fluorescence in the phloem cells under UV light. Amplification of 1.2 kb phytoplasma specific rDNA fragment from diseased tissue confirmed the presence of the pathogen. Further characterization of the pathogen through virtual rDNA–RFLP pattern and rDNA sequence based phylogeny suggested that the pathogen, Enicostemma witches’ broom phytoplasma, belonged to 16SrII–C group of ‘Ca Phytoplasma aurantifolia’.

Detection and Identification of 16SrII Group Phytoplasmas Infecting Stylosanthes in China

AbstractIn July 2008, Stylosanthes guianensis with witches’‐broom symptoms and Stylosanthes capitata with yellows symptoms were observed in Hainan Province, China. From the symptomatic S. guianensis, the phytoplasma‐specific 16S rRNA gene fragment of 1.4 kb was amplified by a direct PCR, and a fragment of 16S rRNA gene of 1.2 kb was obtained from the symptomatic Stylosanthes capitata by a nested PCR. Sequencing results and NCBI BLASTn analysis showed that the two phytoplasmas belonged to group 16SrII. Polygenetic analysis and virtual RFLP classified the two phytoplasmas into subgroup 16SrII‐A.

‘Candidatus Phytoplasma ziziphi’ associated with Sophora japonica witches’ broom disease in China

Chinese scholar tree (Sophora japonica) with witches’ broom symptoms was observed in Shandong Province in China. Phytoplasmas were detected in the diseased plants using 16S rDNA amplification with phytoplasma-specific universal primer pairs. On the basis of the results of 16S rDNA sequencing, virtual restriction fragment length polymorphism patterns and phylogenetic analyses, the phytoplasma found in S. japonica with witches’ broom symptoms was confirmed as a ‘Candidatus Phytoplasma ziziphi’-related strain belonging to the Elm yellows group 16SrV. This is the first report of ‘Ca. P. ziziphi’ infecting S. japonica plant with witches’ broom symptoms.