Potato Witches Research Articles

Cas12a-Based Diagnostics for Potato Purple Top Disease Complex Associated with Infection by ‘Candidatus Phytoplasma trifolii’-Related Strains

'Candidatus Phytoplasma trifolii' is a cell wall-less phytopathogenic bacterium that infects many agriculturally important plant species such as alfalfa, clover, eggplant, pepper, potato, and tomato. The phytoplasma is responsible for repeated outbreaks of potato purple top (PPT) and potato witches' broom (PWB) that occurred along the Pacific Coast of the United States since 2002, inflicting significant economic losses. To effectively manage these phytoplasmal diseases, it is important to develop diagnostic tools for specific, sensitive, and rapid detection of the pathogens. Here we report the development of a DNA endonuclease targeted CRISPR trans reporter (DETECTR) assay that couples isothermal amplification and Cas12a transcleavage of fluorescent oligonucleotide reporter for highly sensitive and specific detection of 'Candidatus Phytoplasma trifolii'-related strains responsible for PPT and PWB. The DETECTR assay was capable of specifically detecting the 16S-23S ribosomal DNA intergenic transcribed spacer sequences from PPT- and PWB-diseased samples at the attomolar sensitivity level. Furthermore, the DETECTR strategy allows flexibility to capture assay outputs with fluorescent microplate readers or lateral flow assays for potentially high-throughput and/or field-deployable disease diagnostics.

Potato witches' broom phytoplasma (yellows-type disease)

Potato witches' broom phytoplasma (yellows-type disease)

Sweet potato little leaf phytoplasma (sweet potato witches' broom)

sweet potato little leaf phytoplasma (sweet potato witches' broom)

First Report of 'Candidatus Phytoplasma ziziphi' in Sweet Potato in China.

Sweet potato (Ipomoea batatas; family Convolvulaceae) is an important food crop and serves as a ground cover in orchards of jujube trees. In August 2020, sweet potatoes growing under jujube trees showed obvious jujube witches' broom phytoplasma disease symptoms in an abandoned jujube orchards in Xinzheng county, Henan province, China (GPS：113°49'56″N， 34°35'54″E). The sweet potato plants were showing symptoms such as small, yellowing leaves and witches' broom. (Figure 1). The incidence of symptomatic sweet potato plants in the orchard (60× 100 m2 ) was about 60%. Leaf samples of sweet potatoes and jujube trees exhibiting disease symptoms were collected to confirm the presence of JWB phytoplasma by PCR. The phytoplasma primers R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008) were used. Leaf samples of three sweet potato plants and three jujube trees exhibiting disease symptoms were collected to confirm the presence of JWB phytoplasma by PCR. Two healthy sweet potatoes collected from the experimental station at Henan Agricultural University and two healthy, in vitro-grown jujube plantlets were used as negative controls. A JWB-diseased in vitro-grown jujube plantlets was set as the positive control. The total DNA of 3 leaves from each sample was extracted using the Hi-DNA secure Plant Kit (DP350, TianGen, Beijing, China). The phytoplasma amplicon was detected in all leaf samples (Figure 2). The expected 16S rDNA (GenBank accession number MW990090) amplicon is 1,348 bp, and the expected secA (GenBank accession number MZ292648) amplicon is 842 bp. These two fragments were detected in the sweet potato samples (Figure 2a, 2b). The DNA fragment amplified from the diseased sweet potato and the diseased jujube leave samples exhibiting disease symptoms using the 16S rRNA primers was then sequenced (Sangon Biotech, Shanghai, China) and was 100% identical to that of 23 JWB phytoplasma strains through BLAST analysis. The DNA fragment amplified using the secA primers was sequenced and was 100% identical to that of JWB phytoplasma strain ZQ secA gene (GU471770.1). RFLP analysis of the 16S rRNA sequence fragment by the online tool iPhyClassifier (Wei et al., 2007) indicated that the pathogen strain was a member of subgroup 16SrV-B and a 'CandidatusPhytoplasmaziziphi'-related strain. Sweet potato witches' broom disease was reported by Gundersen (1994) and Lee, I. M. (2004). Sweet potato little leaf disease was reported and classified as a phytoplasma in the 16 SrII group (Tairo et al., 2006). According to the evolutionary analysis, the 16S rRNA nucleotide sequences found in sweet potato in this study were quite different from that detected in sweet potato little leaf disease (Figure 2c). Taken together, the results indicated that the phytoplasma associated with sweet potato in the present study is a 'Ca. Phytoplasma ziziphi' strain. To our knowledge, this is the first report of its presence in sweet potato in China.

Development of molecular markers and a diagnostic tool for investigation of coinfections by and interactions between potato purple top and potato witches'-broom phytoplasmas in tomato

Columbia Basin potato purple top (PPT) phytoplasma and Alaska potato witches'-broom (PWB) phytoplasma are two closely related but mutually distinct pathogenic bacteria that infect potato and other vegetable crops. Inhabiting phloem sieve elements and being transmitted by phloem-feeding insect vectors, both pathogens are affiliated with ‘Candidatus Phytoplasma trifolii’ and are members of the clover proliferation phytoplasma group (16SrVI). The polyphagous nature and wide geographic distribution of their insect vectors make mixed infection inevitable. In this study, we experimentally constituted a simultaneous PPT and PWB phytoplasma infection in tomato (Solanum lycopersicum) and developed a sensitive diagnostic tool to investigate mixed infections by and in planta interactions of the two phytoplasmas. The distribution and relative abundance of the two co-infecting phytoplasmas were monitored over a 45-day post-infection time course and for three serial passages in planta. Our results revealed that dual infections of the two phytoplasmas induce a new symptom unseen in infection by either phytoplasma alone. Our results also raised an interesting question as to whether the two phytoplasmas differ in ability of competitive dominance under co-infection conditions. The molecular markers and the diagnostic tool devised in this study should be useful for further investigations of the interactions between the two closely related phytoplasmas in their hosts.

Molecular Characterization of a Phytoplasma Associated with Potato Witches’-broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’-broom disease including witches’-broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’-broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’-broom disease in Iran. This is the first report of potato witches’-broom disease in Iran.

Molecular Detection and Identification of Group 16SrI and 16SrXII Phytoplasmas Associated with Diseased Potatoes in Russia

Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. Symptoms resembling stolbur are most prevalent, but round leaf disease, potato witches'-broom, and potato purple top wilt also occur (1). The phytoplasma etiologies of these diseases have never been verified by molecular means. During the summer of 2006, 33 potato plants exhibiting various symptoms including purple top, round leaves, and stolbur-like symptoms characterized by purple top, stunting, bud proliferation, and formation of aerial tubers were randomly collected from the Volga River Region, Central Region, and Northern Caucasian Region in Russia. DNA extracts were prepared from 1.0 g of petioles and leaf mid veins according to a modified procedure with the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as previously described (2). A nested PCR with primer pair P1/P7 in the first amplification followed by R16F2n/R16R2n in the second amplification was performed to detect phytoplasmas in infected potato samples (4). Potato plants maintained in the greenhouse were used as healthy controls. A negative control devoid of DNA templates was included in all PCR assays. One microliter of diluted (1:30) PCR product from the first amplification was used as the template in the nested PCR. Eight of 33 potato samples tested positive in the first PCR. Twelve of 33 potato samples tested positive in nested PCR. Nine of the 12 potato samples that tested positive for phytoplasma exhibited stolbur-like symptoms; the other three samples exhibited round leaves, stunting, or proliferation. The remaining symptomatic samples that exhibited nonspecific purple or yellow discoloration of terminal leaves, without other specific stolbur-like symptoms, may be infected by other pathogens. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products (R16F2n/R16R2n amplicons, 1.2 kb) was performed. PCR products (6 μl) were digested singly with the restriction enzymes AluI, HaeIII, HhaI, HpaII, KpnI, MseI, RsaI, and Tsp509I. Comparison of RFLP profiles with published profiles (3) was used for identification of the putative phytoplasmas detected. Among the 12 PCR positive potato samples, 10 showed very similar or identical RFLP profiles to stolbur phytoplasma, a strain belonging to stolbur phytoplasma group (16Sr XII), subgroup 16SrXII-A and closely related strains, and two showed RFLP profiles similar to those of aster yellows phytoplasma group (16SrI). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession Nos. EU344884-EU344890 and EU333396-EU333400) confirmed the results of the RFLP analyses and also indicated that the two samples showing 16SrI profiles were simultaneously infected with two phytoplasma strains belonging to subgroups 16SrI-A and 16SrI-B. To our knowledge, this is the first confirmation by molecular procedures that stolbur phytoplasma (16SrXII-A) is prevalent in several potato-growing regions and is the first report of 16SrI-A and 16SrI-B phytoplasmas associated with potatoes in Russia.

Clover proliferation phytoplasma: 'Candidatus Phytoplasma trifolii'.

Clover proliferation phytoplasma (CPR) is designated as the reference strain for the CP phylogenetic group or subclade, on the basis of molecular analyses of genomic DNA, the 16S rRNA gene and the 16S-23S spacer region. Other strains related to CPR include alfalfa witches'-broom (AWB), brinjal little leaf (BLL), beet leafhopper-transmitted virescence (BLTV), Illinois elm yellows (ILEY), potato witches'-broom (PWB), potato yellows (PY), tomato big bud in California (TBBc) and phytoplasmas from Fragaria multicipita (FM). Phylogenetic analysis of the 16S rRNA gene sequences of BLL, CPR, FM and ILEY, together with sequences from 16 other phytoplasmas that belong to the ash yellows (AshY), jujube witches'-broom (JWB) and elm yellows (EY) groups that were available in GenBank, produced a tree on which these phytoplasmas clearly clustered as a discrete group. Three subgroups have been classified on the basis of sequence homology and the collective RFLP patterns of amplified 16S rRNA genes. AWB, BLTV, PWB and TBBc are assigned to taxonomic subgroup CP-A, FM belongs to subgroup CP-B and BLL and ILEY are assigned to subgroup CP-C. Genetic heterogeneity between different isolates of AWB, CPR and PWB has been observed from heteroduplex mobility assay analysis of amplified 16S rRNA genes and the 16S-23S spacer region. Two unique signature sequences that can be utilized to distinguish the CP group from others were present. On the basis of unique properties of the DNA from clover proliferation phytoplasma, the name 'Candidatus Phytoplasma trifolii' is proposed for the CP group.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Dry Bean Phyllody Disease in Washington.

During 2003, a new disease, dry bean phyllody (DBPh), was observed in the Columbia Basin of Washington in dry bean (Phaseolus vulgaris L.) cultivars of Andean origin grown in Mattawa and Paterson, WA that caused great reduction in dry bean production. Symptoms of DBPh became apparent during mid-to-late pod development and were characterized by leafy petals (phyllody) and aborted seed pods resembling thin, twisted, and corrugated leaf-like structures. Deformed sterile pods that were small, sickle-shaped, upright, and leathery were also observed. The infected plants generally exhibited chlorosis, stunting, or bud proliferation from leaf axils. Symptoms of DBPh were indicative of possible infection by phytoplasmas. Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Four symptomatic bean plants were analyzed and tested positive for phytoplasma infection on the basis of results of initial polymerase chain reaction (PCR) and subsequent nested-PCR amplifications (2). RFLP analyses of 16S rDNA sequences with restriction enzymes, MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma strains associated with DBPh belonged to the clover proliferation group (16SrVI) subgroup A (16SrVI-A) (2). This subgroup currently consists of three members, clover proliferation (CP; GenBank Accession No. AY500130), potato witches'-broom (PWB; GenBank Accession No. AY500818), and vinca virescence (VR; GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasmas (1,2). The taxonomic affiliations of the DBPh phytoplasma strains were confirmed by phylogenetic analysis of cloned 16S rRNA gene sequences (GenBank Accession Nos. DBPh2, AY496002; DBPh3, AY496003). Among the existing members of subgroup 16SrVI-A, the four DBPh strains were closely related to the VR strain with 99.7% 16S rDNA sequence homology and to the CP strain with 99.2% sequence homology. To gain further evidence on the role of 16SrVI-A phytoplasma strains in DBPh disease, a modified test of Koch's postulates was conducted. Infected tissue from one phytoplasma-positive dry bean sample was grafted onto three Pinto UI-114 bean seedlings in the greenhouse. Within 60 days, the bean seedlings exhibited corrugated leaf-like structures from aborted seedpods, a lack of flower formation, general chlorosis, and stunting similar to the original diseased plants. The lower leaves of the inoculated bean plants became epinastic and leathery. The transmitted phytoplasma was detected in each of the grafted symptomatic seedlings, and the RFLP patterns of its 16S rRNA gene sequences were identical to those of the phytoplasmas in the scions. A high correlation between the presence of disease symptoms and the presence of subgroup 16SrVI-A phytoplasmas in the bean plants suggests that these phytoplasmas play an etiological role in DBPh disease. To our knowledge, these findings provide the first confirmed case of phytoplasma-associated DBPh in the United States.

Clover Proliferation Group (16SrVI) Subgroup A (16SrVI-A) Phytoplasma is a Probable Causal Agent of Potato Purple Top Disease in Washington and Oregon.

An epidemic of purple top disease of potato (Solanum tuberosum) occurred in the Columbia Basin Region of Washington and Oregon in 2002 and 2003, causing great economic loss in the potato industry (1). Symptoms of potato purple top (PPT) were characterized by upright terminal shoots, upward leaf rolling, chlorosis, red or purplish discoloration of new leaves, proliferation of axillary shoots with basal swelling, and the formation of aerial tubers. Preliminary studies on PPT disease suggested phytoplasma as a possible cause (1). In this study, 78 potato samples (including five asymptomatic) were collected from five fields throughout the region. A nested polymerase chain reaction (PCR) with primer pair P1/P7 in the first amplification followed with primer pair R16F2n/R16R2 was performed to detect the presence of phytoplasmas in infected plants (2). Restriction fragment length polymorphism (RFLP) and phylogenetic analyses of amplified 16S rDNA sequences were used for phytoplasma identification. Eighty-four percent (63% in the first amplification) of the symptomatic samples and 60% (0% in the first amplification) of the asymptomatic samples tested positive. Low phytoplasma titers and the presence of PCR inhibitors accounts for the low detection rate in the first PCR amplifications. RFLP analyses of 16S rDNA with enzymes MseI, AluI, HhaI, RsaI, and HpaII indicated that the phytoplasma associated with PPT belonged to the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) (2). 16SrVI-A currently consists of three members, CP (GenBank Accession No. AY500130), potato witches'-broom (GenBank Accession No. AY500818), and vinca virescence (VR) (GenBank Accession No. AY500817), a strain of beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (2). The taxonomic affiliation of PPT phytoplasma was confirmed by phylogenetic analysis of cloned 16S rDNA (GenBank Accession Nos. PPT4, AY496004; PPT8, AY496005). The 16S rDNA sequences of the PPT strains were closely related to VR with 99.7% sequence homology compared with 99.2% with CP. A high correlation between the symptoms and the presence of 16SrVI-A phytoplasmas in the potato plants suggests that these phytoplasmas play an etiological role in PPT disease. To gain further evidence, a modified test of Koch's postulates was conducted. Infected tissues from four phytoplasma-positive potato samples (including PPT4 and PPT8) were grafted onto healthy potato seedlings. Within 60 days after grafting, the potato seedlings developed symptoms similar to those in the original diseased samples. The newly infected plants were maintained through cuttings. RFLP analysis of 16S rDNA indicated that the phytoplasmas detected in each of the seedlings and cuttings were identical to those in the scions. These results confirmed the probable etiological role of CP group, subgroup 16SrVI-A phytoplasma strains in PPT disease in Washington and Oregon. There are two other confirmed cases of phytoplasmas (BLTVA and aster yellows phytoplasma) associated with PPT disease in Utah (4) and Mexico (3).

Characterization of a phytoplasma associated with witches' broom disease of potatoes in Korea

Symptoms of witches' broom disease caused by phytoplasma, including general stunting and yellowing, were observed in potatoes (Solanum tuberosum L.) in a storehouse on Jeju Island, Korea in 1998. Based on sequence analysis of DNA products from polymerase chain reaction (PCR)-amplified 16S ribosomal DNA and 16S–23S spacer region using universal phytoplasma primers, the phytoplasma associated with potato witches' broom disease (PWB) was identified as a member of 16S-group VIII. It was most closely related to elm AH phytoplasma (99.7% similarity, accession no. AF268895), which is in the clover proliferation (CP) subgroup. This report is the first from the East Asian continent of a plant pathogenic phytoplasma belonging to the CP subgroup and includes the nucleotide sequence of most of the potato phytoplasma 16S rDNA.

Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.

Use of Heteroduplex Mobility Assay for Identification and Differentiation of Phytoplasmas in the Aster Yellows Group and the Clover Proliferation Group

ABSTRACT This paper describes the identification and differentiation of phytoplasmas by a highly sensitive diagnostic technique, DNA heteroduplex mobility assay (HMA). Closely related phytoplasma isolates of clover proliferation (CP), potato witches'-broom (PWB), and alfalfa witches'-broom (AWB) were collected from the field from 1990 to 1999. The entire 16S rRNA gene and 16/23S spacer region were amplified by polymerase chain reaction (PCR) from the field samples and standard CP, PWB, and AWB phytoplasmas and were subjected to restriction fragment length polymorphism (RFLP) analysis and HMA. Two subgroups (I and II) of phytoplasmas in the CP group were identified by HMA but not by RFLP analysis. The results were confirmed by 16/23S spacer region sequence data analysis. After HMA analyses of the PCR-amplified 16/23S spacer region, 14 phytoplasma isolates from field samples were classified into two aster yellows subgroups: subgroup I, phytoplasma isolates from China aster (Callistephus chinensis) yellows, French marigold (Tagetes patula) yellows, cosmos (Cosmos bipinnatus cv. Dazzler) yellows, clarkia (Clarkia unguiculata) yellows, California poppy (Eschscholzia californica cv. Tai Silk) yellows, monarda (Monarda fistulosa) yellows, and strawflower (Helichrysum bracteatum) yellows; and subgroup II, phytoplasma isolates from zinnia (Zinnia elegans cv. Dahlia Flower) yellows, Queen-Annes-Lace (Daucus carota) yellows, scabiosa (Scabiosa atropurpurea cv. Giant Imperial) yellows, Swan River daisy (Brachycombe multifida cv. Misty Pink) yellows, pot marigold (Calendula officinalis) yellows, purple coneflower (Echinacea purpurea) yellows, and feverfew (Chrysanthemum parthenium) yellows. The results indicate that HMA is a simple, rapid, highly sensitive and accurate method not only for identifying and classifying phytoplasmas but also for studying the molecular epidemiology of phytoplasmas.

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.

Development of Cloned DNA Probes for Phytoplasma Associated with Loofah Witches’ Broom

AbstractDNA was isolated from periwinkle (Catharanihus roseus) infected with a phytoplasma that originated in loofah witches’ broom affected by loofah. Cloned DNA inserts from six LfWB‐phytoplasma specific recombinant plasmids were not only labelled with digoxigenin, but also used as probes. Probes hybridized with DNA derived from LfWB‐phytoplasma affected periwinkle and loofah, but not with DNA from healthy plants or plants infected with phytoplasmas associated with elm yellows, red bird cactus, peanut witches’ broom, paulownia witches’ broom, Ipomoea obscura witches’ broom, aster yellows (two isolates), and sweet potato witches’ broom obtained with DNA from different phytoplasmas experimentally maintained in periwinkle. The probes could detect LfWB‐phytoplasma DNA with as little as 16 ng and 32 ng of DNA from periwinkle and loofah, respectively. The method proposed herein provides a means for specifically detecting and identifying of loofah witches’ broom phytoplasma, as well as confirming the notion that this phytoplasma represents a distinct strain cluster,

An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom.

A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches' broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches' broom (PNWB) and the two showed homology based on the results of Western blot analysis, Southern hybridization, Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma and PNWB phytoplasma were not found in other phytoplasmas tested.

Identification of a phytoplasma causing yellows of Monarda

Monarda yellows occurring in southern Alberta was found to be associated with a phytoplasma. Using two pairs of universal primers, 16S ribosomal DNA fragments (about 1.5 and 1.2 kb) were amplified separately by polymerase chain reaction (PCR) from DNA samples that had been extracted from infected monarda. No such DNA bands were observed using DNA samples from uninfected monarda. The DNA fragment (1.2 kb) amplified by nested‐PCR was analysed and compared with western aster yellows (AY27, Canada), eastern aster yellows (EAY, USA), French hydrangea aster yellows (AYHF), Belgium hydrangea aster yellows (AYHB), clover proliferation (CP, Canada) and potato witches'‐broom (PWB, Canada) by means of restriction fragment length polymorphism (RFLP) using endonucleases AluI, MseI, HpaII, Sau3AI, KpnI and RsaI. The results showed that monarda yellows phytoplasma belongs to the aster yellows subclade and is different from CP and PWB. This is the first report of aster yellows phytoplasma infecting monarda.

Abstracts, The Canadian Phytopathological Society, Annual Meeting, Saskatoon, Saskatchewan, June 1996

Abstracts, The Canadian Phytopathological Society, Annual Meeting, Saskatoon, Saskatchewan, June 1996

Two Groups of Phytoplasmas from Japan Distinguished on the Basis of Amplification and Restriction Analysis of 16S rDNA.

Phytoplasmas (mycoplasmalike organisms, MLOs) associated with mitsuba (Japanese hone-wort) witches'-broom (JHW), garland chrysanthemum witches'-broom (GCW), eggplant dwarf (ED), tomato yellows (TY), marguerite yellows (MY), gentian witches'-broom (GW), and tsu-wabuki witches'-broom (TW) in Japan were investigated by polymerase chain reaction (PCR) amplification of DNA and restriction enzyme analysis of PCR products. The phytoplasmas could be separated into two groups, one containing strains JHW, GCW, ED, TY, and MY, and the other containing strains GW and TW, corresponding to two groups previously recognized on the basis of transmission by Macrosteles striifrons and Scleroracus flavopictus, respectively. The strains transmitted by M. striifrons were classified in 16S rRNA gene group 16SrI, which contains aster yellows and related phytoplasma strains. Strains GW and TW were classified in group 16SrIII, which contains phytoplasmas associated with peach X-disease, clover yellow edge, and related phytoplasmas. Digestion of amplified 16S rDNA with HpaII indicated that strains GW and TW were affiliated with subgroup 16SrIII-B, which contains clover yellow edge phytoplasma. All seven strains were distinguished from other phytoplasmas, including those associated with clover proliferation, ash yellows, elm yellows, and beet leafhopper-transmitted virescence in North America, and Malaysian periwinkle yellows and sweet potato witches'-broom in Asia.

Development and Application of Cloned DNA Probes for a Mycoplasmalike Organism Associated with Sweetpotato Witches'-broom

EcoRI restriction fragments of genomic DNA from the mycoplasmalike organism (ML0) associated with sweet potato witches'-broom (SPWB) were cloned in plasmid pGEM3Zf(+). Cloned inserts from 14 SPWB-MLO-specific recombinant plasmids were EcoRI-excised, labeled with digoxigenin, and used as probes. Probes hybridized with DNA derived from SPWB-MLO-affected periwinlike and sweetpotato but not with DNA from healthy plants or plants infected with MLOs associated with loofah, paulownia, and Ipomoea obscura witches'-broom, elm and aster yellows, rice yellow dwarf, and bamboo little leaf disease. Twelve of 14 probes also hybridized with the serologically related peanut witches'-broom (PNWB) MLO [...]