N. A. Harrison

Detection and Characterization of a Lethal Yellowing (16SrIV) Group Phytoplasma in Canary Island Date Palms Affected by Lethal Decline in Texas

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in Canary Island date (Phoenix canariensis) palms displaying symptoms similar to lethal yellowing (LY) disease in Corpus Christi, TX. An rDNA product (1.8 kb) was amplified consistently from 10 of 11 palms by PCR employing phytoplasma universal rRNA primer pair P1/P7. Also, AluI endonu-clease digests and sequencing of P1/P7 products revealed that nontarget Bacillus megaterium-related rDNA sequences of similar size were co-amplified along with phytoplasma rDNA from 10 palms. A 1,402-bp product was obtained from all 11 symptomatic palms when initial P1/P7 products were reamplified by PCR employing nested LY phytoplasma group-specific 16S rRNA primer pair LY16Sf/LY16Sr. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products revealed that palm-infecting phytoplasmas were uniform and most similar to strains composing the coconut lethal yellowing phytoplasma (16SrIV) group. Sequence analysis of 16S rDNA determined the Texas Phoenix palm decline (TPD) phytoplasma to be phylogenetically closest to the Carludovica palmata leaf yellowing (CPY) phytoplasma. rDNA profiles of strains TPD and CPY obtained with AluI were co-identical and distinct from other known 16SrIV group phytoplasmas. On this basis, both strains were classified as members of a new subgroup, 16SrIV-D.

Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences

The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

Comparative investigation of MLOs associated with Caribbean and African coconut lethal decline diseases by DNA hybridization and PCR assays.

Mycoplasmalike organisms (MLOs) associated with lethal decline diseases of the coconut palm (Cocos nucifera) in eastern and western Africa were detected by dot hybridizations using (P-32)dATP-labeled cloned DNA probes. Two probes, each consisting of a genomic DNA fragment of the palm lethal yellowing (LY) MLO from Florida, hybridized at moderate stringency to DNAs from four coconut cultivars with lethal disease (LD) in Tanzania and from a solitary West African Tall coconut palm with symptoms of Awka disease in Nigeria. Neither probe hybridized to DNA of the LD-affected hybrid coconut PB121 or to DNA of symptomless palms. Conserved, mollicute-specific oligonucleotide sequences used for polymerase chain reactions (PCR) primed the amplification of near full-length MLO 16S rRNA genes from all decline-affected palms. No restriction fragment length polymorphisms were observed when rDNA amplified from both LY- and LD-affected coconut palms were singly digested with the restriction endonucleases AluI, BamHI, DraI, EcoRI, HpaI, HpaII, RsaI, and ScaI. Polymorphisms were evident after digestion of MLO rDNA with TaqI. These data establish the existence of genetic relationships between MLOs associated with coconut lethal decline diseases in the western Caribbean region and in Africa and provide further evidence indicating that the LY and LD MLOs, although very similar, are not genetically identical pathogens.

PCR Assay for Detection of the Phytoplasma Associated with Maize Bushy Stunt Disease

NA amplification by polymerase chain reaction (PCR) was used to detect the phytoplasma associated with maize bushy stunt (MBS) disease. A pair of oligonucleotide primers was synthesized according to partial sequences of a cloned 1-kb fragment of genomic DNA of the MBS phytoplasma (Texas isolate) maintained in sweet corn. PCR performed for 30 cycles with primer annealing at 61°C amplified a DNA product of about 740-bp in reaction mixtures containing template DNA derived from symptomatic corn singly infected with MBS phytoplasma isolates from either Texas, Florida, Costa Rica, or Mexico. No comparable product was amplified from DNAs of healthy corn, plants affected by various other phytoplasmal diseases, or from Spiroplasma kunkelii. Forty cycles of PCR enabled detection of a Florida isolate of the MBS phytoplasma in all leaf and stalk samples tested from presymptomatic plants 12 days after plants were fed upon by inoculative vector Dalbulus maidis leafhoppers and in the majority of nonvector Peregrinus maidis planthoppers after 1 to 5 days of exposure to symptomatic plants.

Phylogenetic Relationships among Members of the Class Mollicutes Deduced from rps3 Gene Sequences

A gene for a ribosomal protein, rps3, was amplified by PCR and sequenced from representatives of the class Mollicutes. Alignments of the deduced amino acid sequences allowed the construction of a phylogeny that is consistent with the phylogenetic trees created from 5S and 16S rRNA comparisons, including the position of the former Acholeplasma florum on the Mycoplasma branch, rather than with the classical Acholeplasmataceae. Additional confirmation of the phylogeny comes from the deduction that the UGA triplet encodes tryptophan in the rps3 gene from Mesoplasma florum, as it does in the mycoplasmas and spiroplasmas. The sequence data from Acholeplasma axanthum 743 and Acholeplasma sp. strain J233 allow refinements to the phylogenetic tree within the Acholeplasmataceae, providing evidence that the sterol requirement of Anaeroplasma abactoclasticum (order Anaeroplasmatales) is a derived trait. It was also evident that the nonhelical plant-pathogenic members of the class Mollicutes, referred to as mycoplasma-like organisms or phytoplasmas, are more closely related to the true acholeplasmas (Acholeplasma laidlawii and strain J233) than to other members of the Mollicutes.

Detection and diagnosis of lethal yellowing

Lethal yellowing (LY) is one of the most important diseases of the coconut palm (Cocos nucifera L.) in the American tropics because it spreads rapidly, kills palms quickly and is incurable at present. Presently, LY is restricted in its distribution to the western Caribbean region (McCoy et al., 1983) where epidemics of disease in recent decades have destroyed millions of palms in Jamaica, Florida and southern Mexico. Further spread into Belize (Eden-Green, 1997) and Honduras (Ashburner et al., 1996) in recent years threatens coconut production in Central America because the Atlantic Tall (also known as Jamaica Tall), the most common coconut ecotype found throughout the Caribbean and along the Atlantic coast of the Americas, is highly susceptible to LY (Howard, 1983).

Detection and differentiation of phytoplasmas associated with diseases of strawberry in Florida.

Strawberry (Fragaria × ananassa Duchesne) plants with symptoms suggestive of phytoplasmal disease were identified in commercial fields and a breeders plot in west central Florida during the 1995 to 1996 winter growing season. Affected plants were all conspicuously stunted and unproductive. Primary symptoms on cvs. Rosa Linda and Carlsbad and on a breeders accession resembled those of strawberry green petal (SGP). Plants displayed sparse clusters of virescent flowers with enlarged sepals and phylloid receptacles that failed to develop fully into fleshy structures or redden on ripening. Symptoms on cv. Oso Grande were more typical of multiplier disease and included a proliferation of branch crowns producing numerous small leaves with spindly petioles. Oso Grande and Carlsbad originated as transplants from a nursery in Montreal, Canada, whereas Rosa Linda transplants were from Nova Scotia. Plants were assessed for phytoplasma infection by polymerase chain reaction with total DNAs from leaves and petioles as template and phytoplasma-specific ribosomal RNA primers P1 and P7 (3), or mollicute-specific ribosomal protein (rp) gene primers rpF1 and rpR4 (2). Amplification of a 1.8-kb rDNA or 1.2-kb rp gene product, respectively, confirmed infection of Rosa Linda (7 of 7 plants), Carlsbad (3 of 7), Oso Grande (4 of 4), and a single breeders accession. No products were amplified from DNAs of healthy plants. Restriction fragment length polymorphism patterns of rDNA digested with AluI, EcoRI, HaeIII, HhaI, HpaII, KpnI, ScaI, or Tru9I endonucleases, or of rp gene products digested with AluI, DraI, RsaI, TaqI, or Tru9I, revealed no differences among phytoplasma strains affecting both Rosa Linda and Carlsbad. Collectively, patterns were comparable to those of clover phyllody and SGP phytoplasmas, two Canadian strains previously classified as members of phytoplasma 16S rRNA (rr)-ribosomal protein (rp) group 16S rI, subgroup C (16S rI-C (rr-rp)) (1). Similarly, no differences were evident among phytoplasmas associated with all four diseased Oso Grande plants. Both rDNA and rp fragment profiles associated with this cultivar were characteristic of strains such as tomato big bud and eastern aster yellows delineated as 16S rI-A (rr-rp) subgroup members (1). However, AluI rDNA and TaqI rp fragment patterns were unique, identifying Oso Grande-infecting strains as representatives of a new subgroup within the larger 16S rI (rr-rp) group. Cumulative rDNA and rp fragment profiles of the phytoplasma associated with the breeders accession matched those of the Mexican periwinkle virescence phytoplasma, identifying this strain as a 16S rI-I (rr-rp) subgroup member (1) and a second possible etiological agent of SGP. This is the first report of phytoplasmas infecting strawberry in Florida. References: (1) D. E. Gundersen et al. Int. J. Syst. Bacteriol. 46:64, 1996. (2) P.-O. Lim and B. B. Sears. J. Bacteriol. 174:2602, 1993. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Occurrence of a 16SrIV Group Phytoplasma not Previously Associated with Palm Species in Yucatan, Mexico

The occurrence of 16SrIV group phytoplasmas in palm species Sabal mexicana and Pseudophoenix sargentii is reported here for the first time. Palm trees showed leaf decay and leaf yellowing syndromes, respectively. An amplification product (1.4 kb) was obtained in symptomatic S. mexicana (18 of 21) and symptomatic P. sargentii (1 of 1) palm trees sampled in different locations in Yucatan State, Mexico; five of the positive S. mexicana and the positive P. sargentii trees died. The identity of the phytoplasmas from these species was determined by restriction fragment length polymorphism profiling with restriction enzymes AluI and HinfI, showing there could be two phytoplasma strains of the 16SrIV group. In one S. mexicana palm, the profile was the same as observed with these enzymes for phytoplasmas of 16SrIV-A subgroup, previously associated with Cocos nucifera palm trees and, in the rest of the trees, including the P. sargentii palm, the profile was for phytoplasmas of the 16SrIV-D subgroup. These identities were supported by analyses of the amplicons obtained by nested polymerase chain reaction by nucleotide-nucleotide BLAST analysis. Geographical distribution of the association S. mexicana/16SrIV group phytoplasmas was found widely dispersed in Yucatan State. A potential role of S. mexicana palm trees as a permanent source of phytoplasma inoculum is suggested. In addition to P. sargentii, other palm species (Thrinax radiata and C. nucifera) coexisting with S. mexicana trees were also sampled and analyzed.

Detection and Characterization of an Elm Yellows (16SrV) Group Phytoplasma Infecting Virginia Creeper Plants in Southern Florida

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

Fusarium oxysporum f. sp. palmarum, a Novel Forma Specialis Causing a Lethal Disease of Syagrus romanzoffiana and Washingtonia robusta in Florida

A new disease of Syagrus romanzoffiana (queen palm) and Washingtonia robusta (Mexican fan palm) has spread across the southern half of Florida during the past 5 years. The initial foliar symptom is a one-sided chlorosis or necrosis of older leaf blades, with a distinct reddish-brown stripe along the petiole and rachis and an associated discoloration of internal tissue. Within 2 to 3 months after onset of symptoms, the entire canopy becomes desiccated and necrotic but the leaves do not droop or hang down around the trunk. Based on pathogenicity and morphological and molecular characterization, the etiological agent has been identified as a new forma specialis of Fusarium oxysporum, designated f. sp. palmarum. Sequence analysis of a portion of the translation elongation factor 1-α gene (EF-1α) separated 27 representative isolates into two EF-1α groups, which differed by two transition mutations. Members of both EF-1α groups are pathogenic on both species of palm. A phylogenetic analysis inferred from partial EF-1α sequences from a genetically diverse set of F. oxysporum isolates, including three other formae speciales pathogenic on palm (i.e., f. sp. albedinis, f. sp. canariensis, and f. sp. elaeidis), suggested that f. sp. palmarum and f. sp. albedinis may be more closely related to one another than either is to the two other palm pathogens.