Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr‐IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr‐IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead‐end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.     

Direct PCR detection of phytoplasmas in experimentally infected insects

Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)-infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl-trimethyl-ammonium-bromide (CTAB), and the other was boiling in Tris-EDTA buffer. The chromosomal primers amplified phytoplasma-specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20^oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.     

Relative quantification of chrysanthemum yellows (16Sr I) phytoplasma in its plant and insect host using real-time polymerase chain reaction

A real-time polymerase chain reaction (PCR) method for the quantification of chrysanthemum yellows (CY) phytoplasma DNA in its plant (Chrysanthemum carinatum) and insect (Macrosteles quadripunctulatus) host is described. The quantity of CY DNA was measured in each run relative to the amount of host DNA in the sample. Primers and a TaqMan probe for the specific PCR amplification of phytoplasma DNA were designed on a cloned CY-specific ribosomal fragment. Primers and TaqMan probes were also designed on sequences of the internal transcribed spacer region of the insect’s ITS1 rDNA and of the plant’s 18S rDNA for amplification from C. carinatum and M. quadripunculatus, respectively. Absolute quantification of CY DNA was achieved by comparison with a dilution series of the plasmid containing a CY 16S rDNA target sequence. Absolute quantification of plant and insect DNAs was achieved by comparison with a dilution series of the corresponding DNAs. Quantification of CY DNA in relation to host DNA was finally expressed as genome units (GU) of phytoplasma DNA per nanogram of host (plant or insect) DNA. Relative quantification avoided influences due to different yields during the DNA extraction procedure. The quantity of CY DNA was about 10,000–20,000 GU/ng of plant DNA and about 30,000–50,000 GU/ng of insect DNA. The method described could be used to phytoplasma multiplication and movement in different plant and insect hosts.     

Study of the transmission of stolbur phytoplasma to different crop species, by Macrosteles quadripunctulatus

In an epidemiological study conducted on commercial agricultural plots affected by stolbur phytoplasma in Northern and Central Spain, different species of leafhoppers and planthoppers were identified as potential vectors of the phytoplasma. They included individuals of Macrosteles quadripunctulatus infected by stolbur phtytoplasma in most of the locations. The potential of this species as a vector of stolbur was evaluated in this work. The transmission trials were carried out on healthy plants of Catharanthus roseus (periwinkle), Lycopersicon esculentum (tomato), Daucus carota (carrot), Lactuca sativa (lettuce) and Vitis vinifera (grapevine). The first symptoms of infection in these plants were observed 2 weeks after the inoculation period in tomato and periwinkle, and after 4 weeks in carrot. Only one of five grapevines showed phytoplasma symptoms. PCR analysis was used to verify the ability of M. quadripunctulatus in transmitting stolbur phytoplasma in the plant species tested. The phytoplasma was not detected in lettuce or in the healthy control plants. Studies of stolbur transmission to insect‐feeding medium were also conducted and indicated that M. quadripunctulatus acquires and was capable of transmitting the phytoplasma after it fed during a single day on infected plants followed by a 19‐day latent period on healthy plants.     

Molecular data reveals California as the potential source of an invasive leafhopper species, Macrosteles sp. nr. severini, transmitting the aster yellows phytoplasma in Hawaii

A species of aster leafhopper ( Macrosteles sp.) became established in 2001 on Oahu, Hawaii, and through the transmission of the aster yellows phytoplasma, caused devastating losses to the island's watercress industry. DNA sequence data were analysed from two mitochondrial genes [cytochrome oxidase subunit 1( CO1 ) and nicotinamide adenine dinucleotide 1 ( NADH1 )] and one nuclear gene (wingless, Wg ) (combined total of 1874 bp) to reconstruct phylogenetic relationships between putative US mainland source populations of aster leafhoppers and those introduced to Hawaii. These data were applied to elucidate the origin(s) and identity of Hawaiian infestations and the amount of genetic diversity within introduced invasive populations. Both phylogenetic search criteria (Bayesian and maximum likelihood models) converged onto similar tree topologies for all three gene regions and suggested that Hawaii infestations represent a single undescribed leafhopper species unrelated to the common aster leafhopper, Macrosteles quadrilineatus . An exact haplotype match was found from a specimen intercepted from watercress shipped to Hawaii from Los Angeles, California, suggesting this region as the potential source for Hawaiian infestations. Two mitochondrial haplotypes were identified in Hawaii suggesting two or perhaps just a single introduction of more than one female.     

中国二叉叶蝉属三新种记述(半翅目,叶蝉科,殃叶蝉亚科)

记述二叉叶蝉属 Maaosteles Fieber 3 新种,即西藏二叉叶蝉 Macrosteles tibetensis sp.nov.与M.heiseles Kuoh外形相似,主要区别在于新种下生殖板端部细,且弯曲,端部外缘锯齿状.新种宽胫二叉叶蝉Macrasteles latiaedeagus sp.nov.与M.vilbastei Hamilton外形相似,与后者区别在于:1)个体明显大;2)阳茎基部管状弯曲,端区分叉较宽.双斑二叉叶蝉Macrosteles bimaculatus sp.nov.与M.Brumnescens Anufriev相似,不同点在于;1)阳基侧突细长;2)尾节侧瓣近似方形,外缘突起锯齿状;3)阳茎于端部1/3处分为二叉,此二叉又于叉的近端部相交叉而后分开.     

Detection, characterisation and transmission by Macrosteles leafhoppers of watercress yellows phytoplasma in Hawaii

A new yellows disease of watercress (Nasturtium officinale) in Hawaii has symptoms of reduced leaf size, leaf yellowing and crinkling, and occasionally witches’ brooms. This disease is found on all watercress farms on Oahu but has not yet been found on other Hawaiian islands. Watercress plants were tested for phytoplasma infection by polymerase chain reaction assays using phytoplasma‐specific primers. Amplicons of the expected sizes were produced from all symptomatic plants but not from healthy plants raised from seed. Phylogenetic analysis of the 16S rRNA gene indicated that watercress yellows was caused by a phytoplasma in the aster yellows group, with sequence similarity to onion yellows from Japan. Six weed species collected from the vicinity of affected watercress farms, Amaranth sp., Eclipta prostrata, Emilia sonchifolia, Plantago major, Myriophyllum aquaticum and Sonchus oleraceus, were also determined to be hosts of this phytoplasma. Leafhoppers, identified as Macrosteles sp. (Hemiptera, Cicadellidae), collected from symptomatic watercress transmitted this phytoplasma to watercress, plantain and lettuce (Lactuca sativa) in greenhouse experiments.     

Identification of four distinct ‘Candidatus Phytoplasma’ species in pomegranate trees showing witches' broom,little leaf and yellowing in Jordan,and preliminary insights on their putative insect vectors and reservoir plants

During field surveys conducted in northern Jordan from June to November 2020, phytoplasma-like symptoms, including leaf yellowing/reddening and rolling, little leaf and witches' broom were observed in pomegranate. Disease incidence in 22 surveyed orchards ranged from 30% to 65%. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 17% of collected symptomatic pomegranate trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’, ‘Ca. P. aurantifolia’, ‘Ca. P. asteris’ and ‘Ca. P. ulmi’. These phytoplasmas were found in plants showing specific symptoms and differentially distributed in the considered locations. Additionally, three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata and Psammotettix striatus) and two non-crop plants (Plantago major and Capsicum annuum) resulted hosting ‘Ca. P. asteris’ strains, and one cicadellid (Balclutha incisa) was carrying a ‘Ca. P. solani’ strain. A new pomegranate disease complex associated with multiple phytoplasmas, including ‘Ca. P. aurantifolia’ and ‘Ca. P. ulmi’, never reported before in this host plant, is described here. Moreover, preliminary indications are provided on its possible epidemiology in Jordan, involving two putative insect vectors (M. sexnotatus, B. incisa) first reported in the Country.