Differences in Virulence and Genomic Features of Strains of 'Candidatus Phytoplasma mali', the Apple Proliferation Agent

ABSTRACT Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with 'Candidatus Phytoplasma mali' were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52 degrees C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in 'Ca. P. mali'. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.     

Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.     

Translocation of apple proliferation phytoplasma via natural root grafts – a case study

Apple proliferation (AP), a phytoplasma-induced disease of apple trees, was proven to be transmitted through infected grafting material and sap-sucking insects. To date there are little firm data on disease propagation in the field via natural root grafts. This question was thus addressed in the present case study by investigating trees of a 24-year old commercial apple orchard (‘Red Chief’ on MM 111), where the existence of root connections was discovered accidentally. After having displayed specific AP symptoms, nine trees were cut down and the stubs were infiltrated or brushed with glyphosate. Herbicide injury, however, remained not only restricted to the treated stubs, but also spread to approximately 50 neighbouring trees. Surprisingly, none of the pollinators (‘Granny Smith’ on M 9) growing interjacently and alternating between herbicide-damaged main crop trees was affected. Respective to the position of the nine AP-infected and glyphosate-treated cut stumps, four sections in the orchard were defined, from which a total of 122 trees was sampled and analysed using qualitative real-time PCR for detection of AP phytoplasma. The pathogen was found in 71.4% of ‘Red Chief’ trees with severe herbicide damage and 18.8% of trees with partial herbicide damage. None of the 31 investigated pollinators was AP-infected. Our data indicate that root connections seem to play a role for the spread of AP phytoplasma at least in older orchards and between trees on vigorous rootstocks.     

Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction

ABSTRACT For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 x 10(6). Phytoplasma concentrations of 2.2 x 10(8) to 1.5 x 10(9) cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.     

Seasonal colonisation of apple trees by ‘Candidatus Phytoplasma mali’ revealed by a new quantitative TaqMan real-time PCR approach

Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’, is an economically important disease affecting many apple-growing areas in Europe. A new TaqMan real-time PCR assay was established for absolute quantification of ‘Ca. P. mali’ by using a single-copy gene of the host plant as a reference, which is amplified with the pathogen DNA in a single-tube reaction. Normalised estimates of phytoplasma concentration are ultimately expressed as the number of phytoplasma cells per host plant cell. The assay was used to monitor the ‘Ca. P. mali’ titre over the course of two growing seasons in roots and branches of symptomatic and asymptomatic but AP-positive apple trees. All 252 root samples from symptomatic and asymptomatic trees tested positive, with an average number of 59.8 ± 5.68 (standard error) and 55.1 ± 9.83 ‘Ca. P. mali’ per host cell, respectively. From the 378 shoot samples analysed, 81% of the symptomatic and only 20% of the asymptomatic samples were AP-positive with an average number of 9.4 ± 1.04 and 0.7 ± 0.13 ‘Ca. P. mali’ per host cell, respectively. This strengthens evidence that not the pathogen occurrence alone but the presence of a certain quantity of ‘Ca. P. mali’ in the aerial tree sections is involved in symptom expression. In addition, pronounced seasonality of the phytoplasma concentration was found, not only in branches, but also for the first time in roots of symptomatic and asymptomatic apple trees. Highest phytoplasma levels in roots were detected from December to May.     

Genetic diversity of Czech ‘Candidatus Phytoplasma mali’ strains based on multilocus gene analyses

In several European countries apple trees are affected by apple proliferation disease, which is usually associated with the presence of ‘Candidatus Phytoplasma mali’. During 2010, samples from several apple trees displaying proliferation symptoms were collected throughout the Czech Republic to verify identity of phytoplasmas detected in association with the disease. The majority of the 74 apple trees examined using molecular tools were positive for ‘Ca. P. mali’ presence. The 16S–23S ribosomal genes, the ribosomal protein genes and the nitroreductase and rhodonase like genes were then studied to verify phytoplasma strain variability on multigenic bases. Two RFLP profiles and correspondingly two genetic lineages were found in the PCR-amplified fragments covering the 16S–23S rDNA spacer region. ‘Ca. P. mali’ strains belonging to rpX-A subgroup were identified in the majority of the apple tree sampled, whereas phytoplasmas belonging to the rpX-B subgroup were distributed sporadically. The apple proliferation subtypes AP-15 and AT-2 exhibited nearly equal occurrence; the AT-1 subtype and a mixture of the two or all three of the AP subtypes were infrequently found. The PCR/RFLP results were confirmed by nucleotide sequence analyses of selected ‘Ca. P. mali’ strains.     

Detection and characterization of Plum pox virus: serological methods

Tremendous progress has been made in the research and development of Plum pox virus (PPV) serological reagents and methods in recent years. Two facts have revolutionised the serological detection and characterization of the virus: the development of the ELISA method in 1977, and the later emergence of specific monoclonal antibody technology. The availability of commercial kits has popularised PPV diagnosis, now making diagnosis possible at large scale for quarantine purposes, eradication programmes and control of the disease in nurseries. The use of the universal monoclonal antibody 5B-IVIA, used in DASI-ELISA, is the most accurate system for routine PPV detection. Likewise, the use of typing monoclonal antibodies gives exact characterization of the main PPV types described: 4DG5 for PPV-D, AL for PPV-M, EA24 for PPV-EA, and TUV and AC for PPV-C. There is, in general, an excellent correlation between serological data obtained with PPV specific monoclonal antibodies and data obtained by molecular PCR based methods. ELISA using a single or a mixture of monoclonal antibodies will remain the preferred method for universal detection and routine screening of PPV for years to come. Today, other serological methods and reagents are also recommended in the EPPO Diagnostic Protocol, increasing the number of reliable tests available for PPV detection. These developments have helped to control sharka disease in recent years. International co-operation in this field has been crucial to the improvement and validation of serological tools for PPV detection and characterization.     

Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins1

Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non-structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture-PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA-I and immunoprinting-ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D-specific monoclonal antibodies can be used in routine tests with high affinity.     

Recovery in apple trees infected with the apple proliferation phytoplasma: an ultrastructural and biochemical study

ABSTRACT Localization of hydrogen peroxide (H(2)O(2)) and the roles of peroxidases, malondialdehyde, and reduced glutathione in three apple cultivars were compared in healthy trees, trees infected with apple proliferation phytoplasma (APP), and trees that had recovered from the infection. In recovered apple trees, symptoms of the disease and the pathogen had disappeared from the canopy, but phytoplasmas remained in the roots. H(2)O(2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides.H(2)O(2) occurred in the plasmalemma of the phloem of leaves of recovered apple trees, but not in healthy or APP-infected leaves. In all cultivars, the peroxidase activity detected in tissue from APP-diseased trees was greater than or equal to that of tissue from recovered trees, which equaled or exceeded that of tissue from healthy trees, at two sampling times (May and September). In contrast, the glutathione content of leaves decreased in the reverse order. More malondialdehyde was observed in leaves from recovered trees than in leaves from healthy or APP-infected trees in three of six cultivar-date combinations; in the other three combinations, the malondialdehyde contents of leaves from healthy, infected, and recovered trees were not significantly different from one another. The results suggest that some components of the oxidant-scavenging system in recovered leaves are not very active, leading to an overproduction of H(2)O(2) and, possibly, to a membrane lipid peroxidation.The production of H(2)O(2) appears to be involved in counteracting pathogen virulence.     

On the apple proliferation symptom display and the canopy colonization pattern of “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma mali” in apple trees

Notwithstanding the availability of several different real time PCR protocols for “Candidatus Phytoplasma mali”, it is still unclear how informative is the estimation of the concentration of phytoplasma cells in the leaves of apple proliferation infected trees, and how the reliability of the estimations may be affected by an erratic and uneven distribution of the pathogen in the host. Here we investigated these issues systematically and showed that phytoplasma concentration varies significantly among seasons, but not between two cultivars that appeared to have different degree of susceptibility on the basis of the symptoms displayed. In fully symptomatic trees sampled at the end of the season the phytoplasmas were detectable in most leaves, but in more than half of the leaves at low concentrations. Both the pattern of colonization of the canopy and the amount of phytoplasmas varied greatly in trees that show symptom remission, although a direct relation between symptom severity and colonization could not be established. The sampling of the apple canopy for the purpose of evaluation of concentration of “Candidatus Phytoplasma mali” should take into consideration the complex pattern of colonization and seasonal variation.     

Association of phytoplasmas with the decline of European hazel in southern Italy

In the Campania region of southern ltaly. commercial orchards of European hazel ( Corylus avellana ) are severely affected by yellowing and decline. To determine whether phytoplasmas are associated with the disorder, stem samples from diseased trees were examined using polymerase chain reaction assays. No visible products were obtained by amplification of sample DNA with universal and group-specific phytoplasma primers. However, when the products obtained with universal primers were re-amplified with nested primers that were specific for the fruit tree phytoplasmas of the apple proliferation group, most samples tested positively. Restriction site analysis revealed that the trees were infected with the apple proliferation, pear decline, and European stone fruit yellows phytoplasmas in about the same proportion. Some of the trees were doubly infected with one of the fruit tree phytoplasmas and the aster yellows agent. Most of the infected trees were also identified by hybridization of the products obtained in the initial amplification with suitable oligonucleotide probes.     

泡桐丛枝植原体抗原膜蛋白抗血清的制备及应用

 根据报道的泡桐丛枝植原体(Paulownia witches’-broom phytoplasma,PaWB)抗原膜蛋白(antigenic membrane pro-tein,AMP)基因的核苷酸序列设计引物,提取发病泡桐总DNA,经PCR扩增并成功克隆泡桐丛枝植原体amp基因。序列分析表明,amp基因由696个核苷酸组成,编码231个氨基酸残基,与GenBank中登录的2个泡桐丛枝植原体的膜蛋白核苷酸序列同源性为100%。将amp基因91-604 nt部分序列(命名为ampd)克隆到原核表达载体pGEX-4T-3,诱导后,经SDS聚丙烯酰胺凝胶电泳分析,表明融合蛋白在大肠杆菌BL21(DE3)中得到了高效表达。以纯化的带GST标签的AMPD融合蛋白为抗原免疫德国大白兔,制备了PaWB-AMPD抗血清。利用该血清,通过Western印迹、点印迹、ELISA、间接免疫荧光和免疫捕获PCR试验均能在发病泡桐组织中特异检测到泡桐丛枝植原体。     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Long-term maintenance of nonculturable apple-proliferation phytoplasmas in their micropropagated natural host plant

Phytoplasmas associated with apple proliferation (AP) disease of apple trees have been maintained in their micropropagated natural host plant Malus pumila since 1985. Different isolates of these nonculturable plant pathogens could thus be studied in vitro . Amplification of a pathogen-specific DNA fragment by polymerase chain reaction (PCR) confirmed the presence of AP phytoplasmas in the diseased plants even after 10 years of in-vitro propagation. Restriction fragment length polymorphism analysis of the amplified chromosomal DNA fragments revealed no genetic difference between the AP phytoplasma isolates. Growth parameters, symptom expression and phytoplasma concentration were examined to compare the in-vitro behaviour of four different AP phytoplasma isolates and to compare different subculture conditions. A comparison of these data obtained after 2 or 8 years of micropropagation revealed no essential differences. Eight years after culture initiation, diseased shoots still exhibited typical symptoms like witches' broom, small leaves with large stipules and stunted growth. The use of phytoplasma-diseased micropropagated plants to establish a 'type culture collection' of these otherwise nonculturable plant pathogens is discussed.     

Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: Serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas

Eleven stable hybridoma cell lines secreting monoclonal antibodies specific for FD-phytoplasma, the pathogenic agent of grapevine Flavescence dorée, were produced by fusing a non-secreting myeloma cell line with spleen cells from Balb/c mice immunized with Flavescence dorée phytoplasma purified by immunoaffinity. These monoclonal antibodies were characterized for their recognition of phytoplasma proteins by western blot. Six of eleven reacted specifically in ELISA and immunoblotting with Elm-yellows phytoplasma. These antibodies did not react either in ELISA or in western blot with preparations from periwinkles infected with phytoplasmas that cause GYU (Grapevine Yellows from Udine), AP (Apple Proliferation), EAY (European Aster Yellows) and StolC (Stolbur from France). Two of these hybridoma lines were used routinely for the immunodiagnosis of Flavescence dorée phytoplasma in diseased grapevines.Abbreviations ELISA Enzyme linked immunosorbent assay - FD Flavescence dorée - MLO Mycoplasmalike organism - EY Elm yellows     

Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.     

Plum pox virus detection in dormant plum trees by PCR and ELISA

An immunocapture polymerase chain reaction (IC-PCR) protocol and ELISA were compared for their effectiveness in detecting plum pox virus (PPV) in dormant plum material. Although the IC-PCR was about one thousand times more sensitive than ELISA, PPV was detected by ELISA in 71–80% of bark samples collected in December, January and March 1996/97 from pot-grown rootstock trees inoculated with PPV the previous March, compared with 85–86% detection in the same samples by IC-PCR. In similar samples from one-year-old shoots taken from infected branches of orchard trees, 66–81% were positive by ELISA compared with 81–87% by IC-PCR. With bulked samples taken from the fibrous roots of the pot-grown trees, PPV was detected in 92–100% of samples by IC-PCR in winter compared with only 38–65% by ELISA. These results were confirmed in samples from the roots and shoots of the same trees in 1997/98. Three samples per shoot would have been sufficient to detect PPV by ELISA in 87 of the 88 infected shoots tested during the two winters. However, infected shoots are irregularly distributed in diseased trees and PCR assays of root samples offer the potential for improving the reliability of identifying trees infected with PPV.     

Detection of Xanthomonas campestris pv. pelargonii in geranium and greenhouse nutrient solution by serological and PCR techniques

Specificity of a new monoclonal antibody, 2H5, to Xanthomonas campestris pv. pelargonii, causal agent of geranium bacterial blight, was determined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence tests on 14 strains of X. c. pelargonii, 12 strains of other X. campestris pathovars, 3 strains of other Xanthomonas spp., 3 strains of other plant pathogens, and 43 saprophytic bacteria isolated from geranium. X. c. pelargonii was detected in tissue from symptomatic and asymptomatic geraniums sampled from commercial growers, and artificially inoculated plants, by monoclonal antibody-based tests. The intensity of response in ELISA was only moderately correlated (r = 0.56) with symptom severity, while symptom severity was not correlated (r = 0.16) with the number of fluorescing cells in immunofluorescence. The bimodal frequency distribution of ELISA and immunofluorescence results served to validate arbitrarily chosen positive/negative threshold values. Most positive ELISA and immunofluorescence test results were confirmed by the polymerase chain reaction (PCR) using published primers (Manulis et al., 1994. Appl. Environ. Microbiol 60, 4094-4099). In contrast to plant tissue, the bacterium was detected in greenhouse nutrient solution with greater sensitivity by immunofluorescence and PCR than by ELISA. Sensitivity of detection was enhanced 100-fold by concentration of the bacteria by centrifugation.