Desert rose witches’ broom disease associated with ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma aurantifolia’

In March 2011, witches’ brooms comprising many small shoots were observed on desert rose plants, Adenium obesum, in PyinOoLwin, Myanmar. The causal agent of the symptomatic leaves was diagnosed as a phytoplasma by polymerase chain reaction (PCR) analysis. Sequence analysis of the PCR product (1.8 kbp) showed the closest phylogenic relationships with members of the peanut witches’ broom phytoplasma group. Additionally, phylogenetic analyses revealed the phytoplasma is a member of ‘Candidatus Phytoplasma aurantifolia’. This is the first report of desert rose plant as a new host for ‘Ca. P. aurantifolia’.     

Molecular identification of a phytoplasma associated with Russian olive witches’ broom in Iran

Russian olive trees (Elaeagnus angustifolia) showing witches’ broom symptoms typical of phytoplasma infection were observed in the Urmia region of Iran. A phytoplasma named Russian olive witches’ broom phytoplasma (ROWBp-U) was detected from all symptomatic samples by amplification of the 16S rRNA gene and 16S/23S rDNA spacer region using the polymerase chain reaction (PCR) which gave a product of expected length. DNA from symptomless plants used as a negative control yielded no product. The sequence of the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U showed 99% similarity with the homologous genes of members of the aster yellows group. We also detected a phytoplasma in neighboring alfalfa plants (AlWBp-U) showing severe witches’ broom symptoms. An 1107 bp PCR product from the 16S rRNA gene showed 99% homology with the corresponding product in ROWBp-U, suggesting the presence of the same phytoplasma actively vectored in the area. Further observations showed that Russian olive trees with typical ROWB symptoms were present in an orchard near Tehran which is located over 530 km south-east of the original Urmia site. The corresponding sequence of this phytoplasma (ROWBp-T) showed 99% homology to that of the ROWBp-U. A sequence homology study based on the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U and other phytoplasmas showed that ROWBp-U is most closely related to the 16SrI group. To our knowledge, this is the first report of a phytoplasma infection in a member of the Elaeagnaceae.     

Molecular identification of a phytoplasma associated with Ajwain (<Emphasis Type="Italic">Trachyspermum ammi</Emphasis>) in India

A new disease of Trachyspermum ammi, commonly known as Ajwain in India, was observed in Lucknow, India. The symptoms included small chlorotic leaves, highly proliferating shoots, witches’ broom appearance, shortened internodes and an overall stunted growth. Poor flower heads and fruit setting caused considerable yield losses for farmers. In transmission electron microscopy, pleomorphic bodies were detected in the phloem cells of diseased plants but not in those of healthy plants. The disease etiology was investigated using direct and nested polymerase chain reaction with phytoplasma-specific primers, DNA sequencing, and phylogenetic analysis. Phylogenetic analysis indicated that this phytoplasma clustered in the 16SrVI group. A 1,249 bp sequence (FJ970035) of the 16S rRNA gene from the phytoplasma showed 99% homology with the 16S rRNA gene, (FJ427295) of Ca. Phytoplasma trifolii strain A6 belonging to the phytoplasma group VI (reported from Iran). This is the first report of phytoplasma infection affecting Ajwain (T. ammi).     

A disease associated with phytoplasma in <Emphasis Type="Italic">Parthenium hysterophorus</Emphasis>

A disease on parthenium weed (Parthenium hysterophorus L.) was observed in June 2008 in Danzhou of Hainan Province. Infected weeds showed phytoplasma-like associated symptoms such as severe stunting, excessive proliferation of shoots, inflorescence-clustering, green petal, small leaves and witches’-broom. The original cause of phytoplasma was further confirmed by polymerase chain reaction (PCR). PCR products of 1.8 kb were obtained using the universal primers pair (P1/P7) designed to amplify the entire 16S rDNA and the 16/23S intergenic spacer region in a direct PCR assay. The primers pair R16F2n/R2 was used to amplify a PCR product of 1.2 kb. Restriction fragment length polymorphism (RFLP) was used to analyze the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA digested with five endonucleases (Kpn I, Hpa II, Taq I, Rsa I, EcoR I). The RFLP patterns of the strain were found to be identical with that of the reference peanut witches’-broom phytoplasma. Based on the RFLP data, it is suggested that the phytoplasma strain belongs to subgroup 16SrII-A. This is the first demonstration of a 16SrII-A group phytoplasma associated with parthenium weed.     

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real‐time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real‐time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R² > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real‐time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.     

Susceptibility of grapevine tissues to Neofusicoccum luteum conidial infection

This study investigated the ability of Neofusicoccum luteum to infect wounded shoots, trunks, pruned cane ends, leaf surfaces, buds, berries and roots, and its further progression into stem tissues. All tissue types were susceptible to infection except roots, with highest incidences in trunks (100%), cane ends (100%), shoots (92%) and buds (88%), indicating that in New Zealand, N. luteum is primarily a trunk and shoot pathogen. In trunks, there were no external symptoms, although N. luteum could be reisolated from 60 to 70 cm acropetally from the inoculation site after 4 months, by which time the pathogen had progressed into side shoots which became necrotic. Wounded and non‐wounded buds became infected; most were killed, with basipetal progression of the pathogen into the supporting shoots. Berries wounded and inoculated at the pre‐bunch closure stage were susceptible to N. luteum infection, with isolation incidence increasing over the season and peaking at harvest, when infected berries became mummified and produced pycnidia with many conidia. The pathogen was also able to progress from berries into bunch stems and supporting canes. Results from this research have indicated that N. luteum infection can occur in all aerial grapevine tissues and progress to young stem tissues where it causes wood necrosis. Growers should remove mummified berries from vineyard trash to ensure that pruning and trimming times do not coincide with rainy periods when conidia are released and dispersed. Furthermore, the susceptibility of buds to N. luteum infection indicates the need for fungicide sprays before budburst in spring.     

<Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> occurs asymptomatically on sweet cherry leaves

Leaves of sweet cherry, exposed to either paraquat or freezing to quickly senesce the leaf tissue, were incubated in about 100% RH at 25°C for 6 d. Sporulating colonies of Colletotrichum acutatum, the cause of anthracnose, developed on up to 100% of the paraquat-treated and frozen leaves, and on none of the untreated controls. Number of leaves and leaf area containing C. acutatum on naturally infected leaves increased over time from May to September. Mean incidence of C. acutatum on leaf blades on fruit spurs and vegetative shoots from eight orchard/year samplings were 41 and 33%, respectively. Secondary conidiation (formation of short hyphae and new conidia) from conidia applied to detached leaves took place 6 h after inoculation, but only up to 3% of the conidia formed new conidia. It may be concluded that asymptomatic sweet cherry leaves frequently host C. acutatum and may be a potential inoculum source for cherry fruit.     

Occurrence of rocket larkspur witches’ broom caused by “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma asteris” in Japan

In October 2001, a disease of rocket larkspur (Cosolida ambigua (L.) P. W. Ball et Heyw), characterized by witches’ broom, yellows and virescence of flowers, was found in Yakage Town in Okayama Prefecture. Electron microscopy revealed the presence of phytoplasma-like bodies in the phloem of diseased plants. The causal phytoplasma was identified as “Candidatus Phytoplasma asteris” based on 16S rDNA sequence analysis, and demonstrated to be acquired by the leafhopper Macrosteles striifrons. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB258330.     

Direct detection of <Emphasis Type="Italic">Taphrina deformans</Emphasis> on peach trees using molecular methods

The ascomycetous fungus Taphrina deformans is the agent of peach leaf curl, a worldwide disease of peach potentially devastating to both crop yields and tree longevity. Conspicuous leaf curl symptoms result from the invasion of host tissue by the strictly parasitic mycelial phase of the T. deformans dimorphic life-cycle. Successful isolation of the fungus in pure culture is cumbersome and limited to late spring/early summer (time of ascospore discharge from infected leaves) and only rarely has the asymptomatic yeast phase been isolated from buds. Molecular methods, namely those based on the hybridisation of nucleic acids, are advantageous for diagnostic purposes since they do not require isolation of the fungus on culture media. Direct amplification using the polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) were tested for diagnosis of peach leaf curl disease in order to provide a fast and reliable method for disease risk assessment. Specific primers and probes were designed based on available ribosomal DNA sequence data. Positive and specific diagnoses of peach leaf curl were achieved with primer TDITS1, using PCR-detection, and probe TDE634, using FISH, both on infected leaves and in washings of asymptomatic peach buds.     

Phenological dynamics of the invasive plant Acacia longifolia in Portugal

Phenological shifts may play an important role in the success of invasive species. In the coastal regions of continental Portugal, Acacia longifolia is one of the most widespread invasive plants, but there are significant gaps in our understanding and interpretation of its phenology. This study is the first to investigate the phenological variation of A. longifolia and its association with climate in populations representing a temperature and precipitation gradient in mainland Portugal. The results highlighted significant variations in the vegetative (leaf initiation) and reproductive phenophases (bud formation, flowering and fruiting) among populations. Overall, leafing was observed throughout the year, bud formation occurred between summer and spring, anthesis between late winter and the beginning of spring, and fruiting started in late winter. Most of the reproductive phenophases varied across elevation or latitudinal gradients, with populations that were subjected to more stressful climatic conditions advancing their flowering and fruiting phenophases. The occurrence of each phenophase was strongly associated with the climatic conditions in the previous 5–12 months, suggesting that plants receive their phenological cues well in advance of their phenological response. Among climatic factors, temperature and irradiance were the most significant predictors of the phenological cycle of A. longifolia, but precipitation also exerted a greater influence on budding and flowering of the species. The phenological response of A. longifolia in a variety of sites represents an important step towards understanding how its phenology may react to the projected climate change in the Mediterranean region.     

Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

Fire blight is the most damaging bacterial disease in apple production worldwide. Cankers and symptomless infected shoots are known as sites for the overwintering of Erwinia amylovora, subsequently providing primary inoculum for infection in the spring. In the present work, further potential sources of inoculum were investigated. Real‐time PCR assays covering a 3‐year‐period classified 19·9% of samples taken from fruit mummies as positive. Bacterial abundance in fruit mummies during autumn, winter and spring was up to 10⁹ cells per gram of tissue and correlated well with later infection rates of blossoms. Blossoms of non‐host plants growing close to infected trees were also shown to be colonized by E. amylovora and to enable epiphytic survival and propagation of bacteria. The results indicate a potential role of fruit mummies and buds in overwintering and as a source of primary inoculum for dissemination of the pathogen early in the growing season. Non‐host blossoms may also serve as an inoculum source in the build‐up of the pathogen population. Both aspects may contribute significantly to the epidemiology of E. amylovora. The significance of infected rootstocks as an inoculum source is also discussed. Fruit mummies might be used to determine pathogen pressure in an orchard before the beginning of the blooming period.     

Host specialization of <Emphasis Type="Italic">Tranzschelia discolor</Emphasis> on stone fruits at the uredinial infection stage

The host range specialization of Tranzschelia discolor (Fckl.) Tranz. & Litv. on stone fruits at the uredinial infection stage was investigated. Aeciospore inocula were collected from the alternate host, Anemone coronaria, at ten different locations of Aydın Province in early spring and were separately inoculated to 1–2-year-old nursery stocks of plum, peach, apricot, almond and sweet cherry under growth chamber conditions. Rust pustules developed only on plums, 15–21 days after inoculations, but no infections occurred on the other stone fruits. Urediniospore inocula were collected from naturally infected plums, apricots, peaches and almonds and were inoculated to four host species as well as sweet cherry in a series of cross-inoculations. Rust pustules developed on plants of the respective original host species from which the urediniospore inoculum was collected. No morphological differences were found among teliospores and urediniospores of T. discolor sampled from the different stone fruit species. Results of this study indicate that there is host specialization of T. discolor on stone fruits at the uredinial infection stage. Aecial inoculum from anemones in Aydin Province was infectious only on plums. The alternate host for the other stone fruits in this province was not found.     

Sprouting capacity from intact root systems of Cirsium arvense and Sonchus arvensis decrease in autumn

Perennial weeds are often controlled by mechanical means, which aim at stimulating axillary and adventitious buds to sprout. This happens when the apical dominance of the main shoot is removed by defoliation or when the underground system is fragmented. By repeating the measures, the result is a depletion of storage compounds, which weakens the plants and reduces their capacity to grow and reproduce. However, timing is critical. Earlier research has indicated that emergence from fragments of Sonchus arvensis cease during a period in autumn, while the seasonal pattern of sprouting in Cirsium arvense appears to be inconsistent. We studied the emergence pattern of defoliated plants with undisturbed root systems, from late summer to early spring. Potted plants grown outdoors were exhumed at regular intervals, put under forcing conditions for 4 weeks, after which shoots above and below soil level were counted and weighed together with the remaining root systems. In both species, the number and weight of emerged shoots decreased during a period in the autumn. In C. arvense, underground shoots were constantly produced during the same period, while fewer underground shoots were present in S. arvensis. For the latter species, apical dominance does not fully explain the effect; thus, endodormancy might be involved. Root weight increased until withering and did not explain the lack of emergence. Our results suggest an impaired sprouting capacity of undisturbed root systems of C. arvense and S. arvensis during September–October, which has implications for the timing and method of control of these species.     

Detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma brasiliense’ in a new geographic region and existence of two genetically distinct populations’

‘Candidatus Phytoplasma brasiliense’, a phytoplasma taxon associated with hibiscus witches’ broom disease was first described in 2001 in Brazil. In September 2007, a peach tree (Prunus persica) displaying yellowing symptoms reminiscent of phytoplasma infection was sampled in Guba region of Azerbaijan. A phytoplasma was detected in the diseased peach tree by nested PCR amplification of its 16S rDNA with universal primers for phytoplasmas. Phylogenetical analyses of the amplified 16S rDNA showed that the phytoplasma infecting the peach tree corresponded to ‘Ca. P. brasiliense’, a species never reported in Euro-Mediterranean area. To set up a detection assay, cloning of a ‘Ca. P. brasiliense’ DNA fragment was undertaken by comparative RAPD. The amplified dnaK-dnaJ genetic locus was used to design a nested PCR assay able to amplify all ‘Ca. P. brasiliense’ isolates of the subgroup 16SrXV-A without amplifying the related members of the group 16SrII. This assay also allowed confirming the first detection of ‘Ca. P. brasiliense’ in diseased basil collected in south Lebanon.     

Molecular characterization of the phytoplasmas associated with toon trees and periwinkle in India

In November 2008 in Himachal Pradesh and Chandigarh regions in India, toon trees and periwinkles were observed to have formed short internodes, small leaves and witches’-broom symptoms, typical of phytoplasma infection. The symptomatic toon and periwinkle samples were tested with universal PCR tests, and the 16S rRNA, rplB-rpsC, secA and secY genes were sequenced. The causal agents belonged to subgroup 16SrI-B of ‘Candidatus Phytoplasma asteris’, based on 16S rDNA, ribosomal protein gene, secA and secY phylogenetic analysis.     

Development of a PCR-based diagnostic tool specific to wheat dwarf bunt,caused by <Emphasis Type="Italic">Tilletia controversa</Emphasis>

Wheat dwarf bunt, one of the important international quarantine diseases, is caused by Tilletia controversa. Tilletia caries is a close relative species of T. controversa and the teliospore morphology and genomic structure of T. caries are very similar to those of T. controversa. In order to distinguish between them, a random amplified polymorphic DNA (RAPD) primer-mediated asymmetric-PCR (RM-PCR) was developed to screen differential sequences between the two pathogens. By RM-PCR, a 1,322 bp DNA fragment (PR32) was selected from 18 T. controversa and 29 T. caries strains. The PR32 genes were specific for T. controversa and almost had no homology to T. caries or other fungi in the present database. With primers designed from PR32, all 18 T. controversa strains were amplified, but no bands appeared in 29 T. caries strains by classical PCR. To identify T. controversa rapidly and accurately, SYBR Green I and TaqMan probe real-time PCR were established based on PR32. With TaqMan Real-Time PCR, different T. controversa strains and T. caries strains were detected. The results showed that all T. controversa strains were amplified with Ct from 19–29 and amplified curves were obtained. In contrast, the amplification of all T. caries strains did not show any signals, without Ct and amplified curves. Moreover, the developed TaqMan real-time PCR was used to detect T. controversa from asymptomatic wheat tissues successfully.     

Ineffectiveness of pruning to control citrus huanglongbing caused by <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter americanus

The huanglongbing (HLB) disease of citrus trees, caused by Candidatus Liberibacter asiaticus and Ca. Liberibacter americanus, was first reported in Brazil in March, 2004. The presence of the disease has caused serious concerns among growers. Pruning experiments were conducted to determine if removal of symptomatic branches or the entire canopy (decapitation) would eliminate infected tissues and save HLB-affected trees. Pruning was done in five blocks on a total of 592 3- to 16 year-old ‘Valência’, ‘Hamlin’ or ‘Pêra’ sweet orange trees showing no symptoms or with two levels of symptom severity. Ten decapitated trees per block were caged and all trees were treated with insecticides to control the psyllid vector, Diaphorina citri. Mottled leaves reappeared on most symptomatic (69.2%) as well on some asymptomatic (7.6%) pruned trees, regardless of age, variety, and pruning procedure. Presence of the pathogen (Ca. Liberibacter americanus) in all symptomatic trees was confirmed by PCR. In general, the greater the symptom severity before pruning the lower the percentage of trees that remained asymptomatic after pruning.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

Temporal and spatial dispersal of Thekopsora areolata basidiospores,aeciospores, and urediniospores

Cherry spruce rust causes huge yield losses in Norway spruce seed production in Fennoscandia. The causal agent, Thekopsora areolata, has three types of spores that disperse during spring: basidiospores are produced on basidia that grow out from teliospores in overwintered bird cherry leaf litter to infect new pistillate spruce cones, aeciospores are released from old diseased spruce cones to infect bird cherry leaves, and urediniospores are produced from new bird cherry leaves for reinfection. No study has examined the dispersal of T. areolata spores, including the basidiospores that cause primary infection in spruce cones. In this study, teliospores of T. areolata were germinated in the laboratory and the morphology of basidiospores was described. T. areolata spores were sampled in Ultuna, Sweden and Joutsa, Finland with 21 spore traps at each site. Peaks in aeciospores were observed from 11 to 25 May and from 2 to 8 June at the Finnish site, and from 4 to 18 May at the Swedish site. Urediniospores were first observed 2–3 weeks after the peaks in aeciospores and they were mainly distributed within 10 m from the bird cherry trees. Peaks of 1–2 weeks in basidiospore detection coincided with multiple rain events. The basidiospore peak overlapped with the spruce pollen peak in Finland but not in Sweden. The quantities of basidiospores from different spore traps within 100 m from the spore source had no gradient. Information on spatial and temporal spore release is important for making decisions on disease management strategies.     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.