Molecular characterization of Pseudomonas syringae isolates from fruit trees and raspberry in Serbia

Infection of fruit trees by Pseudomonas syringae is a potentially serious problem that may limit the establishment and sustained productivity of pome and stone fruit orchards in Serbia. To estimate possible diversity of Pseudomonas syringae fruit trees strains, we collected a set of strains in several areas of Serbia. The samples were taken from infected orchards with raspberry, plum, cherry, sour cherry, peach, pear and apple trees. Genetic diversity of P. syringae strains isolated from fruit trees was determined by using SpeI macrorestriction analysis of genomic DNAs by pulsed-field gel electrophoresis (PFGE) and REP-PCR. Molecular analysis showed that most of isolates had unique profiles, with the exception of isolates from plum and cherry that displayed profiles identical to each other and similar to P. syringae pv. morsprunorum. The study presented here clearly demonstrates the discriminative power of molecular techniques in enabling a detailed analysis of the genetic variations between strains of P. syringae from different pome and stone fruit hosts in Serbia.     

New host for raspberry bushy dwarf virus: arctic bramble (Rubus arcticus)

Raspberry bushy dwarf virus (RBDV) was detected in three new host plants inRubus species,i.e., arctic bramble (R. arcticus ssp.arcticus), Alaskan arctic bramble (R. arcticus ssp.stellatus) and their hybrid (R. arcticus L. nothosubsp.stellarcticus G. Larsson). The virus was identified as RBDV by the symptoms elicited in the test plantsChenopodium quinoa andC. amaranticolor, by sedimentation profile in sucrose density gradient, by RNA banding pattern in agarose gel electrophoresis, by protein analysis of the purified viruses in SDS-polyacrylamide gel electrophoresis, and by Western blotting. There was a high incidence of RBDV-infected plants in the experimental plots. The presence of the virus in arctic bramble did not always induce foliar symptoms. However, yellowing of the leaves around central and lateral veins was quite frequently observed, especially in spring and autumn.     

Phytophthora root and crown rot of raspberry in Bulgaria

Root and crown rot of raspberry (Rubus idaeus L.) was observed in a plantation at the experimental station of small fruits in Kostinbrod, Bulgaria. Isolates ofPhytophthora spp. were obtained from diseased plants. Colony morphology, growth rates, features of asexual and sexual structures were studied and as a result twoPhytophthora species were identified:Phytophthora citricola Saw. andPhytophora citrophthora (R.E. Sm. & E.H. Sm.) Leonian. Their pathogenicity was confirmed in artificial inoculation experiments. The isozyme (-esterase) patterns ofP. citrophthora andP. citricola isolates from raspberry and from the collection of the CBS, Baarn the Netherlands were compared, using micro-gel electrophoresis. Both species are reported for the first time as pathogens of raspberry in Bulgaria. This is only the second report in phytopathological literature ofP. citrophthora on raspberry, the first being from Chile [Latorre and Munoz, 1993].     

Phytophthora species attacking strawberry and raspberry1

Phytophthora frugariae causes red core root rot of strawberries. Although the disease is probably most acute in northern Europe, serious outbreaks have been reported from a number of Mediterranean countries, especially France and Italy. Leather rot of fruit and crown rot, which are caused by P. cuctorum, can also be severe problems in warmer climates. Both fungi survive in soil for long periods, but the most common form of spread is in diseased planting material. Sensitive tests have been developed to detect red core in planting material, and been used to effect in certification schemes. Root rot of raspberry has gained prominence in the last 10 years in Europe as raspberry growing has expanded, but the disease has been known for many years in France. Nine species of Phyfophthora have been recovered from affected plants, but two of these, one with affinities with P. megasperma, and P. cumbiuora, are responsible for most major outbreaks. The other species are only troublesome where drainage is poor. Again, spread is mainly in infected material. Few raspberries are resistant to root rot, but some wild RubuP spp., blackberries and raspberry x blackberry hybrids are resistant and may be useful in breeding programmes.     

Biological,serological and molecular characterisation of <Emphasis Type="Italic">Raspberry bushy dwarf virus</Emphasis> from grapevine and its detection in the nematode <Emphasis Type="Italic">Longidorus juvenilis</Emphasis>

In 2003, Raspberry bushy dwarf virus was found for the first time in grapevine. Since this was the first report of a non-Rubus natural host, information about it is sparse. During this study the grapevine isolates were characterised biologically, serologically and genetically and compared with known information about Rubus isolates. Infected plant material was used for mechanical inoculation of test plants, and for serological characterisation with monoclonal antibodies. Most of RNA 2 was sequenced and compared with other sequences from the database. Grapevine isolates infected Chenopodium murale systemically, which is not known for raspberry isolates. They were differentiated from Slovenian raspberry isolates with three monoclonal antibodies using TAS-ELISA. Phylogenetic analyses clustered grapevine isolates separately from raspberry isolates. Additionally, the virus was detected using nested RT-PCR in Longidorus juvenilis nematodes collected in an infected vineyard. Grapevine isolates of RBDV are distinct from raspberry isolates.     

陕西杨凌番茄褪绿病毒的分子检测

近年来在陕西番茄生产区出现了一种新病害,病株表现为叶片褪绿,叶脉颜色变深及叶片增厚,果实变小发白,严重影响番茄产量及经济效益,2016年发病极为严重,部分产区甚至绝收。该病疑似由番茄褪绿病毒Tomato chlorosis virus(ToCV)引起。利用ToCV特异引物对感病番茄样品进行RT-PCR检测,结果从所采集的4份病样中均检测到ToCV预期大小的特异片段。对ToCV外壳蛋白CP基因和类热激蛋白HSP70h基因进行克隆与序列分析,ToCV-YL1 CP基因774个核苷酸序列与已报道的ToCVCP基因有较高的一致性,与山东青岛和山西晋中分离物同源性为100%。ToCV-YL1 HSP70h基因1 665个核苷酸序列与已报道的ToCV HSP70h基因有较高的同源性,与中国、韩国、日本、美国、希腊分离物同源性达到99%以上。研究表明ToCV已传播至陕西地区。     

Occurrence and distribution of Raspberry bushy dwarf virus in commercial Rubus plantations in England and Wales

Serological surveys for Raspberry bushy dwarf virus (RBDV) made between 1995 and 1997 and covering ≈ 10% of the commercial farms growing Rubus (red raspberry and hybrid berries) in England and Wales showed that this virus was present on approximately one-quarter of all farms and in approximately one-sixth of all plots tested. RBDV was found in all of the four main raspberry cultivars being grown at that time (Autumn Bliss, Glen Moy, Glen Prosen and Leo), in Loganberry and in Tayberry. Fifteen RBDV genotypes (including two that appeared to be mixed) were identified using RT-PCR/RFLPs, but the majority of genotypes were found only rarely. Of the RBDV isolates tested, two genotypes each comprised 12·5% and another 46·4%. None of the three most common genotypes was associated solely with single Rubus cultivars and vice versa . It is suggested that two separate outbreaks of RBDV are occurring in England and Wales. One outbreak comprises the most frequent genotype combined with one of the moderately frequent genotypes; this outbreak is largely confined to the main growing areas and is either spreading between farms or coming from multiple local sources. Circumstantial evidence suggests that these isolates (and hence this first outbreak) are of the RB pathotype. The second outbreak consists of the other moderately frequent genotype and those genotypes which are less common. These genotypes appear to be more scattered across England and Wales and seem more likely to be coming from local sources and not to be spreading naturally between commercial farms.     

Untersuchungen zum Erreger der Ringfleckigkeit an Stieleichen (Quercus robur)

Oak trees showing leaves with chlorotic ring spots have been observed in an area of Berlin (Spandau Forest). Leaves from infested trees were used for investigations on virus purification and mechanical transmission. Electron microscopic visualisation of the virus particles was unsuccessful, as was their mechanical transmission to herbaceous indicator plants. The use of specific primers to detect ring spot disease on mountain ash showed that both ring spot disease forms do not stem from the same causal agent. Whilst the amplified DNA fragment of mountain ash consists of 224 bp, those of diseased oak trees had lengths of 150 bp and 550 bp. These two fragments amplified in diseased oak leaf tissue probably result from an unspecific reaction of the primers with the cDNA. Further investigations are considered necessary.     

Changes in phenolic content induced by infection with Didymella applanata and Leptosphaeria coniothyrium,the causal agents of raspberry spur and cane blight

The phenolic profile of healthy and infected raspberry canes was investigated in three raspberry cultivars: Autumn Bliss, Himbo Top and Polka. The content of total phenols and tannins was determined using spectrophotometric methods, whilst individual phenolic acids, flavan‐3‐ols, ellagic acid derivatives and glycosides of quercetin were analysed using HPLC/MS analysis. The content of secondary metabolites varied considerably among the analysed raspberry cultivars. Moreover, Didymella applanata and Leptosphaeria coniothyrium infection significantly altered the metabolism of phenolic compounds. Flavanols represented the greatest share of all identified phenolics in raspberry canes (90%), followed by glycosides of quercetin (6%), derivatives of ellagic acid (3%) and traces of hydroxycinnamic acid derivatives. Spur and cane blight diseases caused an increase of flavan‐3‐ols and tannins but the levels of hydroxycinnamic acid derivatives, conjugates of ellagic acid and quercetin glycosides were significantly reduced. Cultivars Himbo Top and Polka contained higher levels of hydroxycinnamic acid and ellagic acid derivatives in healthy and infected canes compared to cv. Autumn Bliss. Cultivar Polka also contained the highest level of flavanols and tannins. However, despite high levels of flavanols and total phenols measured in cv. Polka, the canes were highly diseased following infection with D. applanata and L. coniothyrium. The results of the study provide evidence that the level of phenolic compounds in the canes could be causally linked to the differences in disease susceptibility.     

Papierchromatografie als middel voor de diagnose van de ringvlekkenziekte van zoete kers. een voorlopige mededeling

Summary Sap from leaves of healthy and ring spot diseased cherry trees was chromatographed on Whatman No. 1 filterpaper with n-butanol-acetic acid-water as a solvent. On the dried chromatograms spots became visible after spraying with an ammoniacal solution of silver nitrate. Chromatograms of sap from diseased leaves showed some dark spots. These spots were less pronounced in chromatograms of sap from healthy leaves (see figure). This was the case with material from trees growing on clay soil, as well as from those on sandy soil. Sometimes samples from leaves of diseased trees on clay soil gave chromatograms showing a little band between the spots 3 and 4 (B), and samples from healthy specimens a spot between the spots 1 and 2 (A).Further studies are needed to answer the question whether the method is suitable for practical use in cherry orchards in order to secure healthy propagation stocks.     

Association of a Geminivirus with a Leaf Curl Disease of Sunn Hemp (Crotalaria juncea) in India

Natural occurrence of a geminivirus causing severe leaf curl disease on sunn hemp (Crotalaria juncea) was recorded in India. The association of a geminivirus with the disease was demonstrated by whitefly transmission tests and polymerase chain reaction (PCR) amplification of DNA fragments of expected sizes with three pairs of degenerate geminivirus primers. The PCR-amplified viral DNA fragments were further characterized by Southern hybridization with a geminivirus probe consisting of the cloned coat protein (CP) gene of Indian tomato leaf curl virus (ITLCV). Restriction fragment length polymorphism analysis of a PCR-amplified CP fragment revealed that the geminivirus from sunn hemp was different than ITLCV.     

Differentially-regulated defence genes in Malus domestica during phytoplasma infection and recovery

To improve knowledge about plant/phytoplasma interactions and, in particular, about the ‘recovery’ phenomenon in previously-infected plants, we investigated and compared expression levels of several defence-related genes (four pathogenesis-related proteins and three jasmonate-pathway marker enzymes) in apple plants showing different states of health: vigorous (healthy), phytoplasma-infected, and recovered. Real Time-PCR analyses demonstrated that genes are differentially expressed in apple leaf tissue according to the plants’ state of health. Malus domestica Pathogenesis-Related protein (MdPR) 1, MdPR 2 and MdPR 5 were significantly induced in leaves of diseased and symptomatic plants compared to leaves of those plants that were healthy or recovered. On the other hand, levels of all the jasmonate (JA)-pathway marker genes that we selected for this study, were up-regulated in the leaves of recovered plants compared to the diseased ones. In conclusion, our study demonstrated that two different sets of defence genes are involved in the interactions between apple plants and ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) and that these genes are differentially expressed during phytoplasma infection or recovery.     

Colonization of red raspberry flowers and fruit by Botrytis cinerea under commercial production conditions in northwestern Washington,USA

Colonization of red raspberry flowers and fruit by Botrytis cinerea was determined during 2017–2018 growing seasons under commercial fungicide application programmes used for grey mould management in northwestern Washington, USA. Colonization of flowers and fruit was assessed qualitatively (incidence, %) and quantitatively (abundance, number of colonies) by recovering B. cinerea from surface-disinfested samples. Both incidence and abundance of flower colonization were significantly lower than fruit colonization in both untreated and fungicide-treated plots. Incidence of flower colonization did not differ significantly between untreated and fungicide-treated plots (43% vs. 45%, respectively). In contrast, significantly greater colonization incidence was detected at green fruit stage in untreated compared to fungicide-treated plots (96% vs. 77%, respectively). Ripe fruit had the greatest colonization incidence among the three stages sampled and colonization was not significantly different between untreated and fungicide-treated plots (100% vs. 92%, respectively). Similarly, colonization abundance of flowers did not differ significantly between untreated and fungicide-treated plots (1.0 colonies per colonized flower in both treatments), but colonization abundance of green and ripe fruit was decreased 2.3- and 2.1-fold, respectively, in fungicide-treated plots. DNA fingerprinting analysis of the pathogen revealed that different multilocus genotypes colonized flowers and fruit within the same inflorescence and that genotypic diversity increased through time, suggesting independent infection events. Overall, our results demonstrate that under current environmental conditions, raspberry flowers may not be the exclusive or major route of infection for grey mould of red raspberry in northwestern Washington. Implications of current findings for management and further research are discussed.     

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.     

小西葫芦黄花叶病毒北京分离物的鉴定及其基因组3''端特性分析

在北京东郊自然感病的南瓜Cucurbita moschata上获得一病毒分离物（BJ-1），经生物学、血清学和分子生物学鉴定，确定为小西葫芦黄花叶病毒（Zucchini yellow mosaic virus，ZYMV）。为分析其基因组3’端特性，以发病叶片中提取的总RNA为模板，对基因组3’端进行RT-PCR扩增，产物克隆到pMD18-T栽体上进行序列分析，共测定了该病毒分离物包括全部CP基因在内的1269bp。该分离物CP基因由837个核苷酸组成，编码279个氨基酸。对包括该分离物在内的30个序列的760bp（含NIb基因3’端56bp和CP基因中的704bp）片段、NIb蛋白与CP蛋白的切割位点、蚜传必需基序的变异、寄主来源及地域来源进行了分析。结果表明，ZYMV不同分离物的基因分型与上述五个因素无明显关系。