Characterization of Pseudomonas pathovars isolated from rosaceous fruit trees in East Algeria

A survey of bacterial diseases due to Pseudomonas on rosaceous fruit trees was conducted. In forty two orchards located in the Constantine region ( East Algeria). Pseudomonas isolates were identified on the bases of their cultural and biochemical characteristics . A total of fifty nine phytopathogenic bacteria were isolated from diseased pome and stone fruit trees. Thirty one strains comparable to Pseudomonas syringae pv. syringae were isolated from cherry (Prunus avium L.), plum (P. domestica L.), apricot (P. armeniaca L.), almond (P. dulcis L.) and pear trees (Pirus communis L.); sixteen strains comparable to Pseudomonas syringae pv. morsprunorum were obtained from samples of cherry and plum. Twelve strains of Pseudomonas viridiflava were isolated from cherry, apricot and peach (Prunus persica L.).     

Detection of Prunus necrotic ring spot virus in plum,cherry and almond by serological and molecular techniques from India

Stone fruits are cultivated in the temperate and sub-temperate regions of India. During surveys in stone fruit growing areas, viral symptoms were observed in almond, cherry and plum. These samples were brought to the laboratory for further detection at serological and molecular levels to check the presence of virus. In the present study, incidence of PNRSV is reported on plum (Prunus domestica), almond (Prunus dulcis) and cherry (Prunus avium) using serological and molecular techniques. Coat protein gene of PNRSV was amplified from almond, cherry and plum. This is the first molecular evidence of PNRSV on these stone fruits reported from India.     

Comparison of Plant Pathogenic Pseudomonads from Fruit Trees

S ummary . The characteristics of 59 pathogenic pseudomonads isolated from stone fruit trees in south east England, 30 from cherry and 29 from plum, were compared with 41 isolates from pear trees in a wide range of biochemical, physiological and lesion tests. Many characteristics were common, but several consistent and stable differences were found between the stone fruit and pear groups, each of which exhibited a high level of homogeneity. The few atypical isolates in each group deviated from the majority in one or two respects only. Ten citrus and 3 lilac isolates were biochemically and physiologically indistinguishable from the pear isolates but had distinctive phage and bacteriocin sensitivities. The stone fruit isolates correspond to Pseudomonas morsprunorum and the pear isolates to Ps. syringae. The relationship between these two 'species'is discussed.     

Setting confidence limits for the detection of prune dwarf virus in Prunus avium with a monoclonal antibody-based triple antibody-sandwich ELISA†

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) with a monoclonal antibody was developed and evaluated for the detection of prune dwarf virus (PDV) in sweet cherry trees (Prunus avium). An independent reverse transcribed polymerase chain reaction test was also developed to establish, in conjunction with a bioassay, the incidence of PDV in 40 sweet cherry trees and to confirm the absence of virus in 15 control trees. Trees with two-thirds of their leaves positive for PDV would be identified with 99% probability by testing four leaves per tree with TAS-ELISA. The monoclonal antibody did not cross-react with Prunus necrotic ringspot virus in the TAS-ELISA.     

核果类果树自交不亲和性研究进展

综述了核果类果树甜樱桃(Prunus avium L.)、杏(P. armeniaca L.)、扁桃(P. dulcis (Mill.) D. A.Webb)和梅(P. mume Sieb)等自交不亲和性的研究进展。着重讨论了S-RNase基因(S基因)和SLF基因(S-locus　F-box基因,或称SFB基因),S基因在杂交后代群体中的遗传规律,利用S基因的遗传特性选育自交亲和品种和确定S基因型的主要方法及其特点以及自交亲和机制的几种可能的类型。     

核果类果树自交不亲和性研究进展

综述了核果类果树甜樱桃(PFunus avium L.)、杏(P.armeniaca L.)、扁桃(P.dulcis(Mill.)D.A.Webb)和梅(P.mume Sieb)等自交不亲和性的研究进展.着重讨论了S-RNase基因(s基因)和SLF基因(S-locus F-box基因,或称SFB基因),S基因在杂交后代群体中的遗传规律,利用S基因的遗传特性选育自交亲和品种和确定S基因型的主要方法及其特点以及自交亲和机制的几种可能的类型.     

Overwintering form of the causal agent of shot hole disease in Khorasan Razavi, Iran

Shot hole disease of stone fruit trees caused by some plant pathogenic fungi is a major constraint to stone fruit production worldwide where the trees are grown. Identification of the causal agents of the disease and their overwintering forms in stone fruit trees of Khorasan Razavi was necessary for disease management programs. Buds, twigs, fallen leaves and fruits were collected from the infected peach, apricot, nectarine and almond trees in winter 2007. The samples were superficially disinfested in 1% sodium hypochlorite for 2-3 min and then in 70% ethanol for 45 sec. Two to three fragments of 4x4 mm from each tissue were separately cultured on 2% water agar and potato dextrose agar (PDA), and purified on PDA. Just a pathogenic fungal species, Wilsonomyces corpophilus was isolated from the infected buds and twigs. No microorganism was isolated from the fallen leaves and fruits collected from underneath of the infested stone fruit trees. Pathogenicity of the fungus was examined on detached shoots of current year of four varieties of stone fruit trees. Fungal discs were placed under the bark of the bud base. Control shoots were similarly treated with sterile PDA discs. Inoculated shoots were placed in a humid growth chamber at 25 degrees C. Fungal hyphae appeared at 30 days post inoculation. Control shoots were asymptomatic. Pathogenicity intensities or lesion lengths were significantly different among the four varieties tested. A completely randomised design with five replicates was employed to measure the number of spores in infested buds and twigs of each variety of stone fruit tree. The samples were sliced and placed into a glass tube of centrifuge containing 3 ml of sterile distilled water. They were mixed on a vortex mixer for 30-40 min and centrifuged at 3000 rpm for 5 min. Pelleted material from each sample was suspended in 500 microl of sterile distilled water and the spores were counted using a hemocytometre. Results revealed that the fungus overwinters as hyphae and conidia in the infected buds, and as hyphae and globular chlamydospores in twig lesions.     

Study on morphology,pathogenicity and genetic diversity of Wilsonomyces carpophilus isolates,the causal agent of shot hole of stone fruit trees based on RAPD-PCR in Iran

Shot hole disease is one of the most important diseases of stone fruit trees in Iran. The disease is wide spread among orchards of Prunus spp. During spring and summer of 2007, 80 monoconidial isolates of the pathogen were recovered from infected leaves, fruits and twigs of different Prunus spp. in West Azerbaijan, Tehran, Ghazvin and Razavi Khorasan provinces of Iran and were studied taxonomically. Based on morphological and physiological characteristics and growth optimal temperature, all isolates were identified as Wilsonomyces carpophilus. Seedlings of stone fruits (apricot, almond, peach, nectarine, plum, sweet cherry and sour cherry) were used for pathogenicity tests. All seedlings were susceptible to the fungal isolates and showed disease symptoms on twigs, leaves, buds and petioles. Genetic diversity of 28 selected fungal isolates was investigated based on DNA fingerprinting by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), using four random primers. Based on cluster analysis of the PCR results from the four primers, 10 fingerprinting groups (clonal lineages) and 27 haplotypes were identified. Clonal lineages “C”, “D” and “E”, each with six haplotypes formed the biggest clonal lineages, but other clonal lineages (“B”, “F”, “G”, “H”, “I” and “J”) included only one isolate. No correlation was detected among clonal lineages with the location of selected isolates and their host species. A correlation was found between the substrate (fruit, twig or leaf) and clonal lineages, particularly in “C” clonal lineage. The results showed that the fungus population had high genetic diversity which is distributed among the different areas of Iran.     

Host marking pheromone of Rhagoletis cerasi: field deployment of synthetic pheromone as a novel cherry fruit fly management strategy

We tested the efficacy of synthetic host marking pheromone (HMP) of the European cherry fruit fly (R. cerasi) as a fruit-infestation-reducing-agent in an experimental cherry orchard. Two different pheromone deployment strategies were compared: covering entire tree canopies with synthetic HMP or treating only one half (top to bottom or lower half) of the tree canopy. Pheromone application caused a tenfold reduction in fruit infestation if the entire tree canopy was covered (0.226 vs 0.021 pupae/fruit in untreated and treated trees, respectively). Results show, nevertheless, that a significant reduction in fruit infestation can be achieved by treating only one half of tree canopies (top to bottom) (0.021 vs 0.048 pupae/fruit when comparing totally vs partially treated trees). We conclude that synthetic cherry fruit fly HMP has potential as a fruit fly management tool, especially in cases where organically grown fruit reaches high market values.     

Field efficacy of the entomopathogenic nematode Steinernema feltiae against the Mediterranean flat-headed rootborer Capnodis tenebrionis

The Mediterranean flat-headed rootborer, Capnodis tenebrionis (L.) (Coleoptera: Buprestidae), is an economically important pest of stone fruit and seed fruit in Mediterranean areas. The potential control of the entomopathogenic nematode Steinernema feltiae (Filipjev) (strain Bpa), isolated from a dead C. tenebrionis larva, was tested in a cherry tree orchard in Ullastrell, Barcelona (Spain). Nematode infective juveniles (IJs) were applied by drench and injection. In both the treatments, a rate of 1 million IJs was applied per tree every week during 4 or 8 weeks, with a total dose of 4 × 10⁶ IJs/tree and 8 × 10⁶ IJs/tree. Number, stage and localization of insects in each tree trunk were recorded. In both the experiments, S. feltiae significantly reduced the population of C. tenebrionis providing control ranging from 88.3% to 97%. No significant differences were recorded between the different treatments. Persistence of nematodes was recorded until 6 weeks after application. Results indicate that the application of S. feltiae (Bpa) provides adequate control of C. tenebrionis in cherry trees.     

Sampling conditions for reliable routine detection by enzyme-linked immunosorbent assay of three ilarviruses in fruit trees

Enzyme-linked immunosorbent assay (ELISA) was used to test plum trees for prune dwarf (PDV), Prunus necrotic ringspot (NRSV) and apple mosaic (ApMV) viruses, cherry trees for PDV and NRSV, and apple trees for ApMV. Optimum conditions were determined for sampling in large-scale surveys for these viruses. All three viruses were detected throughout the growing season in individual samples of young leaves, or twigs with newly formed buds. However, when single infected leaves were combined with different numbers of healthy leaves, tests were most successful for all three viruses early in the growing season. PDV was detected in 1/40 (infected/total leaves) cherry leaves in April and May and 1/40 plum leaves until July, whereas NRSV was detected in 1/20 cherry leaves until July and 1/20 plum leaves until May. ApMV was detected in 1/20 apple or plum leaves until June but was detected less readily in mature leaves after June than either NRSV or PDV. There was no evidence of uneven distribution of virus infection in the trees. The viruses were detected in leaf samples kept for 8 wk at 3°C but freezing was less reliable for storage especially with ApMV. ApMV was detected in tests on plants held for several weeks at 25°C, and PDV and NRSV in plants held at 30°C.     

Comparison of Methods for Characterizing and Distinguishing Isolates of Prune Dwarf Virus

A virus isolated from sour cherry trees with reduced growth in an orchard near Frankfurt/Main was shown by symptomatology, serology and various other tests to be strain of prune dwarf virus (PDV). A comparison of two isolates of this virus with three other PDV isolates already described by other authors indicated that groupings of the viruses made on the basis of symptomatology or component ratios determined by density gradient centrifugation were inconsistent and not reliable. However, electrophoresis in a sugar gradient or immuno-electrophoresis appeared to offer a satisfactory means of characterizing the viruses and of assigning the isolates to one of three biologically different groups of the virus.     

Evolutionary analysis of S-RNase genes from Rosaceae species

Eight new cDNA sequences for S-RNases were cloned and analysed from almond (Prunus dulcis) cultivars of European origin, and compared to published sequences from other Rosaceae species. Insertions/deletions of 10-20 amino acid residues were detected in the RC4 and C5 domains of S-RNases from almond and sweet cherry. The S-RNases of the Prunus species and those of the genera Malus and Pyrus formed two distinct groups on phylogenetic analysis. Nucleotide substitutions were analysed in the S-RNase genes of these species. The S-genes of almond and sweet cherry have a lower Ka/Ks value than those of apple, pear and wild apple do. The fact that there is no fixed difference between the S-RNase genes of almond and sweet cherry, or between apple and pear, suggests that nucleotide substitutions only introduce transient polymorphism into the two groups, and rarely became fixed and contribute to divergence. Through the comparative study of 17 S-RNase genes from the genus Prunus and 18 from the genera Malus and Pyrus, some fixed nucleotide differences between the two groups were identified. These differences do not appear to be the result of selection for adaptive mutations, since the number of replacement substitutions is not significantly greater than the number of synonymous substitutions. S-RNase genes of almond and sweet cherry, and of apple and pear, showed little heterogeneity in nucleotide substitution rates. However, heterogeneity was observed between the two groups of S-alleles, with the Prunus alleles exhibiting a lower rate of non-synonymous substitutions than alleles from Malus and Pyrus. The evolutionary relationships between these species are discussed.     

Development of microsatellite markers in peach [ Prunus persica (L.) Batsch] and their use in genetic diversity analysis in peach and sweet cherry ( Prunus avium L.)

We report the sequence of 41 primer pairs of microsatellites from a CT-enriched genomic library of the peach cultivar 'Merrill O'Henry'. Ten microsatellite-containing clones had sequences similar to plant coding sequences in databases and could be used as markers for known functions. For microsatellites segregating at least in one of the two Prunus F(2) progenies analyzed, it was possible to demonstrate Mendelian inheritance. Microsatellite polymorphism was evaluated in 27 peach and 21 sweet cherry cultivars. All primer pairs gave PCR-amplification products on peach and 33 on cherry (80.5%). Six PCR-amplifications revealed several loci (14.6%) in peach and eight (19.5%) in sweet cherry. Among the 33 single-locus microsatellites amplified in peach and sweet cherry, 13 revealed polymorphism both in peach and cherry, 19 were polymorphic only on peach and one was polymorphic only on cherry. The number of alleles per locus ranged from 1 to 9 for peach and from 1 to 6 on sweet cherry with an average of 4.2 and 2.8 in peach and sweet cherry, respectively. Cross-species amplification was tested within the Prunus species: Prunus avium L. (sweet cherry and mazzard), Prunus cerasus L. (sour cherry), Prunus domestica L. (European plum), Prunus amygdalus Batsch. (almond), Prunus armeniaca L. (apricot), Prunus cerasifera Ehrh. (Myrobalan plum). Plants from other genera of the Rosaceae were also tested: Malus (apple) and Fragaria (strawberry), as well as species not belonging to the Rosaceae: Castanea (chestnut tree), Juglans (walnut tree) and Vitis (grapevine). Six microsatellites gave amplification on all the tested species. Among them, one had an amplified region homologous to sequences encoding a MADS-box protein in Malus x domestica. Twelve microsatellites (29.3%) were amplified in all the Rosaceae species tested and 31 (75.6%) were amplified in all the six Prunus species tested. Thirty three (80.5%), 18 (43.9%) and 13 (31.7%) gave amplification on chestnut tree, grapevine and walnut tree, respectively.     

Incidence and genetic diversity of raspberry bushy dwarf virus (RBDV) in Rubus spp. in Turkey

Raspberry bushy dwarf virus (RBDV), recently renamed to Idaeovirus rubi, is one of the most common viruses infecting Rubus species worldwide but there is still a limited number of genome sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA-1 and RNA-2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537 Rubus spp. samples were tested by both DAS-ELISA and RT-PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS-ELISA and 67 samples by RT-PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA-1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA-1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those Rubus spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of Rubus species must be taken into account to choose a correct detection protocol and management strategy.     

Comparison between woody indexing and a rapid hybridisation assay for the diagnosis of little cherry disease in cherry trees*

Sweet cherry (Prunus avium) and sour cherry (Prunus cerasus) trees from orchards in the Kootenay and Okanagan Valleys of British Columbia, Canada were assayed for the presence of little cherry disease by three different methods: Northern blot analysis of double-stranded RNA, woody indexing for fruit symptoms on sweet cherry cv. Lambert, and woody indexing for foliar symptoms on cv. Canindex 1. Results of the three methods were in agreement for 85% of the samples. Of the 78 orchard trees tested, double-stranded RNA isolated from 48 trees hybridised with a radiolabeled cloned probe specific for little cherry disease. When the 48 trees were tested by woody indexing, buds from 41 trees induced fruit symptoms on cv. Lambert, but only 32 yielded foliar symptoms on cv. Canindex 1 under the conditions of the experiment. Of the 30 orchard trees that did not yield a positive response to the Northern blot analysis, 26 samples were negative on cv. Lambert and 26 were negative on cv. Canindex 1. Northern blot analysis of the 78 cv. Lambert indicator trees revealed that there was an absolute correlation between the presence of little cherry disease-associated double-stranded RNA and the development of typical little cherry disease symptoms on the indicator trees. Reliability of woody indexing of orchard samples was impaired by poor transmission of the disease from the inoculating bud to the indicator tree. Woody indexing with cv. Canindex 1 was particularly prone to a large number of apparently erroneous negative results. Of the three protocols used, diagnosis of little cherry disease by Northern blot analysis was found to be the most reliable and offered a greatly accelerated means of diagnosis.     

Molecular variability analyses of <Emphasis Type="Italic">Apple chlorotic leaf spot virus</Emphasis> capsid protein

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91–100% and 70–98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.     

Effects of earthworms, nematodes, cultivations and host plants on Verticillium wilt of peach and cherry

Verticillium dahliae but not V. albo-atrum Berth was isolated from eight out of twenty-one stone fruit orchards surveyed for Verticillium wilt disease in western New York. Wilt incidence was related to the cultivation of tomato or legumes as previous or inter-crop with stone fruit trees. A limited cross species inoculation using isolates of V. albo-atrum and V. dahliae from woody and herbaceous plants showed that peach and cherry were susceptible to both species. The effect of V. dahliae on growth of cherry seedlings in the presence of Tylenchorynchus claytoni, Pratylenchus penetrans and Meloidogyne hapla was compared. P. penetrans and M. hapla produced more severe growth reduction than T. claytoni. The adverse effect of Verticillium on the growth of cherry seedlings was greater acting together with any one of the three nematodes than acting alone. V. dahliae was shown to be capable of passage through earthworms without loss of infectivity.