Detection and Identification of Brenneria nigrifluens, the Causal Agent of the Shallow Bark Canker of Walnut by, PCR Amplification

A 1 kb DNA band from strains of Brenneria nigrifluens, as shown by amplification of their genomic DNA by polymerase chain reaction (PCR) using minisatellite primer designed on the minisatellite sequence of the M13 phage, was isolated, cloned and sequenced. Specific oligonucleotides (F1–C3) were selected into this 1 kb DNA sequence and used in a PCR assay to detect and identify strains of B. nigrifluens . Several strains of B. nigrifluens were assessed with F1–C3 primers producing a specific band of approximately 250 bp pairs in length. This target was successfully amplified from purified genomic DNA, from bacterial culture and directly from infected walnut bark tissue. No amplification was obtained when the PCR assay was performed on other plant-pathogenic species from the following genera Brenneria, Erwinia, Agrobacterium, Pseudomonas, Ralstonia, Pectobacterium, Xanthomonas and from walnut-associated bacteria, indicating the specificity of these primers. The PCR assay with the primers described here provides a rapid, specific and sensitive diagnostic method for B. nigrifluens and a useful tool for epidemiological studies.     

Bark morphological and chemical features are differentially correlated with disease resistance and yield in hybrid poplar taxa

In the southeastern United States, the establishment of short-rotation intensively cultured plantations of hybrid poplar has been hindered by its susceptibility to stem cankers. We evaluated the tradeoffs between biomass yield and disease tolerance in hybrid poplar genotypes belonging to P. deltoides × P. maximowiczii (DM), P. deltoides × P. nigra (DN), P. trichocarpa × P. maximowiczii (TM), and P. deltoides × P. deltoides (DD) taxa. We hypothesized that canker resistant genotypes will have thicker bark but bark thickness and biomass yield will be negatively correlated. After two growing seasons, the DD genotypes developed thicker bark compared to the genotypes of other taxa and bark thickness was not correlated with biomass yield in the DD genotypes (R² = 0.002). However, in the TM, DM, and DN genotypes, bark thickness was negatively correlated with biomass yield (R² = 0.33–0.77). Disease incidence studies revealed that the DM genotypes were most susceptible to canker whereas no disease was detected in DD genotypes. Furthermore, bark analysis conducted by Fourier transform infrared spectroscopy coupled with multivariate analysis showed that that DD genotypes to be chemically separate from the three hybrid genotypes and that bark chemistry was correlated with canker disease incidence. Taken together, these results reveal that it is possible to generate hybrid poplar genotypes with thicker bark, disease resistance, and higher biomass yields. This insight should guide further efforts to develop genetically improved hybrid poplar genotypes, both in terms of biomass yield and disease tolerance, for cultivation in the southeastern United States. Hybrid poplar cultivation in southeastern United States is hindered by its susceptibility to stem cankers. We evaluated tradeoffs between yield and canker disease resistance in various hybrid poplar genotypes. After two growing seasons, the DD genotypes showed disease resistance and developed thicker bark that was chemically distinct from the other genotypes. Bark thickness was not correlated with yield in the DD genotypes but was negatively correlated with yield in the other genotypes. These results will guide the development of hybrid poplar genotypes that are both disease resistant and high yielding for cultivation in the southeastern United States.     

Nested PCR Assays for Detection of Monilinia fructicola in Stone Fruit Orchards and Botryosphaeria dothidea from Pistachios in California

Nested polymerase chain reaction (PCR) assays were developed based on microsatellite regions for detection of Monilinia fructicola, the causal agent of brown rot of stone fruits, and Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio. The nested PCR primers specific to M. fructicola were developed based upon the sequence of a species‐specific DNA fragment amplified by microsatellite primer M13. The external and internal primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571‐ and a 468‐bp fragment, respectively, from M. fructicola, but not from any other fungal species present in stone fruit orchards. The nested PCR primer pairs specific to B. dothidea were developed based upon the sequence of a species‐specific 1330‐bp DNA fragment amplified by microsatellite primer T3B. The external and internal primer pairs EBdF + EBdR and IBdF + IBdR amplified a 701‐ and a 627‐bp fragment, respectively, from B. dothidea, but not from any other fungal species associated with pistachio. The nested PCR assays were sensitive enough to detect the specific fragments in 1 fg of M. fructicola or B. dothidea DNA or in the DNA from only two conidia of M. fructicola or B. dothidea. The nested PCR assays could detect small numbers of M. fructicola conidia caught on spore‐trap tapes and detect visible infections of B. dothidea in pistachio tissues. Microsatellite regions with high numbers of copies are widely dispersed in eukaryotic genomes. The results of this study indicate that microsatellite regions could be useful in developing highly sensitive PCR detection systems for phytopathogenic fungi.     

The ecology of Erwinia rubrifaciens and the development of phloem canker of Persian walnut

A requisite for development of bacterial phloem canker caused by Erwinia rubrifaciens is the presence of the highly susceptible Hartley cultivar; when interplanted with Hartley, the Franquette and Payne cultivars are sometimes attacked by the disease. The recently developed cultivars, Gustine and Howe, developed active cankers when inoculated, but not as extensive as those in Hartley. The age of the plant part is important in the disease. One-year-old Hartley trees and twigs of the current season on mature trees did not develop active cankers. The complete disease syndrome occurs only on trunks and primary (scaffold) branches. Extension of the cankers was most rapid during the summer when the temperature was high. This was correlated with the effect of temperature on growth of the bacterium in culture. At midday in summer the temperature of the cambial area of a shaded trunk was as much as 10d?F (5.6 d?C) below ambient temperature but it was commonly a few degrees above ambient at night. Breaks in the thick phelloderm of the trunks and branches are necessary for penetration of the pathogen to the inner bark. Of the several types of breaks commonly occurring, those produced by mechanical harvesting equipment and by sap-sucking birds were found to be infection sites. Bacteria occur in large numbers in a slimy substance which exudes through cracks and accumulates on the bark of infected trees. They survived for at least 123 days in the exudate and were disseminated laterally as far as 20 ft (6.1 m) in wind-blown rain. Bacteria were also transmitted from tree to tree in exudate contaminating the pads of mechanical harvesting equipment.     

Molecular detection of Pectobacterium species causing soft rot of <Emphasis Type="Italic">Amorphophallus konjac</Emphasis>

Soft rot disease of Amorphophallus konjac is caused by Pectobacterium species. Infected corms are considered a primary and important source of inocula. Based on the 16S rDNA sequences of the soft rot pathogens, one pair of specific primers was designed to identify the soft rot disease by real-time PCR and the other two were used to identify the pathogens of Pectobacterium carotovorum subsp. carotovorum. and P. chrysanthemi respectively. According to the results, a single cell of Pectobacterium could be detected by real-time PCR with the designed primer pair, while at least 100 bacteria were required for conventional PCR. Moreover, the two special primers can directly and accurately authenticate to Pectobacterium carotovorum subsp. carotovorum and P. chrysanthemi by the conventional PCR system without testing the pathogenicity, biochemical and phenotypic characterizations and so on. In conclusion, the PCR-based techniques showed several significant advantages in identifying the soft rot pathogens from konjac, such as higher sensitivity, rapidness and precision, and it could be widely used in seed quarantine.     

Comparative Studies on Borrelia afzelii Isolated from a Patient of Lyme Disease,Ixodes persulcatus Ticks,and Apodemus speciosus Rodents in Japan

The genospecies Borrelia afzelii was isolated from a patient of Lyme disease in Hokkaido, Japan, for the first time, by culturing the minced erythema lesion in BSK II medium. Two analytical methods, rRNA gene restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) using the specific primer set to amplify the 16S rRNA gene, revealed that this clinical isolate belongs to the group of B. afzelii. In our culture collection of spirochetes, part of the isolates from Ixodes persulcatus ticks, and from Apodemus speciosus rodents, were also classified as B. afzelii. These results strongly suggest that the agent pathogenic to humans is maintained in “rodent-tick” transmission cycle.     

Genetic dissection of fusiform rust and pitch canker disease traits in loblolly pine

Loblolly pine (Pinus taeda L.) exhibits genetic resistance to fusiform rust disease (incited by the biotrophic fungus, Cronartium quercuum f. sp. fusiforme) and pitch canker disease (incited by the necrotrophic fungus, Fusarium circinatum). In this study, a total of 14,015 loblolly pine cuttings from 1,065 clones were screened in controlled greenhouse conditions to identify phenotypes of clones, families, and parents that guide a genetic dissection of disease traits associated with pitch canker and fusiform rust. A total of 23,373 phenotypic data points were collected for lesion length (pitch canker) and gall score, gall length, and gall width (fusiform rust). We verified heritable fusiform rust and pitch canker traits and calculated parental, clonal, and full-sib family rankings for both diseases. Genetic correlations revealed that traits associated with fusiform rust are genetically distinct from one another, and that the genetic mechanisms underlying pitch canker and fusiform rust resistance are independent. The disease phenotyping described here is a critical step towards identifying specific loci and alleles associated with fusiform rust and pitch canker resistance.     

An Investigation on Strains of Clavibacter michiganensis subsp. michiganensis in North and North West of Iran

Tomato bacterial canker disease was first reported from Urmiyeh in West Azerbaijan province in Iran. The disease causes lesion (canker), wilting and dryness of infected plants, leaf and fruit spots and the decline of the whole plant. Out of 102 isolates obtained from the fields in the major tomato producing areas of understudy regions, 98 were found Gram positive, yellow‐pigmented isolates, identified as Clavibacter michiganensis subsp. michiganensis based on the morphological and biochemical characteristics described in previous studies. Among these strains, 64 were virulent and 34 showed poor virulence. A strain of Cmm (NCPPB382) was used as a check (standard) in all steps of this study. DNA fingerprinting with repetitive‐sequence‐based PCR (rep‐PCR) (BOX primer) carried out among 11 representative strains (eight strains from West Azerbaijan, two from Golestan and one as standard). The most virulent strain was chosen as representative in each location. Dendrograms were prepared using NTSYS‐pc version 2/o2e software, unweighted pair group with arithmetic average method and simple matching similarity coefficient. According to the site of cut‐off line, three groups (clusters) with 82/5% similarity and six groups with 55% similarity were separated based on biochemical and SDS‐PAGE data, and rep‐PCR reactions respectively. Low similarity among groups (55%) can be explained as high genetic diversity among the strains. One strain of west Azerbaijan and the strains of Golestan, clustered in the same group suggesting that they may have been originated from a common source. Other strains of west Azerbaijan were clustered into different groups including II, III, IV, V and VI, suggesting the possibility of occurrence of different populations in a geographical region.     

A Nested Polymerase Chain Reaction Protocol for in planta Detection of Fusicladium eriobotryae,Causal Agent of Loquat Scab

Scab caused by the fungus Fusicladium eriobotryae is the most serious disease affecting loquat in Spain. Isolation of F. eriobotryae from infected tissue on culture media can be difficult due to its slow growth. A polymerase chain reaction (PCR)‐based protocol was developed for F. eriobotryae‐specific identification from pure culture or infected loquat tissues. The primer set was designed in the elongation factor 1‐α gene (EF1‐α), and specificity and sensitivity for single and nested PCR were validated. The nested PCR assay resulted in 100% positive detection of F. eriobotryae in naturally and artificially infected tissues. This protocol can be useful for routine diagnosis, disease monitoring programmes and epidemiological research.     

Multiplex PCR assay for the detection of enterotoxic <Emphasis Type="Italic">Bacillus cereus</Emphasis> group strains and its application in food matrices

Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis are the major concerns for the food safety in terms of frequency and/or seriousness of the disease. Being members of the same group and sharing DNA homology to a larger extent, they do create problems when their specific detection/identification is attempted from different food and environmental sources. Numerous individual polymerase chain reaction (PCR) and few multiplex PCR (mPCR) methods have been employed to detect these organisms by targeting toxin genes but with lack of internal amplification control (IAC). Therefore, we attempted a mPCR with IAC for the detection of enterotoxic B. cereus group strains by selecting hbl A, nhe A and cyt K genes from B. cereus, indicative of the diarrheal potential and cry I A and pag genes, the plasmid borne phenotypic markers specific to B. thuringiensis and B. anthracis strains, respectively. Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays and correlated to the classical conventional and biochemical identification of the organisms. The mPCR was able to detect as low as 10¹–10² organisms per ml following overnight enrichment of spiked food samples (vegetable biriyani and milk) in buffered peptone water (BPW). The presence of these organisms could also be detected by mPCR in naturally contaminated samples of rice based dishes and milk. The high throughput and cost-effective mPCR method described could provide a powerful tool for simultaneous, rapid and reliable detection of enterotoxic B. cereus group organisms.     

Morphactin transport and metabolism in tree bark tissues: comparative studies in vitro

Bark banding of morphaction is an effective means of controling stem elongation in Pinus radiata D. Don (Monterey pine) but not Juglans regia L. (English walnut). Diffusion coefficients of ¹⁴C-labeled morphactin across excised disks of tree bark, measured in specially designed diffusion chambers, were 11 to 85 fold greater in pine than walnut. In seedlings of comparable age, the suberin layer of walnut bark is much thicker than that of pine; if the layer is removed, diffusion of ¹⁴C-morphactin is enhanced 39-fold in pine and 285-fold in walnut. Morphactin applied to the bark as an ester is rapidly hydrolyzed to its carboxylic acid derivative in both pine and walnut. This conversion occurs rapidly in the bark of both species and does not appear to limit the rate of morphactin movement across the bark. These results suggest that diffusion across the suberin layer and not metabolism limits morphactin transport across the bark.     

Development of a SCAR marker by inter-simple sequence repeat for diagnosis of dwarf bunt of wheat and detection of <Emphasis Type="Italic">Tilletia controversa</Emphasis><Emphasis Type="SmallCaps">Kühn</Emphasis>

Dwarf bunt of wheat, caused by Tilletia controversa Kühn, is a destructive disease on wheat as well as an important internationally quarantined disease in many countries. The primer ISSR818 generated a polymorphic pattern displaying a 867-bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF3/TCKSR3) designed for use in PCR detection assays; they amplified a unique DNA fragment in all isolates of T. controversa but not in the related pathogens. The detection limit with the primer set (TCKSF3/TCKSR3) was 5 ng of DNA which could be obtained from 5.5 μg of teliospores in a 25-μL PCR reaction mixture.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

Morphological,cultural and pathogenic characteristics of Coniothyrium zuluense isolates from different plantation regions in South Africa

Coniothyrium canker caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus species in sub-tropical regions of South Africa. This disease is typified by necrotic bark lesions that coalesce to form large kino-impregnated cankers along the stems of trees. The strategy currently used to manage Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Thus, forest managers are interested in the genetic diversity of the pathogen and its potential to overcome disease resistance in planting stock. In this study, 344 isolates of C. zuluense from different plantation regions in South Africa were compared on the basis of colony colour, conidial morphology, growth characteristics on agar and pathogenicity to a susceptible E. grandis clone. Conidia of all C. zuluense isolates measured were similar in size and shape. The fungus is slow growing in culture, which is indicative of its apparent biotrophic habit, with optimum growth observed at 30 °C. Isolates of C. zuluense displayed considerable variation in colony colour and pathogenicity in inoculation trials. Variation in morphology and pathogenicity amongst isolates suggests that C. zuluense has been present in South Africa for an extended period of time, or that it is changing rapidly due to strong directional selection pressures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Simultaneous detection of eight species of tree nut in foods using two tetraplex polymerase chain reaction assays

ABSTRACT

Tree nuts comprise a category of food allergens that must be included in the food labels in several countries. We developed a polymerase chain reaction (PCR) method using eight specific primer pairs to detect eight representative tree nuts (almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, and walnut) under the same experimental conditions. The specificity of the eight primer pairs was confirmed by PCR testing against a variety of plant and animal samples. The detection limit of the method ranged from 1 fg to 1 pg DNA of individual tree nuts. The method detected tree nut DNA in processed and unprocessed food. In addition, the primer pairs could be combined into two sets of tetraplex PCR system. The developed method is specific, sensitive, and efficient, making it useful for detecting trace amounts of eight species of tree nut in foods.     

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Acquisition of flanking sequence adjacent to a known DNA site is an important task in microbial genome-related research. In this study, we developed a new method containing two rounds of PCR followed by cloning and sequencing. Firstly, specific primer (SP) is added into the reaction system for primary locus-specific linear amplification, and then a complex long primer (CLP) is added into the cooled reaction system for only one cycle. Amplification products from the first round of PCR are directly purified without electrophoresis, diluted, and used as the templates of the second PCR. Secondly, one long specific primer (LSP) and one long base-fixed primer (LFP) are adopted. The amplicons are purified for cloning and sequencing. The achievement of specific amplification for long flanking region mainly depends on ingenious and precise settings of PCR programs, structure design of CLP primer, adding of CLP primer after specific linear amplification, concentration ratio of CLP and SP primer, applying long primers, etc. Through this method, we successfully performed the long PCR walkings (>1.5 Kb) on rpoB gene of Vibrio vulnificus, transposon-like gene of V. alginolyticus, and sto gene of V. cholerae. The method provides a robust and simple strategy for rapid amplification of long unknown DNA fragments from microbes.     

Microbiological quality of walnut kernels and apple juice concentrate

In the present study, we have evaluated the microbiological quality of walnut kernels and pasteurized apple juice concentrate and the application of PCR for quality control of these important horticultural products. PCR assays for the detection of Bacillus cereus, Salmonella, Escherichia coli and E. coli O157:H7 were standardized using minimum time for each step of the reaction. The protocols were effective for their detection in these products after pre-enrichment for 6–12 h. 2, 68 and 30% of the samples of walnut kernels were respectively found satisfactory, acceptable and unsatisfactory on the basis of their viable count. Only 15% of the samples of pasteurized apple juice concentrate were found to possess the desired viable count of less than 100 c.f.u./ml. The predominant contaminants of walnut kernels were found to be the species of Bacillus, Klebsiella, Enterobacter and Staphylococcus. Samples of apple juice concentrate were predominantly found contaminated with species of Bacillus, Staphylococcus and Micrococcus. However, B. cereus, Salmonella and E. coli were also isolated from some of the samples of walnut kernels. Bacillus cereus was also obtained from some of the samples of pasteurized apple juice concentrate in high numbers. Among the moulds Penicillium, Aspergillus, Cladosporium, Rhizopus and Mucor were isolated from these products.     

Biochemical and molecular identification of Xanthomonas translucens pv. undulosa causing bacterial leaf streak of wheat in Punjab,Pakistan

Xanthomonas translucens pv. undulosa, (Xtu.), causal agent of Bacterial leaf streak (BLS) of wheat, was characterised through pathogencity, hypersensitivity, biochemical and molecular assays. Fifty symptomatic leaves of wheat were collected from eight agro-ecological zones of Punjab out of which 25 were isolated and purified. Maximum incidence and severity in Faisalabad were followed by Multan and Rahim Yar Khan. The pathogen isolated from diseased leaves was identified on the basis of colonies pattern, colour, biochemical and pathogencity test as X. translucens pv. undulosa and confirmed its pathogencity through pathogencity test. For molecular characterization, the bacterial 16S–23S rDNA spacer fragments were amplified by PCR with conserved primers (C1 and C2) and then in combination with specific primers (T1 & T2). 300?bp product amplified by C1 and C2 primer pair confirmed the presence of Xanthomonas, while specific primers T1 and T2 amplified a product of 200?bp, confirmed the presence of X. translucens pv. undulosa. This work will be quite helpful for wheat pathologist and breeders for future management strategy for this disease.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Metabarcoding of fungal communities associated with bark beetles

Many species of fungi are closely allied with bark beetles, including many tree pathogens, but their species richness and patterns of distribution remain largely unknown. We established a protocol for metabarcoding of fungal communities directly from total genomic DNA extracted from individual beetles, showing that the ITS3/4 primer pair selectively amplifies the fungal ITS. Using three specimens of bark beetle from different species, we assess the fungal diversity associated with these specimens and the repeatability of these estimates in PCRs conducted with different primer tags. The combined replicates produced 727 fungal Operational Taxonomic Units (OTUs) for the specimen of Hylastes ater, 435 OTUs for Tomicus piniperda, and 294 OTUs for Trypodendron lineatum, while individual PCR reactions produced on average only 229, 54, and 31 OTUs for the three specimens, respectively. Yet, communities from PCR replicates were very similar in pairwise comparisons, in particular when considering species abundance, but differed greatly among the three beetle specimens. Different primer tags or the inclusion of amplicons in separate libraries did not impact the species composition. The ITS2 sequences were identified with the Lowest Common Ancestor approach and correspond to diverse lineages of fungi, including Ophiostomaceae and Leotiomycetes widely found to be tree pathogens. We conclude that Illumina MiSeq metabarcoding reliably captures fungal diversity associated with bark beetles, although numerous PCR replicates are recommended for an exhaustive sample. Direct PCR from beetle DNA extractions provides a rapid method for future surveys of fungal species diversity and their associations with bark beetles and environmental variables.