bluuebunny/crossref_metadata_2025 huggingface dataset:
Variation in Albicidin Biosynthesis Genes and in Pathogenicity ofXanthomonas albilineans, the Sugarcane Leaf Scald Pathogen
Authors: Champoiseau P. , Daugrois J.-H. , Girard J.-C. , Royer M. , Rott P. C.
Published: May 2007

Total genomic DNA from 137 strains of Xanthomonas albilineans from worldwide locations was hybridized with two DNA probes that together harbor the entire 49-kb albicidin biosynthesis gene cluster and two additional 3-kb genomic regions required for albicidin production. Fourteen haplotypes and two major genetic groups (albicidin [ALB]-restriction fragment length polymorphism [RFLP] A and ALB-RFLP B) were identified, and strains that were isolated after recent outbreaks of leaf scald disease belonged to group ALB-RFLP B. Albicidin genetic diversity was very similar to the previously described genetic diversity of the pathogen based on the whole genome. No relationship was found between variability of albicidin biosynthesis genes and the amount of albicidin produced in vitro by X. albilineans. Leaf scald-susceptible sugarcane cv. H70-144 was inoculated with 20 strains of the pathogen belonging to different ALB-RFLP haplotypes. Among them, 10 strains from Guadeloupe belonged to the same ALB-RFLP group but differed in the amount of albicidin produced in vitro. Strains were distributed in at least three different pathogenicity groups based on symptom severity and pathogen population density in the stalk. These two pathogenicity factors varied concurrently; however, no relationship between variation in albicidin biosynthesis genes, variation in the amount of albicidin produced in vitro, and variation in pathogenicity of X. albilineans was found. Further investigation is necessary to identify other genes involved in pathogenicity of X. albilineans.
Genetic Diversity in Australian Populations of Puccinia graminis f. sp. avenae
Authors: Keiper F. J. , Haque M. S. , Hayden M. J. , Park R. F.
Published: May 2007

Sequence-tagged microsatellite profiling was used to develop 110 microsatellites for Puccinia graminis f. sp. tritici (causal agent of wheat stem rust). Low microsatellite polymorphism was exhibited among 10 pathogenically diverse P. graminis f. sp. tritici isolates collected from Australian cereal growing regions over a period of at least 70 years, with two polymorphic loci detected, each revealing two alleles. Limited cross-species amplification was observed for the wheat rust pathogens, P. triticina (leaf rust) and P. striiformis f. sp. tritici (stripe rust). However, very high transferability was revealed with P. graminis f. sp. avenae (causal agent of oat stem rust) isolates. A genetic diversity study of 47 P. graminis f. sp. avenae isolates collected from an Australia-wide survey in 1999, and a historical group of 16 isolates collected from Australian cereal growing regions from 1971 to 1996, revealed six polymorphic microsatellite loci with a total of 15 alleles. Genetic analysis revealed the presence of several clonal lineages and subpopulations in the pathogen population, and wide dispersal of identical races and genotypes throughout Australian cereal-growing regions. These findings demonstrated the dynamic population structure of this pathogen in Australia and concur with the patterns of diversity observed in pathogenicity studies.
Influence of Environmental Conditions on Infection of Peach Shoots by Taphrina deformans
Authors: Rossi V. , Bolognesi M. , Languasco L. , Giosuè S.
Published: May 2007

The effect of weather conditions on the infection of peach shoots by Taphrina deformans was investigated both under orchard conditions and in controlled-environment experiments. Leaf curl incidence and severity were related to rainfall, length of wet periods, and temperature during wetness and during the incubation period, as well as to the development stage of shoots. Surface wetness was more important than rainfall for infection to occur. Minimum rainfall for infection was 3 mm, with a wet period of at least 12.5 h; higher amounts of rainfall did not cause infection when the wet period they triggered was shorter. Wet periods initiated by dew or fog were too short for infection to occur. Infection occurred only when air temperature was <16°C during the wet period and <19°C during incubation. Logistic equations relating relative disease incidence and either duration of wetness or temperature were developed under controlled-environment conditions, with asymptotes at ≥48 h of wetness and ≤8°C for temperature, respectively. Rate of shoot and leaf development during the season influenced disease development, so that three different periods of susceptibility were defined, maximum susceptibility being between bud break and petal fall due to a low development rate of shoots.
The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants
Authors: Woo S. L. , Scala F. , Ruocco M. , Lorito M.
Published: May 2007

Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations today available as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex, three-way cross-talk between the fungal antagonist, the plant, and microbial pathogens. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced resistance, proteins responsible for a gene-for-gene avirulent interaction between Trichoderma spp. and plants, mycoparasitism-related inducers, plant proteins specifically induced by Trichoderma, etc. We have transgenically demonstrated the ability of Trichoderma spp. to transfer heterologous proteins into plant during root colonization, and have used green fluorescent protein and other markers to study the interaction in vivo and in situ between Trichoderma spp. and the fungal pathogen or the plant.
Interference Between D and M Types of Plum pox virus in Japanese Plum Assessed by Specific Monoclonal Antibodies and Quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction
Authors: Capote Nieves , Gorris M. Teresa , Martínez M. Carmen , Asensio Margarita , Olmos Antonio , Cambra Mariano
Published: May 2007

The dynamics of virus interference between two isolates of Plum pox virus (PPV) belonging to the main PPV types, D and M, were analyzed in Japanese plum (Prunus salicina) by challenge inoculations. To assess the consequences of a PPV-M infection on plum already infected with PPV-D, and vice versa (predominance of one of the strains, recombination, synergism, symptoms aggravation, and so on), 30 Japanese plum trees were graft inoculated with PPV-D or PPV-M isolates in quarantine conditions. One year postinoculation, in the event that the inoculated isolates were detected in the whole plant, a second challenge inoculation (PPV-M or PPV-D, respectively) was performed by grafting. The presence of PPV-D, PPV-M, or both was monitored for 7 years by double-antibody sandwich indirect enzyme-linked immunosorbent assay using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction (RT-PCR) with D- and M-specific primers confirmed the serological typing. Real-time RT-PCR assays were performed using D- and M-specific fluorescent 3′ minor groove binder-DNA probes, which were able to detect and quantify PPV populations in the inoculated plants with greater precision. The presence of PPV-D in Japanese plum did not cross-protect the trees against PPV-M infection. In PPV-D-infected plants, the PPV-M strain used as challenge inoculum behaved differently depending on the plum cultivar assayed. In cv. Black Diamond, PPV-M invaded the plant progressively, displacing the previous PPV-D population; whereas, in cv. Sun Gold, both PPV isolates coexisted in the plant. In contrast, the PPV-D isolate used was unable to infect plants of both cultivars in which a PPV-M population already was established. After 7 years, no synergism was observed and no recombination event between PPV-D and PPV-M genomes was detected.
Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions
Authors: Tooley Paul W. , Martin Frank N. , Carras Marie M. , Frederick Reid D.
Published: May 2007

A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10-5 dilution of extracted tissue of artificially infected rhododendron ‘Cunningham's White’, and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.
Biological Control of Fusarium Seedling Blight Disease of Wheat and Barley
Authors: Khan Mojibur R. , Fischer Sven , Egan Damian , Doohan Fiona M.
Published: May 2007

Fusarium fungi, including F. culmorum, cause seedling blight, foot rot, and head blight diseases of cereals, resulting in yield loss. In a screen for potential disease control organisms and agents, Pseudomonas fluorescens strains MKB 100 and MKB 249, P. frederiksbergensis strain 202, Pseudomonas sp. strain MKB 158, and chitosan all significantly reduced the extent of both wheat coleoptile growth retardation and wheat and barley seedling blight caused by F. culmorum (by 53 to 91%). Trichodiene synthase is a Fusarium enzyme necessary for trichothecene mycotoxin biosynthesis; expression of the gene encoding this enzyme in wheat was 33% lower in stem base tissue coinoculated with Pseudomonas sp. strain MKB 158 and F. culmorum than in wheat treated with bacterial culture medium and F. culmorum. When wheat and barley were grown in soil amended with either chitosan, P. fluorescens strain MKB 249, Pseudomonas sp. strain MKB 158, or culture filtrates of these bacteria, the level of disease symptoms on F. culmorum-inoculated stem base tissue (at 12 days post- F. culmorum inoculation) was ≥31% less than the level on F. culmorum-inoculated plants grown in culture medium-amended soil. It seems likely that at least part of the biocontrol activity of these bacteria and chitosan may be due to the induction of systemic disease resistance in host plants. Also, in coinoculation studies, Pseudomonas sp. strain MKB 158 induced the expression of a wheat class III plant peroxidase gene (a pathogenesis-related gene).
Relating Epidemic Progress from a General Disease Model to Seasonal Appearance Time of Rusts in the United States: Implications for Soybean Rust
Authors: Pivonia Shimon , Yang X. B.
Published: May 2007

Soybean rust, Phakopsora pachyrhizi, has been considered a threat to the production of the U.S. soybean, Glycine max. During the past decade, this disease gradually spread to Africa, South America, and recently to the United States. Previous soybean rust risk assessments with an assumption of availability of spores early in a season showed that weather conditions (dew and temperature) during a growing season, in general, are suitable for disease development in U.S. soybean-growing regions. Predicting the time of rust appearance in a field is critical to determining the destructive potential of rusts, including soybean rust. In this study, comparative epidemiology was used to assess likely rust incipient time in four locations within the U.S. Soybean Belt from south to north: Baton Rouge, LA; Charlotte, NC; Indianapolis, IN; and Minneapolis, MN. Temperature effects on the infection cycle of five rusts occurring in the Midwest were evaluated using a general disease model. The likely incipient times were examined with the modeling results. Among the rusts studied, early-appearing rusts had suitable conditions for development earlier in a season. However, a lag period of several weeks to more than 3 months was found from the time when conditions are suitable for a rust to develop or when hosts are available to the time when the rust was detected in fields. Length of the lag period differed among the rust species examined. If nature of long-distance dispersal is not significantly different among the rusts, implications of our study to the expected seasonal soybean rust incipience in fields lead to two possible scenarios: (i) average appearance time of soybean rust across the Soybean Belt should be somewhere between appearance times of common corn rust and southern corn rust, and (ii) with late appearance of the disease, late-planted soybean in the south has greater risk.
Evaluation of Bacterial Antagonists for Biological Control of Broccoli Head Rot Caused by Pseudomonas fluorescens
Authors: Cui Xiaohui , Harling Rob
Published: May 2007

Pectolytic strains of Pseudomonas fluorescens are opportunistic pathogens of broccoli, causing head rot in temperate regions of the world. In this study, we investigated the potential of two bacterial isolates, P. fluorescens m6418 and Bacillus sp. A24, for biological control of broccoli head rot caused by P. fluorescens 5064, isolated from diseased broccoli in Scotland, UK. P. fluorescens m6418, a Tn5 mutant of wild-type 5064, is nonpathogenic and overproduces an extracellular metabolite with strong antimicrobial activity. In this study, we identified the anti-microbial metabolite produced by strain m6418 as pyrrolnitrin. P. fluorescens m6418 had significant inhibitory effects against strain 5064 both in culture and on broccoli leaves. In an excised broccoli head pathogenicity test, strain m6418, when coinoculated with P. fluorescens 5064, reduced disease by 41%. Bacillus sp. A24 produces an enzyme that can degrade N-acyl homoserine lactones, signaling molecules employed by bacteria for quorum sensing. Bacillus sp. A24 was capable of out-competing P. fluorescens 5064 when grown together in culture, and could degrade the quorum sensing signal of P. fluorescens 5064 (and thereby attenuate its virulence gene production). However, Bacillus sp. A24 had only a limited biocontrol effect on P. fluorescens 5064 in the excised broccoli head assay.
Biological and Molecular Characterization of a Novel Carmovirus Isolated from Angelonia
Authors: Adkins Scott , Hammond John , Gera Abed , Maroon-Lango Clarissa J. , Sobolev Irena , Harness Andrea , Zeidan Mohammad , Spiegel Sara
Published: May 2007

A new carmovirus was isolated from Angelonia plants (Angelonia angustifolia), with flower break and mild foliar symptoms, grown in the United States and Israel. The virus, for which the name Angelonia flower break virus (AnFBV) is proposed, has isometric particles, ≈30 nm in diameter. The experimental host range was limited to Nicotiana species, Schizanthus pinnatus, Myosotis sylvatica, Phlox drummondii, and Digitalis purpurea. Virions were isolated from systemically infected N. benthamiana leaves, and directly from naturally infected Angelonia leaves, using typical carmovirus protocols. Koch's postulates were completed by mechanical inoculation of uninfected Angelonia seedlings with purified virions. Isometric particles were observed in leaf dips and virion preparations from both Angelonia and N. benthamiana, and in thin sections of Angelonia flower tissue by electron microscopy. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 38 kDa was observed. Virion preparations were used to produce virus-specific polyclonal antisera in both Israel and the United States. The antisera did not react with Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), or Saguaro cactus virus (SgCV) by either enzyme-linked immunosorbent assay or immunoblotting. In reciprocal tests, antisera against PFBV, CarMV, and SgCV reacted only with the homologous viruses. The complete nucleotide sequence of a Florida isolate of AnFBV and the coat protein (CP) gene sequences of Israeli and Maryland isolates were determined. The genomic RNA is 3,964 nucleotides and contains four open reading frames arranged in a manner typical of carmoviruses. The AnFBV CP is most closely related to PFBV, whereas the AnFBV replicase is most closely related to PFBV, CarMV, and SgCV. Particle morphology, serological properties, genome organization, and phylogenetic analysis are all consistent with assignment of AnFBV to the genus Carmovirus.
Distribution of Mycelial Colonies and Lesions in Field-Grown Barley Inoculated with Fusarium graminearum
Authors: Lewandowski Sharon M. , Bushnell W. R. , Evans C. Kent
Published: May 2007

External surfaces of barley florets have thick-walled epidermal cells resistant to direct penetration by the head blight pathogen, Fusarium graminearum. Surfaces within the floral cavity have thin-walled, susceptible cells. How the fungus gains access to the floral cavity, causing head blight, has not been determined. To investigate pathways of entry, field-grown plants were sprayed with macroconidial inoculum after heads emerged from the flag leaf sheath and then were mist irrigated daily in the morning and evening. On selected days, 1 to 8 days after inoculation (DAI), 80 to 190 florets per day were harvested, dissected, and examined for presence and location of mycelial colonies. At 1 to 12 DAI, 57 to 100 florets likewise were examined for lesions. Patterns of colonization indicated that the fungus entered florets principally through crevices between the overlapping lemma and palea or through the apical floret mouth. The crevices were open for entry until ≈8 days after heads emerged. Most florets had mycelial colonies on the external surface in a sheltered pocket near the base of the ventral furrow of the palea. Mycelia spread laterally from the furrow to the crevice between lemma and palea. Anther colonization had only a minor role in invasion of florets. Hyphal penetration of stomates was not seen. Lesions usually developed first within 3 mm of the floret apex or 3 mm of the floret base. Within florets, lesions often were contiguous between lemma and palea, palea and caryopsis, or in all three floret parts. However, lesions in the caryopsis developed later and were fewer in number than in the lemma and palea and always were associated with lesions in the palea. The results show the importance of initial mycelial colonization of floret outer surfaces, pathways of entry via lemma or palea crevices or floret mouth, and spread of lesions within the floret at interfaces between lemma, palea, and caryopsis.
Quantitative Trait Loci Mapping for Adult-Plant Resistance to Powdery Mildew in Bread Wheat
Authors: Liang S. S. , Suenaga K. , He Z. H. , Wang Z. L. , Liu H. Y. , Wang D. S. , Singh R. P. , Sourdille P. , Xia X. C.
Published: May 2007

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is a major disease to wheat (Triticum aestivum) worldwide. Use of adult-plant resistance (APR) is an effective method to develop wheat cultivars with durable resistance to powdery mildew. In the present study, 432 molecular markers were used to map quantitative trait loci (QTL) for APR to powdery mildew in a doubled haploid (DH) population with 107 lines derived from the cross Fukuho-komugi × Oligoculm. Field trials were conducted in Beijing and Anyang, China during 2003-2004 and 2004-2005 cropping seasons, respectively. The DH lines were planted in a randomized complete block design with three replicates. Artificial inoculation was carried out in Beijing with highly virulent isolate E20 of B. graminis f. sp. tritici and the powdery mildew severity on penultimate leaf was evaluated four times, and the maximum disease severity (MDS) on penultimate leaf was investigated in Anyang under natural inoculation in May 2004 and 2005. The heritability of resistance to powdery mildew for MDS in 2 years and two locations ranged from 0.82 to 0.93, while the heritability for area under the disease progress curve was between 0.84 and 0.91. With the method of composite interval mapping, four QTL for APR to powdery mildew were detected on chromosomes 1AS, 2BL, 4BL, and 7DS, explaining 5.7 to 26.6% of the phenotypic variance. Three QTL on chromosomes 1AS, 2BL, and 7DS were derived from the female, Fukuho-komugi, while the one on chromosome 4BL was from the male, Oligoculm. The QTL on chromosome 1AS showed high genetic effect on powdery mildew resistance, accounting for 19.5 to 26.6% of phenotypic variance across two environments. The QTL on 7DS associated with the locus Lr34/Yr18, flanked by microsatellite Xgwm295.1 and Ltn (leaf tip necrosis). These results will benefit for improving powdery mildew resistance in wheat breeding programs.
Insect Injection and Artificial Feeding Bioassays to Test the Vector Specificity of Flavescence Dorée Phytoplasma
Authors: Bressan Alberto , Clair Denis , Sémétey Olivier , Boudon-Padieu Elisabeth
Published: May 2007

The specificity of vector transmission of Flavescence dorée phytoplasma (FDP) was tested by injecting FDP, extracted from laboratory-reared infective Euscelidius variegatus, into specimens of 15 other hemipteran insect species collected in European vineyards. Concentrations of viable phytoplasma extracts and latency in vectors were monitored by injection of healthy-reared E. variegatus leafhoppers. Based on these preliminary results, insects were injected by using phytoplasma extracts that ensured the highest rate of FDP acquisition and transmission by E. variegatus. Transmission into an artificial diet through a Parafilm membrane about 3 weeks after insect injection was attempted. FDP-injected insects that belonged to 15 hemipteran species were confined in cages and fed through the membrane for a 4- to 5-day inoculation access period. FDP DNA was detected by polymerase chain reaction (PCR) in the feeding buffer fed upon by Anoplotettix fuscovenosus, Aphrodes makarovi,E. variegatus, and Euscelis incisus. PCR amplification with specific primers detected FDP DNA in injected insects of all test insect species. Band intensity was positively correlated with the transmissibility of FDP. Transmission of FDP to plants by feeding was confirmed for Anoplotettix fuscovenosus, E. variegatus, and Euscelis incisus, but not for Aphrodes makarovi. Our results suggest that vector competency of FDP is restricted to specimens belonging to the family Cicadellidae, subfamily Deltocephalinae.
Predicting Severity of Asian Soybean Rust Epidemics with Empirical Rainfall Models
Authors: Del Ponte E. M. , Godoy C. V. , Li X. , Yang X. B.
Published: May 2007

Although Asian soybean rust occurs in a broad range of environmental conditions, the most explosive and severe epidemics have been reported in seasons with warm temperature and abundant moisture. Associations between weather and epidemics have been reported previously, but attempts to identify the major factors and model these relationships with field data have been limited to specific locations. Using data from 2002-03 to 2004-05 from 34 field experiments at 21 locations in Brazil that represented all major soybean production areas, we attempted to identify weather variables using a 1-month time window following disease detection to develop simple models to predict final disease severity. Four linear models were identified, and these models explained 85 to 93% of variation in disease severity. Temperature variables had lower correlation with disease severity compared with rainfall, and had minimal predictive value for final disease severity. A curvilinear relationship was observed between 1 month of accumulated rainfall and final disease severity, and a quadratic response model using this variable had the lowest prediction error. Linear response models using only rainfall or number of rainy days in the 1-month period tended to overestimate disease for severity <30%. The study highlights the importance of rainfall in influencing soybean rust epidemics in Brazil, as well as its potential use to provide quantitative risk assessments and seasonal forecasts for soybean rust, especially for regions where temperature is not a limiting factor for disease development.
Chimeric cDNA Sequences from Citrus tristeza virus Confer RNA Silencing-Mediated Resistance in Transgenic Nicotiana benthamiana Plants
Authors: Roy Gourgopal , Sudarshana Mysore R. , Ullman Diane E. , Ding Shou-Wei , Dandekar Abhaya M. , Falk Bryce W.
Published: May 2007

RNA silencing has been shown to be an important mechanism for conferring resistance in transgenic, virus-resistant plants. We used this approach to evaluate resistance in Nicotiana benthamiana plants transformed with chimeric coding and noncoding sequences from Citrus tristeza virus (CTV). Several independent transgenic plant lines were generated, using two constructs (pCTV1 and pCTV2) designed to produce self-complementary transcripts. The pCTV1 contained cDNA sequences from the CTV capsid protein (CP), p20, and 3′ untranslated region (UTR); and pCTV2 contained CP, p23, and 3′ UTR sequences. Heterologous recombinant Potato virus X (PVX) containing either homologous or heterologous CTV sequences was used to challenge plants and resistance was evaluated phenotypically and validated with reverse-transcriptase polymerase chain reaction and northern hybridization analysis. Transgenic plants (T1 generation) for each construct showed resistance to recombinant PVX constructs used for challenge experiments when PVX contained p20 or UTR (for CTV1 plants), or p23 or UTR (for CTV2 plants). However, no resistance was seen when plants were challenged with PVX containing the CTV CP. T2 generation plants also showed resistance even when challenged with PVX containing the cognate CTV sequences obtained from heterologous CTV isolates. The presence of transgene-specific small interfering RNAs in the resistant CTV1 and CTV2 plants indicated that resistance was mediated by post-transcriptional gene silencing.
Phylogenetic Analysis of “Candidatus Phytoplasma australiense” Reveals Distinct Populations in New Zealand
Authors: Andersen Mark T. , Newcomb Richard D. , Liefting Lia W. , Beever Ross E.
Published: May 2007

The phytoplasma “Candidatus Phytoplasma australiense” has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16–23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and “Candidatus Phytoplasma australiense”. Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.
TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods
Authors: Hayden Katherine , Ivors Kelly , Wilkinson Carla , Garbelotto Matteo
Published: May 2007

The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5′ fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.
Classification and Identification of Xanthomonas translucens Isolates, Including Those Pathogenic to Ornamental Asparagus
Authors: Rademaker J. L. W. , Norman D. J. , Forster R. L. , Louws F. J. , Schultz M. H. , de Bruijn F. J.
Published: May 2007

In order to confirm and refine the current classification scheme of Xanthomonas translucens and to identify novel strains from ornamental asparagus, a collection of field and reference strains was analyzed. Rep-polymerase chain reaction (PCR) genomic fingerprint profiles were generated from 33 isolates pathogenic to asparagus as well as 61 X. trans-lucens reference strains pathogenic to cereals and grasses. Amplified ribo-somal gene restriction analysis profiles were obtained from most of these and 29 additional Xanthomonas reference strains. Rep-PCR genomic fingerprint profiles of all strains were compared with those in a large Xanthomonas database using computer-assisted analysis. Rep-PCR ge-nomic fingerprinting facilitated the characterization and discrimination of X. translucens, including the pathovars arrhenatheri, graminis, phlei, phleipratensis, and poae, as well as a number of strains received as X. translucens pv. cerealis. Strains received as pathovars hordei, secalis, translucens, undulosa, and other cerealis strains were grouped in two subclusters that correspond to the recently redefined pathovars X. trans-lucens pvs. undulosa and translucens. All 33 novel isolates from ornamental asparagus (tree fern; Asparagus virgatus) were identified as X. translucens pv. undulosa. Moreover, a unique amplified small subunit ribosomal gene MspI/AluI restriction profile specific for all X. translucens strains tested, including those pathogenic to asparagus, allowed discrimination from all other Xanthomonas spp. Although phage tests were inconclusive, the classification of the asparagus strains within the X. translucens complex was supported by pathogenicity assays in which all the isolates from ornamental asparagus induced watersoaking on wheat. Surprisingly, several X. translucens reference strains affected asparagus tree fern as well. That the novel asparagus isolates belong to X. translucens pv. undulosa is extraordinary because all hosts of X. translucens pathovars described to date belong only to the families Gramineae and Poaceae, whereas asparagus belongs to the phylogenetically distant family Liliaceae.
Identification and Molecular Mapping of a Gene Conferring Resistance to Pyrenophora tritici-repentis Race 3 in Tetraploid Wheat
Authors: Singh P. K. , Gonzalez-Hernandez J. L. , Mergoum M. , Ali S. , Adhikari T. B. , Kianian S. F. , Elias E. M. , Hughes G. R.
Published: May 2007

Race 3 of the fungus Pyrenophora tritici-repentis, causal agent of tan spot, induces differential symptoms in tetraploid and hexaploid wheat, causing necrosis and chlorosis, respectively. This study was conducted to examine the genetic control of resistance to necrosis induced by P. tritici-repentis race 3 and to map resistance genes identified in tetraploid wheat (Triticum turgidum). A mapping population of recombinant inbred lines (RILs) was developed from a cross between the resistant genotype T. tur-gidum no. 283 (PI 352519) and the susceptible durum cv. Coulter. Based on the reactions of the Langdon-T. dicoccoides (LDN[DIC]) disomic substitution lines, chromosomal location of the resistance genes was determined and further molecular mapping of the resistance genes for race 3 was conducted in 80 RILs of the cross T. turgidum no. 283/Coulter. Plants were inoculated at the two-leaf stage and disease reaction was assessed 8 days after inoculation based on lesion type. Disease reaction of the LDN(DIC) lines and molecular mapping on the T. turgidum no. 283/Coulter population indicated that the gene, designated tsn2, conditioning resistance to race 3 is located on the long arm of chromosome 3B. Genetic analysis of the F2 generation and of the F4:5 and F6:7 families indicated that a single recessive gene controlled resistance to necrosis induced by race 3 in the cross studied.
Plant Pathogen Culture Collections: It Takes a Village to Preserve These Resources Vital to the Advancement of Agricultural Security and Plant Pathology
Authors: Kang Seogchan , Blair Jaime E. , Geiser David M. , Khang Chang-Hyun , Park Sook-Young , Gahegan Mark , O'Donnell Kerry , Luster Douglas G. , Kim Seong H. , Ivors Kelly L. , Lee, Yong-Hwan , Lee Yin-Won , Grünwald Niklaus J. , Martin Frank M. , Coffey Michael D. , Veeraraghavan Narayanan , Makalowska Izabela
Published: May 2007

Plant pathogen culture collections are essential resources in our fight against plant disease and for connecting discoveries of the present with established knowledge of the past. However, available infrastructure in support of culture collections is in serious need of improvement, and we continually face the risk of losing many of these collections. As novel and reemerging plant pathogens threaten agriculture, their timely identification and monitoring depends on rapid access to cultures representing the known diversity of plant pathogens along with genotypic, phenotypic, and epidemiological data associated with them. Archiving such data in a format that can be easily accessed and searched is essential for rapid assessment of potential risk and can help track the change and movement of pathogens. The underexplored pathogen diversity in nature further underscores the importance of cataloguing pathogen cultures. Realizing the potential of pathogen genomics as a foundation for developing effective disease control also hinges on how effectively we use the sequenced isolate as a reference to understand the genetic and phenotypic diversity within a pathogen species. In this letter, we propose a number of measures for improving pathogen culture collections.
Meta-Analysis of Regression Coefficients for the Relationship Between Fusarium Head Blight and Deoxynivalenol Content of Wheat
Authors: Paul P. A. , Lipps P. E. , Madden L. V.
Published: May 2007

A total of 126 field studies reporting deoxynivalenol (DON; ppm) content of harvested wheat grain and Fusarium head blight index (IND; field or plot-level disease severity) were analyzed to determine the overall mean regression slope and intercept for the relationship between DON and IND, and the influence of study-specific variables on the slope and intercept. A separate linear regression analysis was performed to determine the slope and intercept for each study followed by a meta-analysis of the regression coefficients from all studies. Between-study variances were significantly (P < 0.05) greater than 0, indicating substantial variation in the relationship between the variables. Regression slopes and intercepts were between -0.27 and 1.48 ppm per unit IND and -10.55 to 32.75 ppm, respectively. The overall mean regression slope and intercept, 0.22 ppm per unit IND and 2.94 ppm, respectively, were significantly different from zero (P < 0.001), and the width of the 95% confidence interval was 0.07 ppm per unit IND for slope and 1.44 ppm for intercept. Both slope and intercept were significantly affected by wheat type (P < 0.05); the overall mean intercept was significantly higher in studies conducted using winter wheat cultivars than in studies conducted using spring wheat cultivars, whereas the overall mean slope was significantly higher in studies conducted using spring wheat cultivars than in winter wheat cultivars. Study location had a significant effect on the intercept (P < 0.05), with studies from U.S. winter wheat-growing region having the highest overall mean intercept followed by studies from Canadian wheat-growing regions and U.S. spring wheat-growing regions. The study-wide magnitude of DON and IND had significant effects on one or both of the regression coefficients, resulting in considerable reduction in between-study variances. This indicates that, at least indirectly, environment affected the relationship between DON and IND.
Susceptibility of Wheat to Tilletia indica During Stages of Spike Development
Authors: Goates Blair J. , Jackson Eric W.
Published: May 2007

Karnal bunt of wheat is caused by the fungus Tilletia indica, which partially converts kernels into sori filled with teliospores. Despite minor overall yield and quality losses, the disease is of considerable international quarantine concern. Plant development stages reported susceptible to infection vary considerably. A study was designed to better define the susceptibility period by inoculating wheat spikes at different growth stages with naturally liberated secondary sporidia under optimal conditions for disease development. Spikes of a resistant and susceptible cultivar were inoculated at eight growth stages from awns emerging to soft dough. Spikes became susceptible only after emerging from the boot and continued to be susceptible up to soft dough stage at which low levels of disease occurred. Disease severity in both cultivars peaked when spikes were inoculated after complete emergence, but before the onset of anthesis. Disease levels tapered off gradually in spikes inoculated after anthesis. The results broaden the known susceptibility period of wheat to T. indica to include stages long after anthesis, and indicate that infection from airborne inoculum is not possible during boot or awns emerging stages, which are commonly referred to as the most susceptible stages.
Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum
Authors: Hughes Kelvin J. D. , Tomlinson Jennifer A. , Griffin Ruth L. , Boonham Neil , Inman Alan J. , Lane Charles R.
Published: May 2007

Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.
A Methodology to Detect and Quantify Five Pathogens Causing Potato Tuber Decay Using Real-Time Quantitative Polymerase Chain Reaction
Authors: Atallah Z. K. , Stevenson W. R.
Published: May 2007

Late blight (Phytophthora infestans), pink rot (Phytophthora erythroseptica), leak (Pythium ultimum), dry rot (Fusarium sambucinum), and soft rot (Erwinia carotovora subsp. carotovora and subsp. atroseptica) are particularly damaging diseases of stored potato tubers worldwide. In this study, we present a methodology to detect and quantify the causal agents of the five aforementioned diseases from whole potato tubers, using real-time quantitative-polymerase chain reaction. Six primer pairs were designed to amplify targets smaller than 150-bp DNA in single copy protein-coding gene targets of each of the pathogens and the potato host. Using a large collection of pure culture DNA samples, all primer pairs specifically detected the DNA target in the intended pathogenic species. Amplification efficiencies over a five-log dilution series ranged between 95 and 100% and were unaffected by the presence of large amounts of host DNA. The detection level of the primers reached 0.5 pg of target DNA. Pathogens were detected in 100 pg of total DNA extracted from 170 to 250 g of tubers, 4 days after inoculation, regardless of the presence of symptoms. The presence of P. erythroseptica, Pythium ultimum, or E. carotovora was also detected in 1 ng of DNA extracted from potato tubers collected from a commercial storage facility. This study provides the first step in a methodology to predict the storability of potato tubers following harvest.
The Internal Glycine-Rich Motif and Cysteine Suppress Several Effects of the HpaGXooc Protein in Plants
Authors: Liu Fengquan , Liu Hongxia , Jia Qin , Wu Xiaojing , Guo Xiaojing , Zhang Shujian , Song Feng , Dong Hansong
Published: May 2007

HpaGXooc, produced by Xanthomonas oryzae pv. oryzicola, is a member of harpin group of proteins that stimulate plant growth, hypersensitive cell death (HCD), and pathogen defense. The protein contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Genetic modification generated the pro-tein mutants HpaGXoocMG (MG) by deleting GRMs and HpaGXoocC47T (C47T) by replacing cysteine with threonine. When applied to tobacco plants, C47T and MG were 1.2- and 1.7-fold stronger, respectively, than HpaGXooc in inducing HCD, which occurred consistently with expression of the marker genes hin1 and hsr203. The proteins markedly alleviated infection of tobacco by Tobacco mosaic virus and Arabidopsis and tomato by Pseudomonas syringae. Treating tobacco plants with HpaGXooc, C47T, and MG decreased the viral infection by 58, 81, and 92%, respectively. In Arabidopsis and tomato plants treated with HpaGXooc, C47T, or MG, P. syringae multiplication was inhibited; bacterial population multiplied in 5 days in these plants were ca. 160-, 1,260-, or 15,860-fold smaller than that in control plants. So pathogen defense was induced in both plants. Defense-related genes Chia5, NPR1, and PR-1a were expressed consistently with resistance. In response to HpaGXooc, C47T, and MG, aerial parts and roots of tomato plants increased growth by 15 and 53%, 25 and 77%, and 46 and 106%, relative to controls. The expansin gene, EXP2, involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaGXooc represses the effects of the protein in plants.
Induction, Regulation, and Role in Pathogenesis of Appressoria in Monilinia fructicola
Authors: Lee Miin-Huey , Bostock Richard M.
Published: May 2007

Monilinia fructicola, which causes brown rot in stone fruit, forms appressoria on plant and artificial surfaces. On nectarine, the frequency of appressoria produced by conidial germlings depends to a large degree on the stage of fruit development, with numerous appressoria formed on immature (stage II) nectarine fruit, and no appressoria observed on fully mature fruit (late stage III). On polystyrene surfaces, appressorium formation was increased from <10% of germinated conidia to >95% of germinated conidia when the conidia were washed to remove residual nutrients and self-inhibitors. M. fructicola appressorium formation also appears to be regulated by the topography of the plant surface. On fruit, appressoria formed on stomatal guard cell lips, on the grooves of lateral cells adjacent to stomata or between two epidermal cells, and on the convex surfaces of epidermal cells. Pharmacological effectors indicate that cyclic AMP-, MAP kinase-, and calcium/calmodulin-dependent signaling pathways are involved in the induction and development of appressoria. KN-93, an inhibitor of calmodulin-dependent protein kinase II, did not inhibit conidial germination but did inhibit appressorium formation and brown rot development on flower petals, suggesting that appressoria are required for full symptom development on Prunus spp. petals.
Metallothionein and bZIP Transcription Factor Genes from Velvetleaf and Their Differential Expression Following Colletotrichum coccodes Infection
Authors: Dauch Amélie L. , Jabaji-Hare Suha H.
Published: May 2007

Colletotrichum coccodes is a biocontrol agent of velvetleaf (Abutilon theophrasti), a noxious weed of corn and soybean. Metallothioneins (MTs) and basic region/leucine zipper motif (bZIP) are heavy-metal-binding proteins and transcription factors, respectively, that have been related to several plant processes, including the responses of plants to pathogen attack. Previous investigation of the determinants involved in the velvet-leaf-C. coccodes interaction had shed light on particular plant and fungal genes expressed in this pathosystem. Here, we report on the temporal expression patterns of two distinct types (2 and 3) of MT and bZIP transcription factor genes in velvetleaf leaves following infection with C. coccodes using quantitative reverse-transcription polymerase chain reaction. Gene expression ratios were significantly upregulated 1 day after infection (DAI), a time at which velvetleaf leaves appeared symptomless. At 2 DAI, bZIP and type 3 MT expression ratios dropped to levels significantly lower than those estimated for noninfected plants. Necrotic symptoms appeared 5 DAI and increased with time, during which gene expression levels were maintained either below or at levels observed in the control. These findings indicate that C. coccodes altered the expression of type 2 and 3 MT and bZIP genes. In addition, this is the first report on induction of a type 3 MT in plants in response to a pathogen attack.