bluuebunny/crossref_metadata_2025 huggingface dataset:
Closterovirus Infection and Mealybug Exposure Are Necessary for the Development of Mealybug Wilt of Pineapple Disease
Authors: Sether D. M. , Hu J. S.
Published: May 2007

The roles of Pineapple mealybug wilt-associated viruses (PMWaVs) and mealybug (Dysmicoccus spp.) feeding in the etiology of mealybug wilt of pineapple (MWP) were evaluated. Container-grown pineapple (Ananas comosus) plants from five commercially grown Hawaiian proprietary selections and a field study utilizing a randomized complete block design were used to test four treatments for induction of MWP: PMWaV-1-free and PMWaV-1-infected plants maintained mealybug-free, and PMWaV-1-free and PMWaV-1-infected plants that received monthly applications of nonviruliferous mealybugs. A second PMWaV, PMWaV-2, was identified in some of the test plants during the course of these studies and was shown to be an integral factor in MWP etiology. Typical MWP symptoms developed only in plants infected with PMWaV-2 and exposed to mealybugs. MWP did not develop in PMWaV-1-free or PMWaV-1-infected plants that were exposed to mealybugs, or in mealy-bug-free plants infected with PMWaV-1, PMWaV-2, or both viruses. Plants from all five Hawaiian proprietary selections developed MWP when PMWaV-2 infected plants were exposed to mealybug feeding. A PMWaV-2-specific monoclonal antibody was produced that decorated the particles in immunosorbent electron microscopy and detected the virus in tissue blot immunoassays. PMWaV-2 was acquired and transmitted by pink and gray pineapple mealybugs (Dysmicoccus spp.) to pineapple plants, and these plants subsequently developed MWP symptoms while sustaining mealybug populations.
Recovery of Fusarium oxysporum Fo47 Mutants Affected in Their Biocontrol Activity After Transposition of the Fot1 Element
Authors: Trouvelot Sophie , Olivain Chantal , Recorbet Ghislaine , Migheli Quirico , Alabouvette Claude
Published: May 2007

To investigate the biocontrol mechanisms by which the antagonistic Fusarium oxysporum strain Fo47 is active against Fusarium wilt, a Fot1 transposon-mediated insertional mutagenesis approach was adopted to generate mutants affected in their antagonistic activity. Ninety strains in which an active Fot1 copy had transposed were identified with a phenotypic assay for excision and tested for their biocontrol activity against F. oxysporum f. sp. lini on flax in greenhouse experiments. Sixteen strains were affected in their capacity to protect flax plants, either positively (more antagonistic than Fo47) or negatively (less antagonistic). The molecular characterization of these mutants confirms the excision of Fot1 and its reinsertion in most of the cases. Moreover, we demonstrate that other transposable elements such as Fot2, impala, and Hop have no transposition activity in the mutant genomes. The phenotypic characterization of these mutants shows that they are affected neither in their in vitro growth habit nor in their competitiveness in soil compared with wild-type strain Fo47. These results show that mutants are not impaired in their saprophytic phase and suggest that the altered biocontrol phenotype should likely be expressed during the interaction with the host plant.
Weakening and Delayed Mortality of Fusarium oxysporum by Heat Treatment: Flow Cytometry and Growth Studies
Authors: Assaraf Menachem P. , Ginzburg Chen , Katan Jaacov
Published: May 2007

Survival of Fusarium oxysporum f. sp. niveum following heat treatments was studied using flow cytometric, physiological, and microscopic assays. We exposed germinating conidia to sublethal temperatures from 36 to 42°C for 60 min, followed by rhodamine 123 staining and flow cytometry, and found increasing levels of fluorescence that reflect a change in mitochondrial membrane potential, indicating a weakening induced by stress. Viability of conidia or germinating conidia of the fungus exposed to heat decreased with increasing temperature, as assessed by fluorescent staining. However, viability was higher than that assessed with the 5-day-long plate count method and was further reduced 13 and 24 h after treatment, suggesting delayed mortality of the heat-treated germinating conidia. Delayed mortality was substantiated by observing these conidia with light and fluorescent scanning electron microscopy and by subculturing single germinating conidia that had been previously heated. Programmed cell death was not observed in heat-treated conidia or germinating conidia of F. oxysporum based on the detection of plasma membrane phosphatidylserine translocation, cell-cycle measurements, detection of DNA fragmentation, or microscopic observation of apoptotic bodies. We hypothesize that propagules, which survived the heating and apparently are alive, may undergo further irreversible detrimental processes, eventually leading to their death by yet unidentified mechanisms. These findings suggest that pathogen propagules also might be affected under lower temperatures, possibly facilitating pathogen control by heating.
A Rapid Microbioassay for Discovery of Novel Fungicides for Phytophthora spp.
Authors: Kuhajek Jeanne M. , Jeffers Steven N. , Slattery Marc , Wedge David E.
Published: May 2007

A microbioassay was developed for the discovery of compounds that inhibit Phytophthora spp. This assay uses a 96-well format for high-throughput capability and a standardized method for quantitation of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 × 105 zoospores per ml using percent transmittance (620 nm). Subsequent growth of mycelia was monitored by measuring optical density (620 nm) at 24-h intervals for 96 h. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth [RPMI], and mineral salts medium) and four zoospore concentrations (10, 100, 1,000, and 10,000 zoospores per ml) were evaluated. Both full- and half-strength RPMI were identified as suitable synthetic media for growing P. nicotianae, and 1,000 zoospores per ml was established as the optimum initial concentration. The assay was used to determine effective concentration values for 50% growth reduction (EC50) for seven commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, etridiazole, metalaxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These EC50 values were compared with those obtained by measuring linear growth of mycelia on fungicide-amended medium. The microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of compounds that inhibit spore germination in P. nicotianae and should be effective for other species of Phytophthora as well. The assay requires relatively small amounts of a test compound and is suitable for the evaluation of natural product samples.
A Technique for Precise Inoculation of the Internal Phyllosphere of Cotton with Xanthomonas campestris pv. malvacearum
Authors: Kangatharalingam N. , Pierce Margaret L. , Essenberg Margaret
Published: May 2007

A technique was developed to inoculate uniformly and gently the internal phyllosphere from the upper surface of cotton leaves with the phytopathogenic bacterium Xanthomonas campestris pv. malvacearum. The inoculum consisted of 2 to 3 × 107 CFU/ml in CaCO3-saturated sterile distilled water containing 0.02%, vol/vol, of the wetting agent Silwet L-77. A custom-made inoculation apparatus was employed to immerse a circular area of the adaxial surface of a leaf in inoculum for 90 s. This resulted in uniform, passive entry of bacteria into the substomatal chambers, producing an endophytic bacterial population of 2 × 104 CFU/cm2. Microscopic signs of infection were visible 48 to 72 h after inoculation. In susceptible leaves, uniformly distributed water-soaked spots were observed 7 to 8 days after inoculation. When the technique was used on resistant leaves, the autofluorescence that is characteristic of hypersensitively necrotic cells developed in the guard cells and palisade cells lining substomatal chambers, but not in the underlying spongy mesophyll.
Restricted Spread ofTomato spotted wilt virusin Thrips-Resistant Pepper
Authors: Maris P. C. , Joosten N. N. , Goldbach R. W. , Peters D.
Published: May 2007

Spread of Tomato spotted wilt virus (TSWV) and population development of its vector Frankliniella occidentalis were studied on the pepper accessions CPRO-1 and Pikante Reuzen, which are resistant and susceptible to thrips, respectively. Viruliferous thrips were released on plants of each accession (nonchoice tests) or on plants in a 1:1 mixture of both accessions (choice tests) in small cages containing 8 or 16 plants. Significantly fewer CPRO-1 plants became infected in the primary infection phase in both tests. In the nonchoice test, virus infection of the resistant plants did not increase after the initial infection, but all plants eventually became infected when mixtures of both cultivars were challenged in the secondary infection phase. Secondary spread of TSWV from an infected resistant or susceptible source plant was significantly slower to resistant plants than to susceptible plants, independent of source plant phenotype. The restricted introduction and spread of TSWV in the thrips-resistant cultivar was confirmed in a large-scale greenhouse experiment. The restricted and delayed TSWV spread to plants of the resistant accession in both the cage and the greenhouse experiment was explained by impeded thrips population development. The results obtained indicate that thrips resistance may provide a significant protection to TSWV infection, even when the crop is fully susceptible to the virus.
Genotyping of Serratia marcescens Strains Associated with Cucurbit Yellow Vine Disease by Repetitive Elements-Based Polymerase Chain Reaction and DNA-DNA Hybridization
Authors: Zhang Q. , Weyant R. , Steigerwalt A. G. , White L. A. , Melcher U. , Bruton B. D. , Pair S. D. , Mitchell F. L. , Fletcher J.
Published: May 2007

The bacterium that causes cucurbit yellow vine disease (CYVD) has been placed in the species Serratia marcescens based on 16S rDNA and groE sequence analysis. However, phenotypic comparison of the organism with S. marcescens strains isolated from a variety of ecological niches showed significant heterogeneity. In this study, we compared the genomic DNA of S. marcescens strains from different niches as well as type strains of other Serratia spp. through repetitive elements-based polymerase chain reaction (rep-PCR) and DNA-DNA hybridization. With the former, CYVD strains showed identical banding patterns despite the fact that they were from different cucurbit hosts, geographic locations, and years of isolation. In the phylogenetic trees generated from rep-PCR banding patterns, CYVD strains clearly were differentiated from other strains but formed a loosely related group with S. marcescens strains from other niches. The homogeneity of CYVD strains was supported further by the DNA relatedness study, in that labeled DNA from the cantaloupe isolate, C01-A, showed an average relative binding ratio (RBR) of 99%, and 0.33% divergence to other CYVD strains. Used as a representative strain of CYVD, the labeled C01-A had a RBR of 76%, and a 4.5% divergence to the S. marcescens type strain. These data confirm the previous placement of CYVD strains in S. marcescens. Our investigations, including rep-PCR, DNA-DNA hybridization, and previous phenotyping experiments, have demonstrated that CYVD-associated strains of S. marcescens cluster together in a group significantly different from other strains of the species.
Localizing the Bacterial Blight Resistance Gene, Xa22(t), to a 100-Kilobase Bacterial Artificial Chromosome
Authors: Wang Chuntai , Tan Mingpu , Xu Xin , Wen Guosong , Zhang Duanpin , Lin Xinghua
Published: May 2007

The rice bacterial blight resistance gene, Xa22(t), provides resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae isolates. Here, we localize the gene to a small 100-kb fragment of chromosome 11 by a combination of genetic recombination analysis and physical mapping. Mapping was done with two F2 populations from the cross between Zhachanglong and Zhenzhuai. The first population consisted of 248 random individuals and 404 highly susceptible individuals selected from an F2 population of more than 2,000 individuals and was used to construct a linkage map around the Xa22(t) locus. For the second F2 population, 7,680 plants were examined with simple sequence repeat markers flanking the Xa22(t) locus to identify recombinants useful for fine-genetic mapping. Two large-insert bacterial artificial chromasome (BAC) libraries (from cvs. Teqing and Minghui63) were screened with a marker (R1506) which cosegregated perfectly with Xa22(t) in the first population. Restriction mapping of the resulting BAC clones enabled a physical map of the area to be constructed, and subclones from the BAC clones provided additional restriction fragment length polymorphism probes which could be placed on the fine-structure genetic map using the recombinants from the second mapping population. The Xa22(t) locus was mapped to a ≈ 100-kb interval delimited by the R1506 marker and a subclone from the M3H8 BAC clone.
Ecological Implications from a Molecular Analysis of Phytoplasmas Involved in an Aster Yellows Epidemic in Various Crops in Texas
Authors: Lee I.-M. , Martini M. , Bottner K. D. , Dane R. A. , Black M. C. , Troxclair N.
Published: May 2007

In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.
Barley yellow dwarf virus and Cereal yellow dwarf virus Quantification by Real-Time Polymerase Chain Reaction in Resistant and Susceptible Plants
Authors: Balaji Boovaraghan , Bucholtz Dennis B. , Anderson Joseph M.
Published: May 2007

Reliable detection and quantification of barley and cereal yellow dwarf viruses (YDVs) is a critical component in managing yellow dwarf diseases in small grain cereal crops. The method currently used is enzyme-linked immunosorbent assay (ELISA), using antisera against the coat proteins that are specific for each of the various YDVs. Recently, quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR) has been used to detect bacterial and viral pathogens and to study gene expression. We applied this technique to detect and quantify YDVs using primers specific for Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) coat protein genes because of the higher sensitivity of RT-PCR and the advantage of using a real-time PCR instrument. This Q-RT-PCR was used to detect BYDV and CYDV, and to examine disease development in a resistant wheatgrass, a resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV were detected as early as 2 and 6 h, respectively, in susceptible oat compared with detection by ELISA at 4 and 10 days postinoculation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV RNA in both oat and wheat, which may account for PAV being more prevalent and causing more severe viral disease than CYDV. Q-RT-PCR is reproducible, sensitive, and has the potential to be used for examining yellow dwarf disease and as a rapid diagnostic tool for YDVs.
Bacteriocin-Like Substances from Tomato Race 3 Strains of Xanthomonas campestris pv. vesicatoria
Authors: Tudor-Nelson S. M. , Minsavage G. V. , Stall R. E. , Jones J. B.
Published: May 2007

Tomato race 3 (T3) strains of Xanthomonas campestris pv. vesicatoria are antagonistic in vitro to tomato race 1 (T1) strains of the bacterium. All T1 strains and 11 strains of other X. campestris pathovars tested were inhibited by T3 strains. Sensitivity of tomato race 2 (T2) strains was variable. No strains from other bacterial genera tested were inhibited. Cell-free filtrates from T3 strains were inhibitory to sensitive strains. The inhibitory activity of these filtrates was lost after treatment at temperatures above 80°C and with selected protease enzymes. However, treatment with trypsin or DNase had no effect on their activity. Seven cosmid clones from a genomic library of a T3 strain were selected for their ability to consistently inhibit a sensitive indicator strain in plate assays. Southern hybridization analysis placed these into three bacteriocin (BCN)-producing groups designated BCN-A, BCN-B, and BCN-C. The BCN-like groups could be differentiated by variations in inhibitory spectra and levels of activity in plate assays. Mutations that inactivated expression of each BCN group individually in a wild-type T3 strain had inhibitory activity confirming that multiple BCNs are present in the T3 strain. T3 strains were inhibitory to a sensitive indicator strain in tomato leaf tissue, but this effect was observed only when T3 strains were applied in advance of the sensitive strain. BCN-A was the major BCN-like substance involved in the suppression of the sensitive indicator strain in tomato leaf tissues.
Interactions Between Meloidogyne artiellia, the Cereal and Legume Root-Knot Nematode, and Fusarium oxysporum f. sp. ciceris Race 5 in Chickpea
Authors: Castillo Pablo , Navas-Cortés Juan A. , Gomar-Tinoco David , Di Vito Mauro , Jiménez-Díaz Rafael M.
Published: May 2007

In the Mediterranean Basin, Fusarium oxysporum f. sp. ciceris and the root-knot nematode Meloidogyne artiellia coinfect chickpea. The influence of root infection (after inoculation with 20 nematode eggs and second-stage juveniles per gram of soil) by two M. artiellia populations, from Italy and Syria, on the reaction of chickpea lines and cultivars with partial resistance to Fusarium wilt (CA 252.10.1.OM, CA 255.2.5.0, CPS 1, and PV 61) and with complete resistance to F. oxysporum f. sp. ciceris race 5 (CA 334.20.4, CA 336.14.3.0, ICC 14216 K, and UC 27) was investigated under controlled conditions. In genotypes with partial resistance, infection by M. artiellia significantly increased the severity of Fusarium wilt, irrespective of the fungal inoculum density (3,000 or 30,000 chlamydospores per gram of soil), except in cultivar CPS 1 at the lower fungal inoculum density. In genotypes with complete resistance to Fusarium wilt, infection by M. artiellia overcame the resistance to F. oxysporum f. sp. ciceris race 5 in CA 334.20.4 and CA 336.14.3.0 but not in ICC 14216 K, irrespective of the fungal inoculum density, and overcame the resistance in UC 27 only at the higher inoculum density. Infection by the nematode significantly increased the number of propagules of F. oxysporum f. sp. ciceris race 5 in root tissues of genotypes with complete resistance to Fusarium wilt, compared with roots that were not inoculated with the nematode, irrespective of the fungal inoculum density, except in ICC 14216 K, in which this effect occurred only at the higher inoculum density. Reproduction of an M. artiellia population from Syria in the absence of F. oxysporum f. sp. ciceris race 5 was significantly higher than that of a population from Italy in all tested chick-pea genotypes except ICC 14216 K. However, there was no significant difference between the reproduction rates of the two nematode populations in plants infected with F. oxysporum f. sp. ciceris race 5, irrespective of the fungal inoculum density and the reaction of the genotypes to the fungus.
Molecular Differentiation and Detection of Ginseng-Adapted Isolates of the Root Rot Fungus Cylindrocarpon destructans
Authors: Seifert K. A. , McMullen C. R. , Yee D. , Reeleder R. D. , Dobinson K. F.
Published: May 2007

The soilborne fungus Cylindrocarpon destructans (teleomorph: Neonectria radicicola) causes root rot in a wide range of plant hosts; the disease is of particular concern in ginseng production, and in conifer and fruit tree nurseries. β-Tubulin gene and rRNA gene internal transcribed spacer (ITS) sequence data and pathogenicity assays were used to characterize isolates of C. destructans from ginseng and other hosts. The results of these studies demonstrated a high amount of sequence divergence among strains identified as C. destructans or N. radicicola, suggesting the existence of several phylogenetic species in this complex. Accordingly, we propose that the two varieties of N. radicicola be raised to species status. Certain highly aggressive ginseng isolates from Ontario, Korea, and Japan have identical ITS and β-tubulin sequences, and form a monophyletic clade (designated “clade a”); these strains are identified as C. destructans f. sp. panacis. Other ginseng strains clustered in monophyletic groups with strains from angiosperm and conifers. A subtractive hybridization method was used to isolate genomic DNA sequences with diagnostic potential from the aggressive C. destructans Ontario ginseng isolate 1640. One of these sequences was similar to the rRNA gene intergenic spacer from a Fusarium oxysporum isolate from Pinus ponderosa, and hybridized to DNA from F. oxysporum and all C. destructans isolates tested. Primers were designed that could be used to amplify this sequence specifically from the highly aggressive, ginsengadapted C. destructans isolates from Ontario and Korea and other members of clade a.
Nontoxigenic Strains of Pseudomonas syringae pv. phaseolicola Are a Main Cause of Halo Blight of Beans in Spain and Escape Current Detection Methods
Authors: Rico Arantza , López Ruth , Asensio Carmen , Aizpún M. Teresa , Asensio-S.-Manzanera M. Carmen , Murillo Jesús
Published: May 2007

From a collection of 152 pseudomonads isolated from diseased beans in Spain, 138 (91%) of the strains were identified as Pseudomonas syringae pv. phaseolicola and the rest as P. syringae pv. syringae. The P. syringae pv. phaseolicola strains produced typical water-soaked lesions on bean pods, although 95 of them did not produce phaseolotoxin in vitro. Ninety-four of these isolates did not produce the expected 0.5-kb product after polymerase chain reaction (PCR) amplification using primers specific for open reading frame (ORF) 6 of the phaseolotoxin (tox) gene cluster and did not contain DNA homologous to ORF 6 in Southern hybridization experiments. To our knowledge, this is the first report of the widespread occurrence in the field of strains of P. syringae pv. phaseolicola lacking the tox cluster, which contrasts sharply with the general belief that Tox+ isolates are the only ones with epidemiological importance. Additionally, the tox- isolates were not specifically detected by a commercial polyclonal antisera in an enzyme-linked immunosorbent assay. Accordingly, it is possible that the certification of seed lots as free of the pathogen cannot be reliably done in Spain, or in any other country where tox- strains might occur frequently, using current PCR or serological protocols. The amplification of three avirulence genes by PCR allowed us to make predictions of the P. syringae pv. phaseolicola race structure, as confirmed by plant assays. Six races (races 1, 2, 5, 6, 7, and 9) were identified, with race 7 being the most prevalent (46.1%) followed by races 6 (21.3%) and 1 (9.0%). All the tox- isolates contained gene avrPphF, typical of races 1, 5, 7, and 9.
An Excised-Leaf Inoculation Technique for Evaluating Host-Pathogen Interactions and Quantitative Resistance of Bermudagrass Genotypes to Dematiaceous Hyphomycetes
Authors: Pratt R. G.
Published: May 2007

Excised leaves of bermudagrass were inoculated with mycelium of isolates of Bipolaris, Exserohilum, Curvularia, and Drechslera spp. in water agar plates to evaluate differences in susceptibility of leaf tissue, virulence of pathogens, and quantitative resistance of bermudagrass genotypes. Isolates of nine species of pathogens induced similar symptoms of light- to dark-brown necrosis and bordering chlorosis in excised leaves that were not distinct for individual species or genera. Severity of symptoms induced by most isolates increased progressively from younger to older leaves. Within and across leaf positions, numerous significant differences in virulence of isolates within fungal species and between species were observed. Among 40 randomly selected bermudagrass genotypes, a continuous quantitative gradient was observed for mean scores of disease severity in excised leaves inoculated with E. rostratum. Numerous significant differences were observed within this gradient, and severity of symptoms in the most susceptible genotypes was approximately double that in the most resistant. When intact foliage of genotypes from the resistant and susceptible extremes of the gradient was inoculated with spores of E. rostratum, corresponding differences in severity of symptoms and significant (P = 0.05) correlations between results with excised leaves and intact foliage were observed. However, the range of differences in disease severity between genotypes was more narrow in intact foliage than in excised leaves. Results indicate that the excised leaf inoculation technique can be used to evaluate the relative resistance of bermudagrass genotypes to E. rostratum for use in programs to breed for quantitative host resistance.
Moisture Sources in Relation to Conidial Dissemination and Infection by Cladosporium carpophilum Within Peach Canopies
Authors: Lan Z. , Scherm H.
Published: May 2007

Cladosporium carpophilum, the causal agent of peach scab, overwinters in lesions on 1-year-old twigs, from which conidia infect the developing fruit during spring and early summer. Twig lesions constitute the sole source of initial inoculum; therefore, the mode of dissemination of conidia from such lesions to the fruit is of considerable interest. In a 4-year study, we determined the relative importance of air- versus water-borne conidia and their interaction with different fruit wetness sources (splash, twig runoff, and dew) in a peach orchard with areas that had been treated or not treated with fungicide the previous year. The rareness of scab twig lesions in the previously sprayed trees implied that fruit infection in these trees would occur primarily by airborne conidia from unsprayed trees nearby (located within the same tree row or the adjacent row). In the unsprayed areas, additional infections could occur by short-distance waterborne dissemination of conidia from locally abundant twig lesions via splashing or runoff. Beginning at calyx fall, individual fruit were protected from splash by rain shields, protected from runoff by cotton wicks placed proximal to the peduncle, or left untreated. Rain shields were adjustable, allowing rain or dew to be excluded selectively. Various combinations of the shield and wick treatments were implemented in the previously sprayed and unsprayed areas, and statistical comparison of fruit scab severity between individual treatments by linear contrasts allowed us to untangle the relative contributions of the various sources of inoculum and fruit wetness. Results showed that aerial dissemination of conidia contributed little to fruit scab development, even in the presence of fruit surface wetness caused by splashing, runoff, or dew. In contrast, waterborne conidia contributed considerably and significantly (P < 0.0001) to disease development. This was due primarily to the importance of splash in disseminating conidia from twig lesions to the fruit, given that exclusion of splashing via rain shields decreased disease severity by >90%. Runoff water from the twig to the fruit via the peduncle also contributed to scab development, as evidenced by the fact that exclusion of runoff by cotton wicks reduced disease severity by 31.6 to 44.9%; however, this effect was not always statistically significant. The exclusion of dew did not reduce scab severity (P > 0.4), suggesting that it played a limited role in infection in the presence of other fruit wetness sources.
Genetic Diversity and Pathogenic Variability Among Isolates of Colletotrichum Species from Strawberry
Authors: Denoyes-Rothan Béatrice , Guérin Guy , Délye Christophe , Smith Barbara , Minz Dror , Maymon Marcel , Freeman Stanley
Published: May 2007

Ninety-five isolates of Colletotrichum including 81 isolates of C. acutatum (62 from strawberry) and 14 isolates of C. gloeosporioides (13 from strawberry) were characterized by various molecular methods and pathogenicity tests. Results based on random amplified polymorphic DNA (RAPD) polymorphism and internal transcribed spacer (ITS) 2 sequence data provided clear genetic evidence of two subgroups in C. acutatum. The first subgroup, characterized as CA-clonal, included only isolates from strawberry and exhibited identical RAPD patterns and nearly identical ITS2 sequence analysis. A larger genetic group, CA-variable, included isolates from various hosts and exhibited variable RAPD patterns and divergent ITS2 sequence analysis. Within the C. acutatum population isolated from strawberry, the CA-clonal group is prevalent in Europe (54 isolates of 62). A subset of European C. acutatum isolates isolated from strawberry and representing the CA-clonal and CA-variable groups was assigned to two pathogenicity groups. No correlation could be drawn between genetic and pathogenicity groups. On the basis of molecular data, it is proposed that the CA-clonal subgroup contains closely related, highly virulent C. acutatum isolates that may have developed host specialization to strawberry. C. gloeosporioides isolates from Europe, which were rarely observed were either slightly or nonpathogenic on strawberry. The absence of correlation between genetic polymorphism and geographical origin in Colletotrichum spp. suggests a worldwide dissemination of isolates, probably through international plant exchanges.
Variations in the Phytophthora infestans Population in Nepal as Revealed by Nuclear and Mitochondrial DNA Polymorphisms
Authors: Ghimire S. R. , Hyde K. D. , Hodgkiss I. J. , Shaw D. S. , Liew E. C. Y.
Published: May 2007

Phytophthora infestans isolates collected from potato and tomato crops from various parts of Nepal during the 1999 and 2000 crop seasons were characterized for nuclear and mitochondrial DNA polymorphisms using restriction fragment length polymorphism markers. The nuclear DNA probe RG57 detected 11 multilocus genotypes among 280 isolates. Three genotypes were detected 21 times or more, constituting 94% of the total population, whereas frequencies of other genotypes ranged from 0.004 to 0.014. The overall genotypic diversity as estimated by the Gleason index was 1.78. Most of the overall diversity was present at the highest level (i.e., interregional, 46%), indicating limited gene flow among regions. Cluster analysis of multilocus genotypes derived from RG57 and mating type data for Nepalese isolates and representative isolates worldwide showed Nepalese isolates grouping into four clusters. Characterization of 67 isolates for mitochondrial DNA polymorphisms revealed the presence of two mt-haplotypes, Ia and Ib with the proportions of 0.88 and 0.12, respectively. Polymorphisms in nuclear and mitochondrial DNA revealed a moderate level of diversity in this population. Genotype NP3 had an identical RG57 fingerprint to US1 and had mt-haplotype Ib, confirming the presence of an old population in Nepal. Most of the genotypes had a different RG57 fingerprint than that of US1 and mt-haplotype Ia, the common characteristics of new populations. The presence of a new population at high proportions in Nepal was consistent with the global trend of mt-haplotype distribution, and suggests the displacement of old populations. This study indicates at least three possible introductions of P. infestans to Nepal.
Multiplex Nested Reverse Transcription-Polymerase Chain Reaction in a Single Tube for Sensitive and Simultaneous Detection of Four RNA Viruses and Pseudomonas savastanoi pv. savastanoi in Olive Trees
Authors: Bertolini Edson , Olmos Antonio , López María M. , Cambra Mariano
Published: May 2007

A multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) in a single closed tube was developed for the simultaneous detection of four RNA viruses: Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ringspot virus, and Arabis mosaic virus, and the bacterium Pseudomonas savastanoi pv. savastanoi. The method enabled, for the first time, the sensitive and simultaneous detection of RNA and DNA targets from plant viruses and a bacterium, saving time, decreasing risks of contamination, and reducing costs compared with conventional monospecific nested amplifications. The method was successfully coupled with colorimetric detection of amplicons using specific oligoprobes to simplify routine detection. Two hundred forty-five olive trees from 15 different cultivars were analyzed by multiplex RT-nested PCR coupled with colorimetric detection. Multiplex nested RT-PCR for viral detection increased the identification of positive trees by 8.1%. An uneven distribution of the viruses was observed in the infected trees. The bacterium was detected in 28.7% of the analyzed trees by the developed multiplex nested method and by a nested PCR previously developed. This powerful methodology could be applied to other models for the detection of several pathogens in a single assay.
Variability in Virulence Among Asexual Progenies of Phytophthora infestans
Authors: Samen F. M. Abu-El , Secor G. A. , Gudmestad N. C.
Published: May 2007

One hundred two single zoospore isolates of Phytophthora infestans, derived asexually from four parental isolates of US-8 genotype and one isolate of US-1 genotype, were characterized for their virulence phenotypes to determine changes in virulence during asexual reproduction. Potato differentials, each containing a major gene for resistance to P. infestans (R1 to R11), were used to characterize the virulence patterns. Isolates were also characterized for mating type, glucose-6-phosphate isomerase (Gpi) banding pattern, and DNA fingerprints using probe RG57 to determine any genotypic changes in the single zoospore isolates. A subset of these single zoospore isolates was tested for response to mefenoxam to determine any shifts in sensitivity. Results showed that single zoospore isolates derived from parent PI-1 (US-8, 11 isolates) were identical to their parental virulence. Isolates derived from parent PI-191 (US-8, 29 isolates) showed some differences in virulence, mainly toward R8 and R9. Isolates derived from parent PI-126 (US-8, 14 isolates) demonstrated a higher level of virulence diversity. Isolates derived from parents PI-52 (US-1, 28 isolates) and PI-105 (US-8, 20 isolates) showed the highest level of virulence variability among the single zoospore isolates. Mating type, Gpi banding pattern, and DNA fingerprints for the single zoospore isolates were, in most cases, identical to the parental isolates. Single zoospore isolates showed different levels of sensitivity to mefenoxam. Virulence and other genetic changes during asexual reproduction are likely to play a major role in changing the race structure of P. infestans populations. This continuous change in the race structure is a serious problem and now poses a new challenge for utilization of race-specific resistance to manage late blight of potato.
Role of Host Sensitivity to Ptr ToxA in Development of Tan Spot of Wheat
Authors: Friesen T. L. , Ali S. , Kianian S. , Francl L. J. , Rasmussen J. B.
Published: May 2007

Pyrenophora tritici-repentis race 2 produces Ptr ToxA, a host-selective toxin previously described as a pathogenicity factor for tan spot on wheat. The objective of this research was to evaluate the role of host sensitivity to toxin, conditioned by a single dominant gene on chromosome 5BL, in the disease development by race 2. An F2-derived F6 recombinant inbred population of 108 wheat lines, produced from crosses of toxin-sensitive, disease-susceptible cv. Kulm with the toxin-insensitive, disease-resistant cv. Erik segregated 1:1 for toxin reaction. However, the population was skewed toward resistance to race 2 of the fungus. Toxin reaction accounted for 24.4% of the genetic variance for disease. Heritability estimates suggested the presence of four to five genes that influence disease reaction in the population. Toxin-insensitive mutants, previously derived Kulm, were susceptible to race 2, although disease developed more slowly on the mutants than it did on the wild-type Kulm. The data indicate that sensitivity to Ptr ToxA influences disease severity in some host genotypes without defining susceptibility.
Defense Gene Expression Analysis of Arabidopsis thaliana Parasitized by Orobanche ramosa
Authors: Dos Santos C. Vieira , Letousey P. , Delavault P. , Thalouarn P.
Published: May 2007

The infection of Arabidopsis thaliana roots with the obligate parasite Orobanche ramosa represents a useful model for a study of the molecular events involved in the host plant response to a parasitic plant attack. To avoid analysis problems due to the subterranean development of O. ramosa, we developed two in vitro co-culture systems: O. ramosa seedlings infesting Arabidopsis plantlet roots and callus tissues. We were then able to investigate the expression patterns of some host plant genes selected among genes known to be involved in metabolic pathways and resistance mechanisms activated during several plant-pathogen interactions including ethylene, isoprenoid, phenylpropanoid, and jasmonate biosynthesis pathways, oxidative stress responses, and pathogenesis-related proteins. Molecular analyses were carried out using polymerase chain reaction amplification methods allowing semiquantitative evaluation of transcript accumulation during early (first hours) and late (15 days) stages of infestation, in whole roots or parts close to the parasite attachment site. In A. thaliana, O. ramosa induced most of the general response signaling pathways in a transient manner even before its attachment to A. thaliana roots. However, no salicylic acid-dependent defense is observed because no activation of systemic acquired resistance markers is detectable, whereas genes, co-regulated by jasmonate and ethylene, do display enhanced expression.
Spatial and Temporal Analyses of Citrus Sudden Death as a Tool to Generate Hypotheses Concerning Its Etiology
Authors: Bassanezi Renato B. , Bergamin Filho Armando , Amorim Lilian , Gimenes-Fernandes Nelson , Gottwald Tim R. , Bové Joseph M.
Published: May 2007

Citrus sudden death (CSD), a new disease of unknown etiology that affects sweet orange grafted on Rangpur lime, was visually monitored for 14 months in 41 groves in Brazil. Ordinary runs analysis of CSD-symptomatic trees indicated a departure from randomness of symptomatic trees status among immediately adjacent trees mainly within rows. The binomial index of dispersion (D) and the intraclass correlation (k) for various quadrat sizes suggested aggregation of CSD-symptomatic trees for almost all plots within the quadrat sizes tested. Estimated parameters of the binary form of Taylor's power law provided an overall measure of aggregation of CSD-symptomatic trees for all quadrat sizes tested. Aggregation in each plot was dependent on disease incidence. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to three lag distances; however, significant lag positions discontinuous from main proximity patterns were rare, indicating a lack of spatial association among discrete foci. Some asymmetry was also detected for some spatial autocorrelation proximity patterns, indicating that within-row versus across-row distributions are not necessarily equivalent. These results were interpreted to mean that the cause of the disease was most likely biotic and its dissemination was common within a local area of influence that extended to approximately six trees in all directions, including adjacent trees. Where asymmetry was indicated, this area of influence was somewhat elliptical. Longer-distance patterns were not detected within the confines of the plot sizes tested. Annual rates of CSD progress based on the Gompertz model ranged from 0.37 to 2.02. Numerous similarities were found between the spatial patterns of CSD and Citrus tristeza virus (CTV) described in the literature, both in the presence of the aphid vector, Toxoptera citricida. CSD differs from CTV in that symptoms occur in sweet orange grafted on Rangpur lime. Based on the symptoms of CSD and on its spatial and temporal patterns, our hypothesis is that CSD may be caused by a similar but undescribed pathogen such as a virus and probably vectored by insects such as aphids by similar spatial processes to those affecting CTV.
Role of Blossoms in Watermelon Seed Infestation byAcidovorax avenaesubsp.citrulli
Authors: Walcott R. R. , Gitaitis R. D. , Castro A. C.
Published: May 2007

The role of watermelon blossom inoculation in seed infestation by Acidovorax avenae subsp. citrulli was investigated. Approximately 98% (84/87) of fruit developed from blossoms inoculated with 1 × 107or 1 × 109CFU of A. avenae subsp. citrulli per blossom were asymptomatic. Using immunomagnetic separation and the polymerase chain reaction, A. avenae subsp. citrulli was detected in 44% of the seed lots assayed, despite the lack of fruit symptoms. Furthermore, viable colonies were recovered from 31% of the seed lots. Of these lots, 27% also yielded seedlings expressing bacterial fruit blotch symptoms when planted under conditions of 30°C and 90% relative humidity. A. avenae subsp. citrulli was detected and recovered from the pulp of 33 and 19%, respectively, of symptomless fruit whose blossoms were inoculated with A. avenae subsp. citrulli. The ability to penetrate watermelon flowers was not unique to A. avenae subsp. citrulli, because blossoms inoculated with Pantoea ananatis also resulted in infested seed and pulp. The data indicate that watermelon blossoms are a potential site of ingress for fruit and seed infestation by A. avenae subsp. citrulli.
Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins
Authors: Vivanco Jorge M. , Tumer Nilgun E.
Published: May 2007

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP.
Genetic Diversity of Xanthomonas campestris pv. vitians, the Causal Agent of Bacterial Leafspot of Lettuce
Authors: Barak Jeri D. , Gilbertson Robert L.
Published: May 2007

Bacterial leafspot of lettuce (BLS), caused by Xanthomonas campes-tris pv. vitians, has become more prevalent in many lettuce-growing areas of the world over the past decade. To gain insight into the nature of these outbreaks, the genetic variation in X. campestris pv. vitians strains from different geographical locations was examined. All strains were first tested for pathogenicity on lettuce plants, and then genetic diversity was assessed using (i) gas-chromatographic analysis of bacterial fatty acids, (ii) polymerase chain reaction analysis of repetitive DNA sequences (rep-PCR), (iii) DNA sequence analysis of the internal transcribed spacer region 1 (ITS1) of the ribosomal RNA, (iv) restriction fragment length polymorphism (RFLP) analysis of total genomic DNA with a repetitive DNA probe, and (v) detection and partial characterization of plasmid DNA. Fatty acid analysis identified all pathogenic strains as X. campestris, but did not consistently identify all the strains as X. campestris pv. vitians. The rep-PCR fingerprints and ITS1 sequences of all pathogenic X. campestris pv. vitians strains examined were identical, and distinct from those of the other X. campestris pathovars. Thus, these characteristics did not reveal genetic diversity among X. campestris pv. vitians strains, but did allow for differentiation of X. campestris pathovars. Genetic diversity among X. campestris pv. vitians strains was revealed by RFLP analysis with a repetitive DNA probe and by characterization of plasmid DNA. This diversity was greatest among strains from different geographical regions, although diversity among strains from the same location also was detected. The results of this study suggest that these X. campestris pv. vitians strains are not clonal, but comprise a relatively homogeneous group.