Showing papers in "Phytopathology in 2001"

Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer

Abstract: Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 × 106 conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the...

Biological Control of the Root-Knot Nematode Meloidogyne javanica by Trichoderma harzianum.

Abstract: The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2...

Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew.

Abstract: Ishii, H., Fraaije, B. A., Sugiyama, T., Noguchi, K., Nishimura, K., Takeda, T., Amano, T., and Hollomon, D. W. 2001. Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology 91:1166-1171. Between 1998 and 1999, control failure of powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) by the strobilurin fungicides azoxystrobin and kresoxim-methyl was observed in cucumber-growing areas of Japan. Results from inoculation tests carried out on intact cucumber plants and leaf disks clearly showed the distribution of pathogen isolates highly resistant to azoxystrobin and kresoximmethyl. Fragments of the fungicide-targeted mitochondrial cytochrome b gene were polymerase chain reaction amplified from total pathogen DNA and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochrome b gene, resulting in substitution of glycine by alanine at position 143, was found in resistant isolates of downy mildew. This substitution in cytochrome b seemed to result in high resistance to strobilurins in this pathogen. The same mutation was found in some but not all resistant isolates of powdery mildew. This study suggests that a mutation at position 143 in the target-encoding gene, resulting in an amino acid substitution, was probably a major cause of the rapid development of high strobilurin resistance in these two pathogens.

Combining biocontrol agents to reduce the variability of biological control.

Abstract: Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves. The ...

Antimicrobial Activity of Culture Filtrate of Bacillus amyloliquefaciens RC-2 Isolated from Mulberry Leaves.

Abstract: A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in v...

Phylogenetic analysis of cercospora and mycosphaerella based on the internal transcribed spacer region of ribosomal DNA.

Abstract: Most of the 3,000 named species in the genus Cercospora have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few. Mycosphaerella is an extremely large and important genus of plant pathogens, with more than 1,800 named species and at least 43 associated anamorph genera. The goal of this research was to perform a large-scale phylogenetic analysis to test hypotheses about the past evolutionary history of Cercospora and Mycosphaerella. Based on the phylogenetic analysis of internal transcribed spacer (ITS) sequence data (ITS1, 5.8S rRNA gene, ITS2), the genus Mycosphaerella is monophyletic. In contrast, many anamorph genera within Mycosphaerella were polyphyletic and were not useful for grouping species. One exception was Cercospora, which formed a highly supported monophyletic group. Most Cercospora species from cereal crops formed a subgroup within the main Cercospora cluster. Only species within the Cercospora cluster produced the toxin cercosporin, suggesting...

Suppression of specific apple root pathogens by Brassica napus seed meal amendment regardless of glucosinolate content.

Abstract: The impact of Brassica napus seed meal on the microbial complex that incites apple replant disease was evaluated in greenhouse trials. Regardless of glucosinolate content, seed meal amendment at a rate of 0.1% (vol/vol) significantly enhanced growth of apple and suppressed apple root infection by Rhizoctonia spp. and Pratylenchus penetrans. High glucosinolate B. napus cv. Dwarf Essex seed meal amendments did not consistently suppress soil populations of Pythium spp. or apple root infection by this pathogen. Application of a low glucosinolate containing B. napus seed meal at a rate of 1.0% (vol/vol) resulted in a significant increase in recovery of Pythium spp. from apple roots, and a corresponding reduction in apple seedling root biomass. When applied at lower rates, B. napus seed meal amendments enhanced populations of fluorescent Pseudomonas spp., but these bacteria were not recovered from soils amended with seed meal at a rate of 2% (vol/vol). Seed meal amendments resulted in increased soil populations of total bacteria and actinomycetes. B. napus cv. Dwarf Essex seed meal amendments were phytotoxic to apple when applied at a rate of 2% (vol/vol), and phytotoxicity was not diminished when planting was delayed for as long as 12 weeks after application. These findings suggest that B. napus seed meal amendments can be a useful tool in the management of apple replant disease and, in the case of Rhizoctonia spp., that disease control operates through mechanisms other than production of glucosinolate hydrolysis products.

A Compromised Mlo Pathway Affects the Response of Barley to the Necrotrophic Fungus Bipolaris sorokiniana (Teleomorph: Cochliobolus sativus) and Its Toxins.

Abstract: In search of new durable disease resistance traits in barley to control leaf spot blotch disease caused by the necrotrophic fungus Bipolaris sorokiniana (teleomorph: Cochliobolus sativus), we developed macroscopic and microscopic scales to judge spot blotch disease development on barley. Infection of barley was associated with cell wall penetration and accumulation of hydrogen peroxide. The latter appeared to take place in cell wall swellings under fungal penetration attempts as well as during cell death provoked by the necrotrophic pathogen. Additionally, we tested the influence of a compromised Mlo pathway that confers broad resistance against powdery mildew fungus (Blumeria graminis f. sp. hordei). Powdery mildew-resistant genotypes with mutations at the Mlo locus (mlo genotypes) showed a higher sensitivity to infiltration of toxic culture filtrate of Bipolaris sorokiniana as compared with wild-type barley. Mutants defective in Ror, a gene required for mlo-specified powdery mildew resistance, were also more sensitive to Bipolaris sorokiniana toxins than wild-type barley but showed less symptoms than mlo5 parents. Fungal culture filtrates induced an H2O2 burst in all mutants, whereas wild-type (Mlo) barley was less sensitive. The results support the hypothesis that the barley Mlo gene product functions as a suppresser of cell death. Therefore, a compromised Mlo pathway is effective for control of biotrophic powdery mildew fungus but not for necrotrophic Bipolaris sorokiniana. We discuss the problem of finding resistance traits that are effective against both biotrophic and necrotrophic pathogens with emphasis on the role of the anti-oxidative system of plant cells.

Host Specialization in the Charcoal Rot Fungus, Macrophomina phaseolina.

Abstract: To investigate host specialization in Macrophomina phaseolina, the fungus was isolated from soybean, corn, sorghum, and cotton root tissue and soil from fields cropped continuously to these species for 15 years in St. Joseph, LA. Chlorate phenotype of each isolate was determined after growing on a minimal medium containing 120 mM potassium chlorate. Consistent differences in chlorate sensitivity were detected among isolates from different hosts and from soil versus root. To further explore genetic differentiation among fungal isolates from each host, these isolates were examined by restriction fragment length polymorphism and random amplified polymorphic DNA (RAPD) analysis. No variations were observed among isolates in restriction patterns of DNA fragments amplified by polymerase chain reaction covering the internal transcribed spacer region, 5.8S rRNA and part of 25S rRNA, suggesting that M. phaseolina constitutes a single species. Ten random primers were used to amplify the total DNA of 45 isolates, and banding patterns resulting from RAPD analysis were compared with the neighbor-joining method. Isolates from a given host were genetically similar to each other but distinctly different from those from other hosts. Chlorate-sensitive isolates were distinct from chlorate-resistant isolates within a given host. In greenhouse tests, soybean, sorghum, corn, and cotton were grown separately in soil infested with individual isolates of M. phaseolina that were chosen based on their host of origin and chlorate phenotype. Root colonization and plant weight were measured after harvesting. More colonization of corn roots occurred when corn was grown in soil containing corn isolates compared with isolates from other hosts. However, there was no host specialization in isolates from soybean, sorghum, or cotton. More root colonization in soybean occurred with chlorate-sensitive than with chlorate-resistant isolates.

The citrus canker epidemic in Florida: the scientific basis of regulatory eradication policy for an invasive species.

Abstract: Increasing international travel and trade have rendered US borders much more porous and dramatically increased the risk of introductions of invasive plant pests into agricultural crops (1) Nationally, the responsibility for safeguarding agriculture falls to the US Department of Agriculture, Animal and Plant Health Inspection Service, Plant Protection and Quarantine (USDA, APHIS, PPQ) However, the current system for protecting agricultural industries has broken down allowing for an unprecedented number of introductions of exotic pests, including plant pathogens Such introductions threaten crops and can hinder national and international agricultural markets and trade In 1999, President Clinton announced an initiative for invasive species to address these issues and introduced an Executive Order on Invasive Species (10) The order is intended to coordinate and enhance federal government efforts to prevent introduction of invasive species and to provide for their control The Executive Order calls for the appointment of a council, chaired by the Secretaries of Agriculture, Commerce, and the Interior, and composed of key federal agencies charged with developing an invasive species management plan In response, the National Plant Board initiated a “stakeholder” review of the USDA, APHIS, PPQ safeguarding system in order to identify means to improve pest exclusion and detection of and response to invasive pest introductions (1) Enter citrus canker Currently in Florida, one such invasive species is Xanthomonas axonopodis pv citri (Xac), a bacterial plant pathogen that causes Asiatic citrus canker Citrus canker is an introduced plant disease that has received considerable press attention, has produced far-reaching political and socioeconomic impact in Florida, and has implications for national and international trade (3,4) Although Xac is mostly a leaf and fruit-blemishing pathogen, the disease triggers immediate quarantines of areas with outbreaks in Florida, disrupting movement of fresh fruit (3,11) The importance of citrus canker as a major invasive disease and the political forces that govern attempts to eradicate the disease were recently exemplified by the visit of the US Secretary of Agriculture to the Homestead/Florida City area south of Miami on 22 March 2000 to view a major outbreak of the disease first hand Secretary Glickman declared the South Florida canker outbreak area in Dade and Broward Counties a federal disaster area, opening the door for disaster relief funds and for US Marshals to assist crews in expediting the cutting and removal of trees from homeowners’ residences in metropolitan Miami (26) South Florida was one stop on a national tour conducted by the Secretary of Agriculture to highlight non-native, invasive pests and diseases that threaten US agriculture and natural resources (20) Citrus canker has a long history The disease was first found spread throughout the southeastern United States on imported seedlings from Japan in the early 1910s and was declared eradicated from Florida and the adjacent states in 1933 Citrus canker was discovered again in Manatee County Florida, south of Tampa Bay in the late 1980s, and was thought to be eradicated by 1994 Two years thereafter, the disease re-emerged in the same area on the west coast of Florida where the 1980s outbreak had occurred In the meantime, a new and separate infestation of citrus canker was discovered in urban Miami in 1995, that was estimated to have been introduced in 1992 or 1993 (17) Since that time the disease has spread from an initial area of 14 square miles near the Miami airport, to over 1,005 square miles in the metropolitan area plus an additional 260 square miles of urban and commercial citrus areas throughout the State Genomic analysis of bacterial isolates from both time periods indicates that the latest Manatee County outbreak is a hold over from the 1980s outbreak that escaped the eradication program However, the majority of current outbreaks of citrus canker are caused by the same genotype of Xac as in the Miami area Thus, human-assisted movement of the bacterium from that source appears to have occurred several times In early 2000, a third genomically distinct isolate of Asiatic citrus canker with an attenuated host range was identified in Palm Beach County on the east coast of Florida Thus, there are presently at least three Xac genotypes that have been introduced into Florida in the recent two decades Although this bacterial disease is mostly a leaf and fruit-spotting malady, it results in immediate quarantines, disrupting national and international trade (3,11) Although citrus canker can cause debilitation of trees and losses in fruit quality and yield, it is because of its socioeconomic and political impact that the disease is so devastating If Xac should become endemic in Florida, it will effectively result in a prohibition of interstate commerce of fresh citrus fruit, which comprises approximately 20% of the State’s $8 billion commercial citrus industry (23) In addition, some cultivars of highly susceptible citrus species, such as grapefruit (Citrus paradisi), will have reduced economic viability due to the requirement for multiple bactericidal sprays per year to maintain yields and quality The Florida citrus industry is concentrated predominantly in the southern half of the State, in close proximity to rapidly expanding urban population centers Because the outbreaks originated in urban areas, response to citrus canker, therefore, affects not just the citrus industry Hundreds of thousands of urban homeowners have citrus trees as ornamentals and for dooryard fruit production and have had or will have their trees destroyed The Florida Department of Agriculture and Consumer Services, Division of Plant Industry (DPI) and USDA, APHIS responded to the 1995 Corresponding author: T R Gottwald; E-mail address: TGottwald@ushrlarsusdagov

Genetic Diversity of phlD from 2,4-Diacetylphloroglucinol-Producing Fluorescent Pseudomonas spp.

Abstract: Fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) have biocontrol activity against damping-off, root rot, and wilt diseases caused by soilborne fungal pathogens, and play a key role in the natural suppression of Gaeumannomyces graminis var. tritici, known as take-all decline. Diversity within phlD, an essential gene in the biosynthesis of 2,4-DAPG, was studied by restriction fragment length polymorphism (RFLP) analysis of 123 2,4-DAPG-producing isolates from six states in the United States and six other locations worldwide. Clusters defined by RFLP analysis of phlD correlated closely with clusters defined previously by BOX-polymerase chain reaction (PCR) genomic fingerprinting, indicating the usefulness of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers. Genotypes defined by RFLP analysis of phlD were conserved among isolates from the same site and cropping history. Random amplified polymorphic DNA analyses of genomic DNA revealed a higher degree of polymorphism than RFLP and BOX-PCR analyses. Genotypic diversity in a subset of 30 strains representing all the phlD RFLP groups did not correlate with production in vitro of monoacetylphloroglucinol, 2,4-DAPG, or total phloroglucinol compounds. Twenty-seven of the 30 representative strains lacked pyrrolnitrin and pyoluteorin biosynthetic genes as determined by the use of specific primers and probes.

A rapid polymerase chain reaction-based assay characterizing rhizosphere populations of 2,4-diacetylphloroglucinol-producing bacteria.

Abstract: Pseudomonas species that produce 2,4-diacetylphloroglucinol (2,4-DAPG) play a significant role in the suppression of fungal root pathogens in the rhizosphere of crop plants. To characterize the abundance and diversity of these functionally important bacterial populations, we developed a rapid polymerase chain reaction (PCR)-based assay targeting phlD, an essential gene in the phloroglucinol biosynthetic pathway. The phlDgene is predicted to encode a polyketide synthase that synthesizes mono-acetylphloroglucinol, the immediate precursor to 2,4-DAPG. A major portion of the phlD open reading frame was cloned and sequenced from five genotypically distinct strains, and the sequences were screened for conserved regions that could be used as gene-specific priming sites for PCR amplification. Several new phlD-specific primers were designed and evaluated. Using the primers B2BF and BPR4, we developed a PCR-based assay that was robust enough to amplify the target gene from a diverse set of 2,4-DAPG producers and sensitive enough to detect as few as log 2.4 cells per sample when combined with enrichment from a selective medium. Restriction fragment length polymorphism analysis of the amplified phlD sequence allows for the direct determination of the genotype of the most abundant 2,4-DAPG producers in a sample. The method described was useful for characterizing both inoculant and indigenous phlD(+) pseudomonads inhabiting the rhizosphere of crop plants. The ability to rapidly characterize populations of 2,4-DAPG-producers will greatly enhance our understanding of their role in the suppression of root diseases.

Bacterial populations associated with rice seed in the tropical environment.

Abstract: During the 1995 wet season, harvested rice seed was collected from farmers' fields at different locations in Iloilo, Philippines. Bacterial isolations from crushed seed yielded 428 isolates. The isolates were characterized by BOX-polymerase chain reaction fingerprinting of total genomic DNA and represented 151 fingerprint types (FPT). Most FPTs were found on a single occasion, although matching fingerprints for isolates from different samples also were found. Identifications were made by cellular fatty acid methyl ester analysis and additional use of Biolog GN/GP MicroPlates and API 20E/50CHE systems. The predominant bacteria were Enterobacteriaceae (25%), Bacillus spp. (22%), and Pseudomonas spp. (14%). Other bacteria regularly present were identified as Xanthomonas spp., Cellulomonas flavigena, and Clavibacter michiganense. Of the total number of isolated bacteria, 4% exhibited in vitro antifungal activity against Rhizoctonia solani or Pyricularia grisea. Two percent of isolates were pathogens identified as Burkholderia glumae and Burkholderia gladioli. Five percent of isolates induced sheath necrosis on only 50 to 90% of inoculated plants and were related to Bacillus pumilus, Paenibacillus spp., Pseudomonas spp., and Pantoea spp.

The 14alpha-Demethylasse(CYP51A1) Gene is Overexpressed in Venturia inaequalis Strains Resistant to Myclobutanil.

Abstract: We identified the cytochrome P450 sterol 14alpha-demethylase (CYP51A1) gene from Venturia inaequalis and optional insertions located upstream from CYP51A1 and evaluated their potential role in conferring resistance to the sterol demethylation-inhibitor (DMI) fungicide my-clobutanil. The CYP51A1 gene was completely sequenced from one my-clobutanil sensitive (S) and two myclobutanil-resistant (R) strains. No nucleotide variation was found when the three sequences were aligned. Allele-specific polymerase chain reaction (PCR) analysis indicated that a previously described single base pair mutation that correlated with resistance to DMI fungicides in strains of other filamentous fungi was absent in 19 S and 32 R strains of V. inaequalis from Michigan and elsewhere. The sequencing results and PCR analyses suggest that resistance in these strains was not due to a mutation in the sterol demethylase target site for DMI fungicides. Expression of CYP51A1 was determined for strains from an orchard that had never been sprayed with DMI fungicides (baseline orchard), and the data provided a reference for evaluating the expression of strains collected from a research orchard and from three commercial Michigan apple orchards with a long history of DMI use and a high frequency of R strains. Overexpression of CYP51A1 was significantly higher in 9 of 11 R strains from the research orchard than in S strains from the baseline orchard. The high expression was correlated with the presence of a 553-bp insertion located upstream of CYP51A1. Overexpression of the CYP51A1 gene was also detected in eight of eight, five of nine, and nine of nine R strains from three commercial orchards, but the insertion was not detected in the majority of these strains. The results suggest that overexpression of the target-site CYP51A1 gene is an important mechanism of resistance in some field resistant strains of V. inaequalis, but other mechanisms of resistance also appear to exist.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

Abstract: Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA′-′lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize express...

Rate of Tomato yellow leaf curl virus Translocation in the Circulative Transmission Pathway of its Vector, the Whitefly Bemisia tabaci

Abstract: Whiteflies (Bemisia tabaci, biotype B) were able to transmit Tomato yellow leaf curl virus (TYLCV) 8 h after they were caged with infected tomato plants. The spread of TYLCV during this latent period was followed in organs thought to be involved in the translocation of the virus in B. tabaci. After increasing acquisition access periods (AAPs) on infected tomato plants, the stylets, the head, the midgut, a hemolymph sample, and the salivary glands dissected from individual insects were subjected to polymerase chain reaction (PCR) without any treatment; the presence of TYLCV was assessed with virus-specific primers. TYLCV DNA was first detected in the head of B. tabaci after a 10-min AAP. The virus was present in the midgut after 40 min and was first detected in the hemolymph after 90 min. TYLCV was found in the salivary glands 5.5 h after it was first detected in the hemolymph. Subjecting the insect organs to immunocapture-PCR showed that the virus capsid protein was in the insect organs at the same time as the virus genome, suggesting that at least some TYLCV translocates as virions. Although females are more efficient as vectors than males, TYLCV was detected in the salivary glands of males and of females after approximately the same AAP.

Identification and Origin of Xanthomonas campestris pv. campestris Races and Related Pathovars.

Abstract: One hundred sixty-four isolates of Xanthomonas campestris pv. campestris and other X. campestris pathovars known to infect cruciferous hosts (X. campestris pvs. aberrans, raphani, armoraciae, and incanae) were inoculated onto a differential series of Brassica spp. to determine both pathogenicity to brassicas and race. Of these, 144 isolates were identified as X. campestris pv. campestris and grouped into six races, with races 1 (62%) and 4 (32%) being predominant. Other races were rare. The remaining 20 isolates from brassicas and other cruciferous hosts were either nonpathogenic or very weakly pathogenic on the differential series and could not be race-typed. Five of these isolates, from the ornamental crucifers wallflower (Cheiranthus cheiri), stock (Matthiola incana) and candytuft (Iberis sp.), showed clear evidence of pathovar-like specificity to the hosts of origin. A gene-for-gene model based on the interaction of four avirulence genes in X. campestris pv. campestris races and four matching resistance genes in the differential hosts is proposed. Knowledge of the race structure and worldwide distribution of races is fundamental to the search for sources of resistance and for the establishment of successful resistance breeding programs.

Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum from Strawberry and Other Plants

Abstract: Freeman, S., Horowitz, S., and Sharon, A. 2001. Pathogenic and nonpathogenic lifestyles in Colletotrichum acutatum from strawberry and other plants. Phytopathology 91:986-992. Anthracnose is one of the major fungal diseases of strawberry occurring worldwide. In Israel, the disease is caused primarily by the species Colletotrichum acutatum. The pathogen causes black spot on fruit, root necrosis, and crown rot resulting in mortality of transplants in the field. The host range and specificity of C. acutatum from strawberry was examined on pepper, eggplant, tomato, bean, and strawberry under greenhouse conditions. The fungus was recovered from all plant species over a 3-month period but caused disease symptoms only on strawberry. Epiphytic and endophytic (colonization) fungal growth in the different plant species was confirmed by reisolation from leaf tissues and by polymerase chain reaction (PCR)-specific primer amplification. C. acutatum was also isolated from healthy looking, asymptomatic plants of the weed genera Vicia and Conyza. Isolates that were recovered from the weeds caused disease symptoms on strawberry and were positively identified as C. acutatum by PCR. The habitation of a large number of plant species, including weeds, by C. acutatum suggests that, although it causes disease only on strawberry and anemone in Israel, this fungus can persist on many other plant species. Therefore, plants that are not considered hosts of C. acutatum may serve as a potential inoculum source for strawberry infection and permit survival of the pathogen between seasons.

Endophytic Colonization of Plants by the Biocontrol Agent Rhizobium etli G12 in Relation to Meloidogyne incognita Infection

Abstract: The external and internal colonization of potato and Arabidopsis roots by the biocontrol strain Rhizobium etli G12 containing a plasmidborne trp promoter green fluorescent protein transcriptional fusion, pGT-trp, was studied in the presence and absence of the root-knot nematode Meloidogyne incognita. Plant colonization behavior and biocontrol potential of the marked strain G12(pGT-trp) was not altered compared with the parental strain. Plasmid pGT-trp was stable for more than 80 generations without selection and conferred sufficient fluorescence to detect single bacterial cells in planta. Although bacteria were found over the entire rhizoplane, they preferentially colonized root tips, the emerging lateral roots, and galled tissue caused by Meloidogyne infestation. Internal colonization of potato roots was mainly observed in epidermal cells, especially root hairs. G12(pGT-trp) colonization was also observed in inner Arabidopsis root tissues in areas of vascularization. In the presence of M. incognita, G12(pGT-trp) colonized the interior of nematode galls in high numbers. In some cases, bacterial colonization even extended from the galled tissue into adjacent root tissue. The internally colonized sites in roots were often discontinuous. Fluorescence microscopy of gfp-tagged rhizobacteria was a sensitive and a rapid technique to study external and internal colonization of plant roots by bacteria interacting with nematodes.

The influence of silicon on components of resistance to blast in susceptible, partially resistant, and resistant cultivars of rice.

Abstract: The application of silicon (Si) fertilizers reduces the severity of blast, caused by Magnaporthe grisea, in irrigated and upland rice; however, little research has been conducted to examine the epidemiological and etiological components of this reduction. Four cultivars of rice with differential susceptibilities to race IB-49 of M. grisea were fertilized with three rates of a calcium silicate fertilizer and inoculated with the pathogen to test the effects of Si on the following components of resistance to blast: incubation period, latent period, infection efficiency, lesion size, rate of lesion expansion, sporulation per lesion, and diseased leaf area. For each cultivar, the incubation period was lengthened by increased rates of Si, and the numbers of sporulating lesions, lesion size, rate of lesion expansion, diseased leaf area, and number of spores per lesion were reduced. Lesion size and sporulation per lesion were lowered by 30 to 45%, and the number of sporulating lesions per leaf and diseas...

Evolution of Fusarium oxysporum f. sp. vasinfectum Races Inferred from Multigene Genealogies.

Abstract: Fusarium wilt of cotton is a serious fungal disease responsible for significant yield losses throughout the world. Evolution of the causal organism Fusarium oxysporum f. sp. vasinfectum, including the eight races described for this specialized form, was studied using multigene genealogies. Partial sequences of translation elongation factor (EF-1alpha), nitrate reductase (NIR), phosphate permase (PHO), and the mitochondrial small subunit (mtSSU) rDNA were sequenced in 28 isolates of F. oxysporum f. sp. vasinfectum selected to represent the global genetic diversity of this forma specialis. Results of a Wilcoxon Signed-Ranks Templeton test indicated that sequences of the four genes could be combined. In addition, using combined data from EF-1alpha and mtSSU rDNA, the phylogenetic origin of F. oxysporum f. sp. vasinfectum within the F. oxysporum complex was evaluated by the Kishino-Hasegawa likelihood test. Results of this test indicated the eight races of F. oxysporum f. sp. vasinfectum appeared to be nonmonophyletic, having at least two independent, or polyphyletic, evolutionary origins. Races 3 and 5 formed a strongly supported clade separate from the other six races. The combined EF-1alpha, NIR, PHO, and mtSSU rDNA sequence data from the 28 isolates of F. oxysporum f. sp. vasinfectum recovered four lineages that correlated with differences in virulence and geographic origin: lineage I contained race 3, mostly from Egypt, and race 5 from Sudan; lineage II contained races 1, 2, and 6 from North and South America and Africa; lineage III contained race 8 from China; and lineage IV contained isolates of races 4 and 7 from India and China, respectively.

Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3.

Abstract: Stenotrophomonas maltophilia strain C3, a biocontrol agent of Bipolaris sorokiniana in turfgrass, produced chitinases in broth media containing chitin. Chitinases were partially purified from culture fluid by ammonium sulfate precipitation and chitin affinity chromatography. The chromatography fraction with the highest specific chitinase activity was inhibitory to conidial germination and germ-tube elongation of B. sorokiniana, but it was less inhibitory than the protein fraction or the raw culture filtrate. The fraction exhibited strong exochitinase and weak endo-chitinase activity. Optimum temperature and pH for chitinase activity were 45 to 50 degrees C and 4.5 to 5.0, respectively. Chitinase activity was inhibited by Hg(2+) and Fe(3+), but not by other metal ions or enzyme inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the chromatography fraction revealed the presence of five protein bands of 25, 32, 48, 65, and 75 kDa. Partial amino acid sequences of the 32-, 65-, and 75-kDa proteins indicated that they are homologous to known bacterial chitinases. There was no homology found in the partial amino acid sequences of the 25- and 48-kDa proteins to any known chitinases. Five chitinase-active proteins were detected in the protein and chromatography fractions by activity gels, but when each protein was extracted and re-electrophoresed separately under denaturing conditions, only 32- or 48-kDa proteins were revealed. It was concluded that strain C3 produces at least two chitinases that are antifungal.

Genetic Control and Host Range of Avirulence Toward Brassica napus Cultivars Quinta and Jet Neuf in Leptosphaeria maculans.

Abstract: Leptosphaeria maculans causes blackleg of oilseed rape. Gene-for-gene interactions between race PG3 and Brassica napus cv. Quinta were related to interaction between the fungal avirulence (Avr) gene AvrLm1 and the corresponding resistance gene Rlm1. AvrLm1 isolates were aviru-lent on cvs. Doublol, Vivol, Columbus, and Capitol, and no recombinant phenotypes were observed in the progeny of two AvrLm1 × avrLm1 crosses, suggesting that all of these cultivars may possess Rlm1 or genes displaying the same recognition spectrum, or that a cluster of Avr genes is present at the Avrlm1 locus. In one cross, segregation distortion was observed at the AvrLm1 locus that could be explained by interaction between AvrLm1 and one unlinked deleterious gene, termed Del1. Incompatibility toward cvs. Jet Neuf and Darmor.bzh was governed by a single gene, unlinked to AvrLm1 or Del1. This avirulence gene was termed AvrLm4. Preliminary plant genetic analysis suggested the occurrence of a corresponding dominant resistance...

Detection of rice panicle blast with multispectral radiometer and the potential of using airborne multispectral scanners.

Abstract: Rice reflectance was measured to determine the spectral regions most sensitive to panicle blast infection. Reflectance increased in the 430- to 530-, 580- to 680-, and 1,480- to 2,000-nm regions at the dough stage both in the laboratory and the field as the percentage of diseased spikelets increased. The wavebands of the greatest sensitivity were in the visible region, located near 485 and 675 nm. After the yellow-ripe growth stage, near-infrared rather than visible reflectance responded to panicle blast infections. Ratios of rice reflectance were evaluated as indicators of panicle blast. R470/R570 (reflectance at 470 nm divided by reflectance at 570 nm), R520/R675, and R570/R675 decreased significantly as the incidence of panicle blast increased at the dough stage. At the yellow-ripe stage, R550/R970 and R725/R900 were used to estimate panicle blast severity as measured in terms of the percentage of diseased spikelets. According to the simulation that uses ground-based sensor data, airborne multispectral scanners may be effective in detecting the occurrence of panicle blast using a band combination of 530- to 570- and 650- to 700-nm regions at the dough stage.

Effect of Host Plant Resistance to Tomato yellow leaf curl virus (TYLCV) on Virus Acquisition and Transmission by Its Whitefly Vector.

Abstract: The effect that Tomato yellow leaf curl virus (TYLCV)-infected resistant tomato plants may have on virus epidemiology was studied. Four tomato genotypes that exhibit different levels of viral resistance, ranging from fully susceptible to highly resistant, served as TYLCV-infected source plants. Viral acquisition and transmission rates by white-flies following feeding on the different source plants were evaluated. TYLCV transmission rate by whiteflies that had fed on infected source plants 21 days postinoculation (DPI), shortly after the appearance of TYLCV symptoms, was negatively correlated with the level of resistance displayed by the source plant. Therefore, the higher the resistance, the lower the transmission rate. In addition, TYLCV DNA accumulation was shown to be lower in the resistant source plants compared with the susceptible plants. Whitefly survival rate, following feeding on source plants 21 DPI, was similar for all the cultivars tested. Significant differences in whitefly survival ...

Role of iron in rhizobacteria-mediated induced systemic resistance of cucumber

Abstract: Press, C. M., Loper, J. E., and Kloepper, J. W. 2001. Role of iron in rhizobacteria-mediated induced systemic resistance of cucumber. Phytopathology 91:593-598. Seed treatment with the rhizosphere bacterium Serratia marcescens strain 90-166 suppressed anthracnose of cucumber, caused by Colletotrichum orbiculare, through induced systemic resistance (ISR). When the iron concentration of a planting mix was decreased by addition of an iron chelator, suppression of cucumber anthracnose by strain 90-166 was significantly improved. Strain 90-166 produced 465 ± 70 mg/liter of catechol siderophore, as determined by the Rioux assay in deferrated King’s medium B. The hypothesis that a catechol siderophore produced by strain 90-166 may be responsible for induction of systemic resistance by this strain was tested by evaluating disease suppression by a miniTn5-phoA mutant deficient in siderophore production. Sequence analysis of genomic DNA flanking the mini-Tn5-phoA insertion identified the target gene as entA, which encodes an enzyme in the catechol siderophore biosynthetic pathways of several bacteria. Severity of anthracnose of cucumbers treated with the entA mutant was not significantly different (P = 0.05) from the control, whereas plants treated with wild-type 90-166 had significantly less disease (P = 0.05) than the control. Total (internal and external) population sizes of 90-166 and the entA mutant on roots did not differ significantly (P = 0.05) at any sample time, whereas internal population sizes of the entA mutant were significantly lower (P = 0.05) than those of the wild-type strain at two sampling times. These data suggest that catechol siderophore biosynthesis genes in Serratia marcescens 90-166 are associated with ISR but that this role may be indirect via a reduction in internal root populations.

Cytological Analysis of Defense-Related Mechanisms Induced in Pea Root Tissues in Response to Colonization by Nonpathogenic Fusarium oxysporum Fo47.

Abstract: The ability of nonpathogenic Fusarium oxysporum, strain Fo47, to trigger plant defense reactions was investigated using Ri T-DNA-transformed pea roots. Cytological investigations of strain Fo47-inoculated roots showed that the fungus grew actively at the root surface and colonized a number of epidermal and cortical cells, inducing marked host cell metabolic changes. In roots inoculated with pathogenic F. oxysporum f. sp. pisi, the pathogen multiplied abundantly through much of the tissues, whereas in Fo47-inoculated roots, fungal growth was restricted to the epidermis and the outer cortex. Invading cells of strain Fo47 suffered from serious alterations, a phenomenon that was not observed in control roots in which F. oxysporum f. sp. pisi grew so actively that the vascular stele was invaded within a few days. Strain Fo47 establishment in the root tissues resulted in a massive elaboration of hemispherical wall appositions and in the deposition of an electron-opaque material frequently encircling pathogen hyphae and accumulating in the noninfected xylem vessels. This suggests that the host roots were signaled to defend themselves through the rapid stimulation of a general cascade of nonspecific defense responses. The specific relationship established between strain Fo47 and the root tissues is discussed in relation to other types of plant-fungus interactions, including pathogenic and symbiotic associations.

Biocontrol Potential of Metchnikowia pulcherrima Strains Against Blue Mold of Apple.

Abstract: Eight strains of Metschnikowia pulcherrima isolated over a 4-year period from an unmanaged orchard and selected for their biocontrol activity against blue mold (caused by Penicillium expansum) of apples were characterized phenotypically, genetically, and for their biocontrol potential against blue mold on apples. All strains grew well and only differed slightly in their growth in nutrient yeast dextrose broth medium at 1°C after 216 h, but large differences occurred at 0°C, with strain T5-A2 outgrowing other strains by more than 25% transmittance after 360 h. This strain was also one of the most resistant to diphenylamine (DPA), a postharvest antioxidant treatment. All strains required biotin for growth in minimum salt (MS) medium, although strain ST2-A10 grew slightly in MS medium containing riboflavin or folic acid, as did ST3-E1 in MS medium without vitamins. None of the strains produced killer toxins against an indicator strain of Saccharomyces cerevisiae. Analysis of Biolog data from YT plat...

Germination and Sporulation of Colletotrichum acutatum on Symptomless Strawberry Leaves

Abstract: The germination and sporulation of Colletotrichum acutatum were characterized over time on strawberry leaves (cv. Tristar) and plastic coverslips incubated at 26 degrees C under continuous wetness. Conidia germinated within 3 h after inoculation and formed melanized appressoria with pores by 9 h after inoculation. Host penetration was not observed up to 7 days after inoculation. Production of secondary conidia on conidial and hyphal phialides began within 6 h after inoculation. Secondary conidiation was responsible for up to a threefold increase in the total number of conidia within 7 days after inoculation. Primary conidia and hyphae began to collapse 48 h after inoculation, whereas melanized appressoria remained intact. These findings suggest that appressoria and secondary conidia of C. acutatum produced on symptomless strawberry foliage may be significant sources of inoculum for fruit infections.

Virulence and Molecular Diversity in Cochliobolus sativus.

Abstract: Spot blotch, caused by the fungal pathogen Cochliobolus sativus, is an important disease of barley in many production areas of the world. To assess genetic diversity in this pathogen, a worldwide collection of C. sativus isolates was evaluated for virulence on barley and DNA polymorphism. Three pathotypes (0, 1, and 2) were identified among the 22 isolates tested in this study and the 36 isolates characterized previously on three barley differentials (ND5883, Bowman, and NDB112) that differ in their resistance to C. sativus. Pathotype 2, which exhibits high virulence on cv. Bowman, was only found in North Dakota, whereas the other two pathotypes occurred in many other regions of the world. Genetic diversity of the 58 C. sativus isolates, together with isolates of three related pathogenic Cochliobolus spp. (C. heterostrophus, C. carbonum, and C. victoriae) was analyzed using amplified fragment length polymorphism (AFLP) markers. A total of 577 polymorphic AFLP markers were recorded among the 70 is...